DETERMINATION OF ADSORPTION COEFFICIENT OF ON SOIL USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

OECD Proposal for a New Guideline 121 Estimation of the Adsorption Coefficient (K

oc ) on Soil and on Sewage Sludge Using High Performance Liquid Chromatography (HPLC)- 2 -

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS



### No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA section 10(d)(1)(A), (B) or (C).

Company :

# Wildlife International, Ltd. Project Number 523~-112

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

SPONSOR:

This study was conducted in compliance with OECD Principles of Good Laboratory Practice, (ENVhlCICHEM (98)17) and Title 40 of the Code of Federal Regulations, Parts 160 and 792, Good Laboratory Practice Standards, 1989, with the following exception:

Characterization and stability of the test substance under storage conditions at the test site were not
determined in accordance with Good Laboratory Practice Standards.

Chemist /

SPONSOR APPROVAL:

Sponsor's Representative DATEQUALITY ASSURANCE STATEMENT

This study was examined for compliance with 

### OECD Principles of Good Laboratory Practice, (ENVMCICHEM (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 June 7,2005 June 7,2005 June 22,2005

Test Substance Preparation July 15,2005 July 15,2005 July 18,2005

Raw Data and Draft Report September 6-7,2005 September 7,2005 September 13,2005

Final Report September 14,2005 September 14,2005 September 14,2005

All inspections were study-based unless otherwise noted.

hes H. Coleman Quality Assurance Representative DATE-5 

### -

REPORT APPROVAL

SPONSOR:TABLE OF CONTENTS

Title/Cover Pa ge ............................................................................................................... ...................... 1

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

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

Quality Assurance Statement

.................................................................................................... .............. 4

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

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

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

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

Purpose ........................................................................................................................ ........................... 9

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

Materials and Methods.......................................................................................................... ................ 10

Test Substance................................................................................................................. ............. 10

Reference Standards ............................................................................................................ ......... 10 Solvents and Reagents.......................................................................................................... ........ 11 Mobile Phase Preparation....................................................................................................... ...... 12 Calibration Stocks and Standards Preparation .............................................................................. 12 Test Substance Stock and Solution Preparation............................................................................ 12

Test Procedure................................................................................................................. ............. 12 Data Analysis.................................................................................................................. .............. 13

Results ........................................................................................................................ .......................... 13

Conclusions .................................................................................................................... ...................... 14

References ..................................................................................................................... ....................... 15- 7 -

TABLE OF CONTENTS

- Continued -

TABLES

Table 1 - Preparation of Reference Stocks and Standard Solutions..................................................... 16

Table 2 - Typical HPLC Operational Parameters ................................................................................ 17

Table 3 - Reference Standards Retention Times, Capacity Factors, and Log K

oc Values .................... 18

Table 4 - Adsorption Coefficients (Log K

oc ) for ................................................................. 19

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 acet anilide reference standa rd ................................. 22

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

Figure 5 - A representative chromatogram of an an iline 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 DDT reference standard............................................ 27

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

APPENDICES

Appendix 1 - Protocol and Protocol Amendment ........................................................................... 29

Appendix 2 - Example Calculation................................................................................................. 40

Appendix 3 - Personnel Involved in the Study................................................................................ 41- 8 -

SUMMARY

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

resolved peaks. The corresponding adsorption coefficients (Log K

oc ) for the test substance

were calculated as 1.33, 2.43, 3.83, 4.29, 4.34 and 4.61.INTRODUCTION

Wildlife International, Ltd. determined the adsorption coefficient of the test substance using the high

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

International, Ltd. analytical chemistry facility in Easton, Maryland. The study was performed following

procedures in the OECD Proposal for a New Guideline 121 (adopted January 22, 2001): Estimation of the

Adsorption Coefficient (K

oc ) on Soil and on Sewage Sludge using High Performance Liquid

Chromatography(HPLC) (1).

The adsorption coefficient of the test subtance was determined using high performance liquid

chromatography (HPLC) with ultraviolet detection (UV). The study was identified as Project Number

523C-112. All raw data generated by Wildlife International, Ltd. and a copy of the final report are filed

under Project Number 523C-112 in archives located on the Wildlife International, Ltd. site.

PURPOSE

The purpose of this study was to experimentally determine an estimate of adsorption coefficient

(K

oc ) of the test substance by a reverse-phase HPLC method.

EXPERIMENTAL DESIGN

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

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

oc range of the

HPLC estimation method were separately prepared in either acetonitrile or methanol and diluted in an 55%

methanol:45% HPLC grade water (v/v) HPLC mobile phase. The reference and test substance solutions

were sequentially injected into an HPLC system operating under standardized isocratic, reverse-phase

operating conditions employing a CN-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 K

oc values to establish a linear regression calibration. The capacity factors of the test

substance were used in conjunction with the linear regression equation to calculate the Log K

oc range for

the test substance.- 10 -

MATERIALS AND METHODS

Test Substance

The test substance was received from and was assigned- 11 -

Aniline was received from ChemSe rvice on August 12, 2004, and was assigned

identification number 6782. The reference standard was identified on the label as

Aniline; lot/batch number 320- 36A; CAS number 62-53-3. The reference standard was a clear liquid, had

a reported purity of 99.5%, with an expiration date of April, 2009, and was stored under ambient

conditions.

stored under ambient conditions.Mobile Phase Preparation

The HPLC mobile phase was 55% methanol: 45% HPLC grade water (v/v). The mobile phase was

degassed prior to use.

Calibration Stocks and Standards Preparation

Seven reference standards were used to generate a Log K

oc calibration curve. Calibration stock

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

or methanol. Calibration standard solutions were prepared from these stock solutions of the reference

standards by diluting appropriately in 55% methanol: 45% HPLC grade water (v/v). The concentrations of

the reference standards were adjust ed, as necessary, to give a detect or response of at leas t 270 milliabsorbance 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 as prep ared by accurately weighing 0.500 g of the test substance into a

20-mL glass scintillation vial . The test substance was then qua ntitatively transfe rred 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 aceton itrile stock solu tion yielded a 500 μ g

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

solution gave a detector response of at least 100 m illi-absorbance units for the p eak 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 100 variable wavelength UV detector. Chromatographic separations were achieved using a

Zorbax CN analytical column (150 mm x 4.6 mm, 5- μ m 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.- 13 -

Reference standards (100 μ L 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 analy zed 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 ′ = (t R – t 0 )/t 0

where t R was the retention time of the test substance or reference standard and t 0 was the column dead time

established with thiourea. A correlation graph of Log k ′ versus Log K

oc 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 K

oc for the test substance was calculated by substituting the calculated logarithm of the

capacity factor for the test substance 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 (t 0 ) 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 2.034 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,

phenol, aniline, 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 six resolved

peaks. The mean peak retention times for the test substance ranged from 2.810 ± 0.0993 minutes to- 14 -

11.787 ± 0.568 minutes. The capacity factors (k ′ ) for the minimum and maximum retained test substance

components were then calculated based on the above retention time range. The corresponding adsorption

coefficients (Log K

oc ) for the test substance were calculated to range from 1.33 to 4.61. The calculated

mean retention times, capacity factors, and mean adsorption 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 six

resolved peaks. The corresponding adsorption coefficients (Log K

oc ) for the test substance were calculated

as 1.33, 2.43, 3.83, 4.29, 4.34 and 4.61.- 15 -

REFERENCES

1. Organisation of Economic Cooperation and Development. 2001. Guideline for the Testing of

Chemicals, 121: Estima tion of Adsorption Coefficient (K oc) on Soil and Sew age Sludge using
High Performance Liquid Chromatography (HPLC)

.

2. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01: 45-47.- 16 -

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 ( μ g/mL)

Thiourea 500 100 5.00 0.0500 10.0 25.0

Acetanilid e 500 100 5.00 0.0500 10.0 25.0

Phenol 500 100 5.00 0.200 10.0 100

Aniline 500 100 5.00 0.0500 10.0 25.0

Naphthalene 492 100 4.92 0.0500 10.0 24.6

Phenanthrene 500 100 5.00 0.0500 10.0 25.0

DDT 500 100 5.00 0.100 10.0 50.0

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

2 Initial volumes measured with a gas-tight syringe.

3 Solutions were brought to final volume with 55% methanol:45% HPLC grade water (v/v) HPLC

mobile phase in volumetric flasks.- 17 -

Table 2

Typical HPLC Operational Parameters

INSTRUMENT: Agilent Series 1100 High Performance Liquid Chromatograph
(HPLC) with an Agilent Series 1100 Variable Wavelength (UV)

Detector

ANALYTICAL COLUMN: Zorbax CN (150 mm x 4.6 mm, 5- μ m particle size)

STOP TIME: 30.0 minutes

FLOW RATE: 1.0 mL/minute

OVEN TEMPERATURE: 40 ° C

MOBILE PHASE:Table 3

Reference Standards Retention Times, Capacity Factors, and Log K

oc Values

Reference

Standard Concentration ( μ g/mL) Retention

Time (minutes) Capacity

Factor (k ′ ) 1 Log k ′ 2 Log K

oc 3y = 0. 3360x

- 0. 8683

R 2

= 0. 9779

- 1.

0 - 0.

5 0.

0 0.

5 1.

0 1.

5

0. 0 1. 0 2. 0 3. 0 4. 0 5. 0 6. 0 7. 0

Log Koc

Figure 1. Reference standard calibration curve.- 21 -

min 0 5 10 15 20 25 mAU

0 500 1000 1500 2000 2500 3000 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\002-0201.D)2.038

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

min 0 5 10 15 20 25 mAU

0 500 1000 1500 2000 2500 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\003-0301.D)2.623

Figure 3. A representative chromatogram of an acetanilide reference standard.min 0 5 10 15 20 25 mAU

0 500 1000 1500 2000 2500 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\004-0401.D)2.724

Figure 4. A representative chromatogram of a phenol reference standard.- 24 -

min 0 5 10 15 20 25 mAU

0 500 1000 1500 2000 2500 3000 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\005-1501.D)3.913

Figure 5. A representative chromatogram of an aniline reference standard.min 0 5 10 15 20 25 mAU

0 500 1000 1500 2000 2500 3000 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\006-0601.D)5.025

Figure 6. A representative chromatogram of a naphthalene reference standard.min 0 5 10 15 20 25 mAU

0 500 1000 1500 2000 2500 3000 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\007-0701.D)8.855

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

0 500 1000 1500 2000 2500 3000 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\008-0801.D)20.595

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

0 500 1000 1500 2000 2500 3000 VWD1 A, Wavelength=210 nm (S:\HPCHEM\1\DATA\OECD002\009-0901.D)2.9054.0177.71710.04910.36312.347

Figure 9. A representative chromatogram of the test substance,Appendix 1

Protocol and Protocol Amendment- 34 -- 35 -- 36 -- 37 -- 38 -Appendix 2

Example Calculation

Mean dead volume retention time (thiourea) = 2.034 minutes. The capacity factor (k ′ ) for the 12.347 minute peak was calculated as:

k ′ = (t R – t 0 )/t 0

where t R was the retention time of the ending peak for the test substance and t 0 was the

column dead time established with thiourea.

k ′ = (12.347 – 2.034)/2.034

= 5.070

The logarithm (base 10) of the capacity factor = Log k ′ = 0.705

The n -octanol/water partition coefficient (Log K OW ) corresponding to the above capacity factor was

calculated from the linear regression equation established for the reference standards under the same

chromatographic conditions:

Regression equation: y = mx + b, where y = Log k ′ x = Log K

oc m = slope = 0.3360 b = y-intercept = -0.8683

0.705 = (0.3360 × Log K

oc ) – 0.8683
Log K

oc = (0.705 + 0.8683) ÷ 0.3360

Log K

oc = 4.68- 41 -

Appendix 3

Personnel Involved in the Study

m

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

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