Document YGjNNmNQmaXbKGv1zB9NJQ69E
AR226-1891
Final Report
Study Title
(14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium Method
Data Requirement
Protocol designed to meet the known requirements of OECD Guideline 106 (January 2000)
Author
S Swales
Sponsor Test Facility
Katie Smythe, Administrator Telomer Research Program The Rand Corporation 1200 South Hayes Street Arlington, VA 22202 USA
Covance Laboratories Ltd Otley Road, Harrogate North Yorkshire HG3 1PY ENGLAND
RECEIVED OPPT NCIC 2004 DEC 22 AM 11:20
Covance Study Number 2304/001
Covance Report Number 2304/001-D2149
Report Issued
16 July 2004
Page Number
1 of 89
Covance Study Number 2304/001 Final Report
STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS
(14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium Method
No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA Sections 10(d)(1)(A), (B) or (C).
Company
Telomer Research Program
Company Agent:
Date:
These data are the property of the Telomer Research Program, and as such are considered to be confidential for all purposes other than compliance with FIFRA 10. Submission of these data in compliance with FIFRA does not constitute a waiver of any right to confidentiality, which may exist under any statue or in any other country.
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Covance Study NumbeFirna2l30R4e1p0o0r1t GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
(*C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium
Method `The study was performed in accordance with the agreed protocol and with Covance Laboratories Limited, Standard Operating Procedures, unless otherwise stated, and the study objectives were achieved. The study was conducted in compliance with the United Kingdom Statutory Instrument No. 3106, The Good Laboratory Practice Regulations 1999, from 27th April 2004 as amended by the Good Laboratory Practice (Codification Amendments Etc.) Regulations 2004, and the OECD Principles on Good Laboratory Practice (revised 1997, Issued January 1998) ENV/MC/CHEM (98)17. Studies performed in compliance with the GLP regulations and standards referenced above will be fully acceptable to the US EPA regulatory authorities.
C S Swe ales e Study Director, Covance Laboratories Ltd
Ho uly oon
|
Date
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Name
Date
Submitter
Department or Company
NSaubmmietter Department or Company Name Submitter Department or Company
Date
Date 3.
Covance Study Number 2304/001 Final Report
FLAGGING STATEMENTS (14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium
Method
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Covance Study NumbeFirna2l3R0e4p0o0r1t
STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT
("*C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium
Method
1, the undersigned, hereby declare that the work was performed by me or under my
supervision and that the findings provide a true and accurate record of the results obtained.
The study was performed in accordance with the agreed protocol and with Covance Laboratories Limited Standard Operating Procedures, unless otherwise stated, and the
study objectives were achieved. The study was conducted in accordance with the following intemational Codes of Good Laboratory Practice:
+ United Kingdom Statutory Instrument 1999 No.3106 the Good Laboratory Practice Regulations 1999, from 27th April 2004 as amended by the Good Laboratory Practice (Codification Amendments Etc.) Regulations 2004
+ The OECD Principles on Good Laboratory Practice (revised 1997, issued Jan 1998) ENV/MC/CHEM(98)17
Soils used for the study were sterilised by gamma irradiation at Isotron (Bradford, West Yorkshire). Isotron is not partof the UK GLP compliance-monitoring program.
== I S Swales BSc MSc PhD Study Director
(3 iy 2004 Date
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`Covance Study Number 23041001
QUALITY ASSURANCE STATEMENT
(C)-8-2 TelomeBr Alcohol: Adsorption/Desorption using a Batch Equilibrium
Method
`This study has been reviewed by the Quality Assurance Unitof Covance Laboratories Ltd. and the report accurately reflects the raw data. The following inspections were conducted and findings reported to the Study Director (SD) and associated `management.
Critical procedures which are performed routinely in an operational area, may be audited as part ofa "process" inspection program. This can be in addition to phases scheduled on an individual study basis. Selected process inspections conducted and considered applicable to this study are included below.
-- Inspection Dates TIFMroamr20 7MaTro2008 2G6MAarN20S03 0326AMparr2200033 2A 7Novg 2B 1237NAouvg22000033 B 18Fe0D 2004 0293DMeacr220000s4
--
TDTueRefoSpDanodSsD d
ProtoPchoalRseeview
Maragement 17Mar2003
Pro"toecsoltAAmveincdemReecnetipRetv.iew
0236AMparr22000033
PPrroottooccooll AAmmeennddmmeennttRReevviieeww
1257NAouvg22000033
DraDoRsepeoArdtmainndDisuattaiRoenview
2039DMeacr22000034
FrIopmosinDusTo 0TMSaMayi22000035 133M0aMayy22000030 32002000m3 230040m2200033
Process iaPPhraocseessing
High Perfomance`LSaimpdleCRehcreioptmatogrAnaaplyhsyis `Sample reparation
TeSDReapSorDt 1M3aMnaagye2m0e0n3t 2304Mjamy22000033 301m2003
T it
{
Section Head, Quality Assurance
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Date 4
7
Covance Study NumbeFirna2l30Re4p/o0r1t RESPONSIBLE SCIENTIST'S STATEMENT
(C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium
Method 1, the undersigned, hereby declare that I have reviewed this report in conjunction with the Study Director and that the interpretation and presentation of the data in the report are consistent with the results obtained.
la AHI BSc PhD Director, Environmental Sciences
16Tuuley 2 Date
7.
Covance Study Number 2304/001 Final Report
RESPONSIBLE PERSONNEL
(14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium Method
The following personnel were responsible for key elements of the study:
STUDY MANAGEMENT Study Director Deputy Study Director Scientific Reviewer Study Co-ordinator
OPERATIONAL SUPERVISION Study Supervisor Formulation Chemical Analysis Data Processing In Life Supervisor
QUALITY ASSURANCE Director, Quality Systems
NAME S Swales C Lewis A Hill C Unsworth
K Dixon K Dixon K Dixon K Dixon P Davies
C Clare
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Covance Study Number 2304/001 Final Report
ARCHIVE STATEMENT (14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium
Method All primary data, or authenticated copies thereof, samples and the final report will be retained in the Covance Laboratories Limited archives for one year after issue of the final report. At this time, the sponsor will be contacted to determine whether data should be returned, retained or destroyed on their behalf. Sponsors will be notified of the financial implications of each of these options at that time. All details supplied with the soils and all raw data generated by National Soil Resource Institute (NSRI) will be archived at CLE. Samples requiring frozen storage are specifically excluded from the above. Samples requiring chromatographic analysis and adsorption/desorption solutions will be retained for as long as the quality of the material permits but for no longer than six months after issue of the draft report or three months after the issue of the final report, whichever is shorter. The study sponsor will be notified before such samples are destroyed on their behalf. All other samples may be disposed of in an ongoing manner.
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Covance Study Number 2304/001 Final Report
CONTENTS
STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS .............................................................2
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ...................................................3
FLAGGING STATEMENTS ...................................................................................................................4
STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT ......................5
QUALITY ASSURANCE STATEMENT ...............................................................................................6
RESPONSIBLE SCIENTIST'S STATEMENT .......................................................................................7
RESPONSIBLE PERSONNEL................................................................................................................8
ARCHIVE STATEMENT........................................................................................................................9
CONTENTS ...........................................................................................................................................10
SUMMARY............................................................................................................................................13
INTRODUCTION ..................................................................................................................................16
MATERIALS AND METHODS............................................................................................................17 Protocol adherence ..........................................................................................................................17 Test article .......................................................................................................................................17 Non-radiolabelled test article...........................................................................................................17 Solvents and reagents ......................................................................................................................18 Test System .....................................................................................................................................18 Test article application.....................................................................................................................20 Solubility test and adsorption to vessels ..........................................................................................21 Ratio of soil to aqueous phase .........................................................................................................22 Equilibrium time determination and stability ..................................................................................23 Adsorption isotherms.......................................................................................................................25 Desorption isotherms.......................................................................................................................26 Mass balance ...................................................................................................................................26 pH measurements ............................................................................................................................26 Analytical Methods and Measurements...........................................................................................27 Radioactivity....................................................................................................................................27 Calculations .....................................................................................................................................28 Expression of Results ......................................................................................................................30
RESULTS ...............................................................................................................................................31 Preliminary tests ..............................................................................................................................31 Adsorption and desorption isotherms ..............................................................................................33
SUMMARY AND CONCLUSIONS .....................................................................................................35
TABLES .................................................................................................................................................36 Table 1 1:1 Ratio of soil to aqueous phase following treatment at 0.025 g/mL ............................37 Table 2 1:5 Ratio of soil to aqueous phase following treatment at 0.025 g/mL ............................37 Table 3 1:25 Ratio of soil to aqueous phase following treatment at 0.025 g/mL ..........................37 Table 4 Recovery of applied radioactivity from the 24 hour equilibrium time samples at a soil to aqueous phase ratio of 1:1 and 20 1C .....................................................................................38 Table 5 Stability over a 24 hour period (20C) at a soil to aqueous phase ratio of 1:1 ...................38
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Covance Study Number 2304/001 Final Report
Table 6 Comparison of the applied radioactivity recovered in the supernatants from soils (soil to aqueous phase ratio of 1:1)..........................................................................................................38 Table 7 Stability over a 3 hour period (4C) at a soil to aqueous phase ratio of 1:1 .......................39 Table 8 Percent applied radioactivity recovered in the supernatants from sterile soils at 4C and stability of 8-2 TBA in the soil extract of sterile soil................................................................39 Table 9 Percent applied radioactivity recovered in the desorption supernatants from sterile soils at 4C following a 3 h equilibrium time..................................................................................39 Table 10 Values for Ce, X/m, C1 and X1/m for 8-2 TBA on soil SK961089 ...................................40 Table 11 Values for Ce, X/m, C1 and X1/m for 8-2 TBA on soil SK566696 ...................................40 Table 12 Values for Ce, X/m, C1 and X1/m for 8-2 TBA on Emperor Son's Lake sediment...........41 Table 13 Values for Ce, X/m, C1 and X1/m for 8-2 TBA on soil SK179618 ...................................41 Table 14 Values for Ce, X/m, C1 and X1/m for 8-2 TBA on Eurosoil 5 ..........................................42 Table 15 Quantities of 8-2 TBA adsorbed to and desorbed from soil SK961089 ...........................42 Table 16 Quantities of 8-2 TBA adsorbed to and desorbed from soil SK566696 ...........................43 Table 17 Quantities of 8-2 TBA adsorbed to and desorbed from Emperor Son's Lake sediment...43 Table 18 Quantities of 8-2 TBA adsorbed to and desorbed from soil SK 179618 ..........................44 Table 19 Quantities of 8-2 TBA adsorbed to and desorbed from Eurosoil 5...................................44 Table 20 Adsorption coefficients for 8-2 TBA on soil SK961089 ..................................................45 Table 21 Adsorption coefficients for 8-2 TBA on soil SK566696 ..................................................45 Table 22 Adsorption coefficients for 8-2 TBA on Emperor Son's Lake .........................................46 Table 23 Adsorption coefficients for 8-2 TBA on soil SK179618 ..................................................46 Table 24 Adsorption coefficients for 8-2 TBA on Eurosoil 5 .........................................................47 Table 25 Freundlich adsorption and desorption constants and regression analysis for 8-2 TBA on soil SK961089 ............................................................................................................................47 Table 26 Freundlich adsorption and desorption constants and regression analysis for 8-2 TBA on soil SK566696 ............................................................................................................................48 Table 27 Freundlich adsorption and desorption constants and regression analysis for 8-2 TBA on Emperor Son's Lake sediment....................................................................................................48 Table 28 Freundlich adsorption and desorption constants and regression analysis for 8-2 TBA on soil SK179618 ............................................................................................................................49 Table 29 Freundlich adsorption and desorption constants and regression analysis for 8-2 TBA on Eurosoil 5....................................................................................................................................49 Table 30 Recovery of radioactivity from all five soils treated with 8-2 TBA at 0.125 g/mL after adsorption, desorption, organic wash and combustion ............................................................50 Table 31 pH of the blank solutions used for the main isotherms test ..............................................50 Table 32 pH of the adsorption supernatants from the main isotherms test following treatment with 8-2 TBA...................................................................................................................................50
FIGURES................................................................................................................................................52 Figure 1 Adsorption equilibrium time data for soil SK961089 following treatment with 82 TBA ..............................................................................................................................................53 Figure 2 Adsorption equilibrium time data for soil SK566696 following treatment with 82 TBA ..............................................................................................................................................54 Figure 3 Adsorption equilibrium time data for Emperor Son's Lake sediment following treatment with 8-2 TBA...................................................................................................................55 Figure 4 Adsorption equilibrium time data for soil SK179618 following treatment with 82 TBA ..............................................................................................................................................56 Figure 5 Stability of 8-2 TBA in the soil extract of non-sterile SK961089 soil following adsorption for 24 h at 20C determined by TLC .............................................................................57 Figure 6 Stability of 8-2 TBA in the soil extract of non-sterile SK566696 soil following adsorption for 24 h at 20C determined by TLC .............................................................................58 Figure 7 Stability of 8-2 TBA in the soil extract of non-sterile Emperor Son's Lake sediment following adsorption for 24 h at 20C determined by TLC.............................................................59 Figure 8 Stability of 8-2 TBA in the soil extract of non-sterile SK179618 soil following adsorption for 24 h at 20C determined by TLC .............................................................................60 Figure 9 Stability of 8-2 TBA in the soil extract of non-sterile SK961089 soil following adsorption for 3 h at 4C determined by TLC .................................................................................61 Figure 10 Stability of 8-2 TBA in the soil extract of non-sterile SK566696 soil following adsorption for 3 h at 4C determined by TLC .................................................................................62
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Covance Study Number 2304/001 Final Report
Figure 11 Stability of 8-2 TBA in the soil extract of non-sterile Emperor Son's Lake sediment following adsorption for 3 h at 4C determined by TLC.................................................................63 Figure 12 Stability of 8-2 TBA in the soil extract of non-sterile SK179618 soil following adsorption for 3 h at 4C determined by TLC .................................................................................64 Figure 13 Stability of 8-2 TBA in the soil extract of sterile SK179618 soil following adsorption for 3 h at 4C determined by TLC .................................................................................65 Figure 14 Stability of 8-2 TBA in the soil extract of sterile Eurosoil 5 soil following adsorption for 3 h at 4C determined by TLC .................................................................................66 Figure 15 Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK961089 at 4C......67 Figure 16 Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK566696 at 4C......68 Figure 17 Adsorption and desorption isotherms for 8-2 TBA on sterile Emperor Son's Lake sediment at 4C................................................................................................................................69 Figure 18 Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK179618 at 4C......70 Figure 19 Adsorption and desorption isotherms for 8-2 TBA on sterile Eurosoil 5 soil at 4C ......71 APPENDICES ........................................................................................................................................72 Appendix 1 Certificates of analysis .................................................................................................73 Appendix 2 Soil certificates of analysis ..........................................................................................75 Appendix 3 Radiochemical purity of (14C)-8-2 TBA and formulation stability ..............................81 Appendix 4 Chromatography of reference standard ........................................................................85 Appendix 5 Minor deviations from protocol ...................................................................................86 Appendix 6 Example calculations ...................................................................................................87
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Covance Study Number 2304/001 Final Report
SUMMARY
The adsorption/desorption characteristics of (14C)-8-2 TBA were examined in four soil types and one sediment. The study was conducted to the OECD Guideline 106 (January 2000).
Preliminary experiments were conducted in the dark at 20 2C. Equilibrium time determination and isotherm tests were conducted in the dark at 4 1C. The air-dried soils were pre-conditioned by mixing overnight with 0.01M calcium chloride.
Preliminary tests were performed to assess the solubility and adsorption to containers. These tests demonstrated that in a sealed vial with minimal headspace 8-2 TBA may not exist fully in solution over a concentration range of 0.01 - 0.1 g/mL. Loss of radioactivity occurred when the vials were opened thereby confirming the volatile nature of 8-2 TBA. No definitive conclusions could be drawn from the preliminary solubility test due to the volatile nature of the compound. The amount of 8-2 TBA adsorbed to the test vessels from aqueous solution was 2% of the applied radioactivity. The concentrations used for the remainder of the study were selected such that they were below the stated water solubility (0.14 mg/L), but were high enough to ensure that the radioactivity could be detected throughout each stage of the study.
Three soils and a sediment from sites in the United Kingdom were used for the study. A fourth soil (Eurosoil 5) was obtained from the Institute for Reference Materials and Measurements in Belgium:
Soil name
SK961089 SK566696 Emperor Son's Lake SK179618 Eurosoil 5
pH (0.01M CaCl2)
7.6
4.2
6.0
Organic carbon content (%)
4.6
0.8
1.2
Organic Matter (%)
7.9
1.4
2.1
Clay content (%)
34
11
17
Soil texture
Clay loam Sandy loam Sandy loam
5.6
3.8
6.6
20
Clay loam
3.1
6.7
11.6
5
Loamy sand
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Further preliminary tests demonstrated that:
Covance Study Number 2304/001 Final Report
The percentage adsorption of 8-2 TBA to the test soils (not including Eurosoil 5) was >80% at ratios of soil to solution of 1:1 w/v and 1:5 w/v. For a soil:solution ratio of 1:25 the percentage adsorption of 8-2 TBA was >60% for the test soils. The recovery of applied radioactivity from the samples was dependent on the proportion of soil present with >90% being recovered at a soil:solution ratio of 1:1 w/v. The 1:1 ratio was, therefore, selected for the remainder of the study.
3 hours was a suitable adsorption and desorption equilibrium time for the study. The 8-2 TBA was not stable in the test system at 20C and therefore, the isotherms test was conducted using sterile soil, sterile 0.01M calcium chloride and at a reduced temperature of 4 1C.
Five dose formulations of (14C)-8-2 TBA were prepared at appropriate concentrations in sterile 0.01M calcium chloride. Aliquots (2.2, 5.2, 6.9 or 8.6 L) of the appropriate formulations were added to the pre-conditioned sterile soils to obtain duplicate units at five concentrations, nominally 0.125, 0.1, 0.075, 0.05 and 0.0125 g/mL.
Tests were performed in sealed glass vials with minimal headspace and aliquots were taken using a Hamilton SGE syringe (glass syringe with stainless steel needle and piston) inserted through the PTFE-lined rubber septum. All samples were mixed continuously for 3 hours on an end-over-end shaker at 4 1C. Each sample was then centrifuged to separate the soil and solution. The radioactivity in the supernatant was determined by assaying duplicate aliquots by LSC and the supernatant was removed.
The supernatant from each sample after the adsorption phase was replaced with an equivalent weighed portion of fresh 0.01M calcium chloride. The tubes were shaken to break up the compacted soil and were then placed back on the end-over-end shaker and mixed continuously for a further 3 hours for the desorption phase. Each sample was centrifuged to separate the soil and solution. The radioactivity in the supernatant was determined by assaying duplicate aliquots by LSC and the supernatant was removed.
The 0.125 g/mL samples were extracted three times with acetone (10 mL) and the supernatants for each sample pooled. The soil extracts for each sample were quantified by LSC. The extracted soil samples (0.125 g/mL) were air-dried then ground prior to combustion in oxygen.
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Covance Study Number 2304/001 Final Report
The Freundlich adsorption constants (Kf) were calculated by linear regression analysis
and the results were as follows:
Soil
SK961089 (clay loam) SK566696 (loamy sand) Emperor Son's lake sediment (sandy loam)
SK 179618 (loam) Eurosoil 5 (loamy sand)
Freundlich constant (Kf)
35 40 20 130 38
The Freundlich adsorption constants related to organic carbon content (Koc) as shown below:
Soil
SK961089 (clay loam) SK566696 (loamy sand) Emperor Son's lake sediment (sandy loam)
SK 179618 (loam) Eurosoil 5 (loamy sand)
(Koc)
765 4941 1667 3414 572
The adsorption and desorption isotherm showed a strong positive monotonic relationship between log Ce and log X/m, indicated by the correlation coefficients being close to 1. These values characterise 8-2 TBA as slightly mobile (Koc 500-4000) to non-mobile (Koc>4000) in the four soils and one sediment used in this study (Hollis, 1991)a
Mass balance for the four soils and one sediment were in the range 86 to 96%.
a Hollis, J M (1991). Mapping the vulnerability of aquifers and surface water to pesticide contamination at the national/regional scale. Pesticides in Soil and Water: Current Perspectives (A Walker Ed), BCPC Monograph 47, pp 169 to 174
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Covance Study Number 2304/001 Final Report
INTRODUCTION
(14C)-8-2 TBA (2-perfluorooctylethanol) used in this study had the following structure:
*Position of the 14C radiolabel
C8F17
* * OH
The objective of the study was to:
determine the adsorption/desorption characteristics of (14C)-8-2 TBA in four soil types and one sediment.
The study was conducted to meet the requirements of the OECD Guideline 106 (January 2000).
The study was initiated on 11 March 2003 (date the protocol was signed by the Study Director) and completed on 16 July 2004 (date the final report was signed by the Study Director). The practical phase of the study was conducted at Covance Laboratories Europe (CLE), Otley Road, Harrogate, North Yorkshire between 4 April 2003 (test article quantification) and 8 January 2004 (combustion of residues).
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Covance Study Number 2304/001 Final Report
MATERIALS AND METHODS
Protocol adherence The study described in this report was carried out according to the agreed protocol and three amendments. Minor deviations that did not affect the integrity or outcome of the study are listed in Appendix 5.
Test article The following physical information on the (14C)-test substance is known:
Common Names: Chemical Name: CAS Name: CAS Number: Lot Number: Radiochemical Purity: Specific Activity: Molecular Weight: Chemical Formula: Solubility: Storage Conditions: Vapor Pressure:
8-2 Telomer B Alcohol 8-2 TBA 2-Perfluorooctylethanol [Ethanol-1,2-14C] 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro678-39-7 3489-169 98.6% (HPLC) 33 mCi/mmole 464.12 g/mole C8F1714CH214CH2OH ca 140 g/L frozen (<-10 C) in the dark 0.023 mmHg
The radiolabelled test article was supplied by Perkin Elmer Life Sciences and was received at Covance on 3 April 2003. It was stored at <-10C in the dark.
The specific activity was not re-determined at Covance but the value stated above was used, where applicable. A Certificate of Analysis for the radiolabelled test article was provided by the supplier (Appendix 1).
Non-radiolabelled test article Non-radiolabelled test article was supplied by the sponsor and was received at Covance on 27 February 2003. It was stored at room temperature in the dark. The following information was supplied:
Name: Batch number: Quantity received:
8-2 Telomer B alcohol Haskell 24691 5.6 g
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Covance Study Number 2304/001 Final Report
A Certificate of Analysis for the non-radiolabelled test article was provided by the sponsor (Appendix 1).
Solvents and reagents Reagents and solvents were of Analytical grade (or a suitable alternative) and were obtained principally from VWR International Limited (formerly Merck, BDH) (Poole, Dorset) or Rathburn Chemicals Limited (Walkerburn, Peebleshire).
StarscintTM, obtained from Perkin Elmer Life Sciences Ltd (Pangbourne, Berkshire) was the liquid scintillant used for general LSC. Quickszint Flow 302 (Zinsser Analytic, Maidenhead, Berkshire) was the liquid scintillant used in HPLC liquid mixing cells.
Permafluor E+ and Carbo-sorb E, obtained from Perkin Elmer Life Sciences Ltd were mixed (2:1 v/v) to form the scintillation cocktail used in the sample oxidiser.
Test System Three soils (SK961089, SK566696 and SK179618) and one sediment (Emperor Son's Lake) from sites in the United Kingdom, and one soil (Eurosoil 5) from Belgium were used for the study. The soils were supplied by Land Research Associates (Derby, Derbyshire) and the Institute for Reference Materials and Measurements (Belgium).
On arrival at Covance the test soils and sediment were thoroughly mixed and passed through a 2 mm mesh sieve. The soils were then air-dried and stored in the dark at room temperature. Information on the sampling site, sampling method and pesticide history was supplied with the soilsa.
The UK soils were characterised for the following parameters by Covance:
pH (0.01 M CaCl2) organic carbon content organic matter content particle size distribution (UK/BBA/USDA) textural class (UK, BBA and USDA)
a Information on the sampling site, sampling method and pesticide history was not supplied with Eurosoil 5 and, therefore, this information has not been reported.
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cation exchange capacity (mmol/kg)
Covance Study Number 2304/001 Final Report
moisture holding capacity at pF 0 (maximum water holding capacity, MWHC) and pF 2.5 (33 kPa)
C/N ratio
Nitrogen content of the soils was determined by The National Soil Resource Institute, formerly known as the Soil Survey and Land Research Centre (Silsoe, Bedford) under their own study number and protocol.
Certificates of Analysis were produced for each soil. These are shown in Appendix 2.
Soil Eurosoil 5 was supplied with a certificate of analysis detailing the pH in water and pH in calcium chloride (CaCl2). Eurosoil 5 also had an unaudited certificate of analysis detailing:
organic carbon content organic matter content particle size distribution (UK/BBA/USDA) textural class (UK/BBA/USDA) cation exchange capacity (mmol/kg)
The soil characteristics are summarised in the following table:
Soil name
pH (0.01M CaCl2)
Organic carbon Content (%)
Clay content (%)
Soil texture (USDA)
SK961089
7.6
4.6
34
Clay loam
SK566696
4.2
0.8
11
Loamy sand
Emperor
6.0
1.2
Son's Lake
17
Sandy loam
SK179618
5.6
3.8
20
Loam
Eurosoil 5*
3.1
6.7
5
Loamy sand
* Eurosoil 5 included after initial equilibrium time test, due to stability problems of (14C)-8,2 TBA with SK179618 soil.
All future references to soil, for the purpose of this report, include soil and sediment.
Prior to dispensing, the moisture contents of the stored soils were determined at Covance by drying a portion at ca 105C. This allowed the dry weight equivalent of
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Covance Study Number 2304/001 Final Report
the dispensed soil to be calculated from the wet weight. All calculations were based upon soil dry weight.
All experiments were conducted in the dark at either 20 2C or 4 1C.
Soils were dispensed into pre-weighed centrifuge tubes and pre-conditioned by mixing with either 0.01M calcium chloride solution or sterile 0.01M calcium chloride solution overnight before the day of the experiment.
Soils and solutions were mixed mechanically using a Stuart Scientific Rotator Drive STR4 end-over-end shaker at a speed that ensured efficient mixing.
Centrifugation was performed in an IEC Centra GP8R centrifuge at 2000 rpm (ca 700 g) for a length of time calculated to be sufficient to remove particles larger than 0.2 m from the portion of the solution from which aliquots were taken.
Test article application The radiochemical purity of (14C)-8-2 TBA was assessed by high performance liquid chromatography (HPLC, page 27) and by thin layer chromatography (TLC, page 27) after receipt at Covance and prior to use.
A stock solution (stock solution 1) was prepared by dissolving one ampoule containing (14C)-8-2 TBA (approx. 4.22 mg) in ethanol (6 mL) to produce a stock solution at a concentration of 0.679 mg/mL (by assay). The radiochemical purity of (14C)-8-2 TBA in stock solution 1 (determined 4 April 2003) was >98% by HPLC and >96% by TLC (Appendix 3).
Stock solution 2 was prepared by removing an aliquot (500 L) from stock solution 1 and adding this to ethanol (4.5 mL). The concentration of stock solution 2 (determined by assay) was 0.07 mg/mL.
Stock solution 1 was used for the preparation of dose formulations for the equilibrium time determination and isotherms tests. Stock solution 2 was used for the solubility and soil:solution ratio tests.
Dose formulations for the isotherms test were prepared through the addition of appropriate amounts of radiolabelled test article, as detailed below. The specific activities of the dose formulations were calculated and the concentration of 8-2 TBA
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Covance Study Number 2304/001 Final Report
in each formulation prior to dosing was checked by removing triplicate aliquots for
LSC.
Test
Formulation
Volume of stock Radiolabelled
number
solution (mL) 8-2 TBA (g)
SSR
SSR
EQT
2
EQT-Rpt
3
Isotherms
4
Isotherms
5
0.3572
25
0.1471
100
0.1911
130
0.1301
88
0.0451
30
1 stock solution 1, 2 stock solution 2 SSR = soil:solution ratio EQT = equilibrium time EQT-Rpt = repeat equilibrium time
Solvent volume (mL)
0.643 0.853 1.109 0.570 0.555
Specific activity (MBq/mg)
2.63 2.63 2.63 2.63 2.63
Tests were initiated by the addition of an appropriate amount of (14C)-8-2 TBA dose formulation. Application of test article complied with OECD Guideline 106 (January 2000).
Due to the low level of radioactivity in the individual formulations, the radiochemical purity of (14C)-8-2 TBA in stock solution 1 was assessed by HPLC and TLC immediately prior to preparing the individual dose formulations. The radiochemical purity was >98% by HPLC and >96% by TLC.
Solubility test and adsorption to vessels Tests were performed in sealed glass vials with minimal headspace after a series of initial tests indicated that there was potentially a problem with the volatile nature of this compound. Aliquots for liquid scintillation counting (LSC) were removed using a Hamilton SGE syringe (glass syringe with stainless steel needle and piston) that was inserted through the PTFE-lined rubber septum to minimise any volatile losses. A control vial containing ethanol only was used to confirm that the dosing procedure was acceptable. The test was performed over the concentration range 0.01 g/mL to 0.1 g/mL.
Solubility tests were inconclusive due to the volatile nature of the compound. The volatile nature of 8-2 TBA was considered less of a problem in the presence of soil. Therefore, for the remainder of the study the concentrations used were selected such that they were below the stated water solubility of the compound (0.14 mg/L), but
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Covance Study Number 2304/001 Final Report
were high enough to ensure that the radioactivity could be detected throughout each stage of the study.
Ratio of soil to aqueous phase Tests were performed in sealed glass vials with minimal headspace. All soils (SK961089, SK566696, Emperor Son's Lake and SK179618) were pre-equilibrated overnight with 0.01M calcium chloride prior to dose application. The test article (dissolved in ethanol) was applied to each of the test units by a syringe inserted through the PTFE-lined rubber septum. The test article was applied such that the target concentration in solution was 0.025 g/mL. The sample vials were then placed on an end-over-end shaker set at a speed sufficient to keep the soils in suspension. After 24 hours the samples were removed from the shaker and centrifuged (ca 1700 g for 10 min). Aliquots for LSC were removed using a Hamilton SGE syringe that was inserted through the PTFE-lined rubber septum to minimise any volatile losses. The centrifuge speed (ca 1700 g) resulted in the loss of a number of the glass vials, therefore subsequent centrifugation was performed at ca 700 g.
Once the aliquots for LSC had been taken the lids were removed from the sample vials, the supernatant removed and acetone added (10 mL). All samples were shaken then centrifuged and the supernatant removed, the process was repeated a further two times and the supernatants for each sample pooled. The soil extracts for each sample were quantified by LSC.
The extracted soil samples were air-dried overnight then ground in a pestle and mortar prior to combustion in oxygen.
The PTFE-lined lids were soaked in acetone prior to quantification by LSC.
The following weights of soil and volumes of 0.01M calcium chloride were used for the test:
1:1 Ratio. 9 g of soil to 9 mL calcium chloride solution
1:5 Ratio. 3 g of soil to 15 mL calcium chloride solution
1:25 Ratio. 0.6 g of soil to 15 mL of calcium chloride solution.
The percentage adsorption of 8-2 TBA to the test soils (not including Eurosoil 5) was >80% at ratios of soil to solution of 1:1 w/v and 1:5 w/v. For a soil:solution ratio of
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Covance Study Number 2304/001 Final Report
1:25 the percentage adsorption of 8-2 TBA was >60% for the test soils. The recovery of applied radioactivity from the samples was dependent on the proportion of soil present with >90% being recovered at a soil:solution ratio of 1:1 w/v. The 1:1 ratio was, therefore, selected for the remainder of the study.
Equilibrium time determination and stability The adsorption equilibrium time was determined by preparation of samples of 8-2 TBA (0.1 g/mL) in 0.01M calcium chloride (9 mL) with 9 g soil (SK961089, SK566696, Emperor Son's Lake and SK179618). The sample vials were placed on an end-over-end shaker set at a speed sufficient to keep the soils in suspension. At intervals of 3, 6, 24 and 48 hours, duplicate samples of each soil type were removed from the shaker and centrifuged. Aliquots for LSC were removed using a Hamilton SGE syringe that was inserted through the PTFE-lined rubber septum to minimise volatile losses. Once the aliquots for LSC had been taken the lids were removed and the supernatant removed. The 24 hour samples were extracted three times with acetone (10 mL) and the supernatants for each sample pooled. The soil extracts for each sample were quantified by LSC and analysed by TLC.
The extracted soil samples were air-dried overnight then ground in a pestle and mortar prior to combustion in oxygen.
It was determined that 3 hours was a suitable adsorption equilibrium time. However, degradation of the 8-2 TBA had occurred over the 24 hour equilibration period.
The equilibrium time was established as 3 hours but the stability of the 8-2 TBA was unknown over this period of time, so further testing was carried out as detailed below.
Duplicate samples of 8-2 TBA (0.1 g/mL) were prepared in 0.01M calcium chloride (9 mL) with 9 g soil (SK961089, SK566696, Emperor Son's Lake and SK179618). The sample vials were placed on an end-over-end shaker (set at a speed sufficient to keep the soils in suspension) in an incubator set at 4 1C. After 3 hours, duplicate samples of each soil type were removed from the shaker and centrifuged. Aliquots for LSC were removed using a Hamilton SGE syringe that was inserted through the PTFE-lined rubber septum to minimise volatile losses. Once the aliquots for LSC had been taken the lids were removed and the supernatant removed. The samples were extracted three times with acetone (10 mL) and the supernatants for each sample pooled. The soil extracts for each sample were quantified by LSC and analysed by TLC.
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Covance Study Number 2304/001 Final Report
The extracted soil samples were air-dried overnight then ground in a pestle and mortar prior to combustion in oxygen.
The level of radioactivity remaining in the supernatants after 3 hours at 4C was similar to that previously obtained at 20C (i.e. <3% of applied radioactivity). This indicated that equilibrium had been established at 3 hours. The stability was checked for all soils and, with the exception of SK179618 soil, (14C)-8-2 TBA was the only component present in the soil extracts.
Due to the ongoing stability problem with SK179618 soil, an additional soil (Eurosoil 5) was obtained. The previous test was repeated using both SK179618 soil and Eurosoil 5. The soils were sterilised by gamma irradiation (Isotron, Bradford, West Yorkshire) and the test was conducted at 4 1C using sterile 0.01M calcium chloride solution. Irradiation of the soils was performed to minimise the microbial degradation of the test substance during the test. 0.01M calcium chloride was sterilised by autoclaving.
Duplicate samples of 8-2 TBA (0.1 g/mL) were prepared in sterile 0.01M calcium chloride (9 mL) with 9 g sterile soil (SK179618 and Eurosoil 5). The sample vials were placed on an end-over-end shaker (set at a speed sufficient to keep the soils in suspension) in an incubator set at 4 1C. After 3 hours, duplicate samples of each soil type were removed from the shaker and centrifuged. Aliquots for LSC were removed using a Hamilton SGE syringe that was inserted through the PTFE-lined rubber septum to minimise volatile losses. Once the aliquots for LSC had been taken the lids were removed and the supernatant removed. The samples were extracted three times with acetone (10 mL) and the supernatants for each sample pooled. The soil extracts for each sample were quantified by LSC and analysed by TLC.
The level of radioactivity remaining in the supernatants after 3 hours at 4C was similar to that previously obtained for SK179618 (i.e. <3% of applied radioactivity). This indicated that equilibrium had been established at 3 hours. The stability was checked for both soils and (14C)-8-2 TBA was the only component present in the soil extracts.
Under sterile conditions the results for SK179618 soil and Eurosoil 5 confirmed that the (14C)-8-2 TBA was stable over the 3 hour equilibrium time at 4 1C.
The results of the adsorption equilibrium time led to the following changes being made to the study design:
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Covance Study Number 2304/001 Final Report
inclusion of an additional soil (Eurosoil 5) all five soils were sterilised by gamma irradiation prior to use 0.01M calcium chloride solutions were sterilised by autoclaving prior to use the temperature at which the test was performed was reduced from 20 2C to
4 1C.
The desorption equilibrium time for each soil type was determined by preparation of samples of 8-2 TBA (0.1 g/mL) in sterile 0.01M calcium chloride (9 mL) with 9 g sterile soil (all five soils).
After mixing for the adsorption equilibrium time of 3 hours, samples were centrifuged and the supernatants removed. The supernatants were replaced by equal weights of fresh sterile 0.01M calcium chloride and the samples were placed back on the shaker.
At intervals of 1.5, 3, 6 and 24 hours, duplicate samples of each soil type were removed from the shaker and centrifuged. Aliquots for LSC were removed using a Hamilton SGE syringe that was inserted through the PTFE-lined rubber septum to minimise volatile losses.
It was determined that 3 hours was a suitable desorption equilibrium time.
Adsorption isotherms Duplicate samples at five concentrations of 8-2 TBA in sterile 0.01M calcium chloride (9 mL) with 9 g sterile soil were prepared. The table below details how the test article formulations were used to achieve the initial concentration of 8-2 TBA in the units:
Formulation
Formulation conc.1
(mg/mL)
Volume formulation
(L)
Weight of 8-2 TBA
(g)
42
0.131
8.6
1.127
42
0.131
6.9
0.904
42
0.131
5.2
0.681
52
0.053
8.6
0.456
52
0.053
2.2
0.117
1concentration 2 refer to table on page 21 for preparation of the formulations.
Volume of 0.01 MCaCl2
(mL)
9 9 9 9 9
Nominal conc.1 (g/mL)
0.125 0.1 0.075 0.05 0.0125
Actual conc.1 (g/mL)
0.125 0.1 0.076 0.05 0.0129
The sample vials were placed on an end-over-end shaker in an incubator set at 4 1C. After mixing for the adsorption equilibrium time of 3 hours, the phases were separated by centrifugation. Aliquots (ca 1 mL or 1.5 mL) for LSC were removed using a Hamilton SGE syringe that was inserted through the PTFE-lined rubber
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Covance Study Number 2304/001 Final Report
septum to minimise potential volatile losses. Once the aliquots for LSC had been taken, as much of the adsorption supernatant as possible was removed.
Desorption isotherms The volume of adsorption supernatant removed was replaced by an equivalent weighed portion of fresh 0.01M calcium chloride. The samples were shaken vigorously to break up the soil packed at the bottom of the tube. The samples were then re-mixed for the desorption equilibrium time of 3 hours at 4 1C. After this time, each sample was centrifuged and the supernatant quantified by LSC. The 0.125 g/mL samples were extracted three times with acetone (10 mL) and the supernatants for each sample pooled. The soil extract for each sample was quantified by LSC. The extracted soil samples (0.125 g/mL) were air-dried then ground in a pestle and mortar prior to combustion in oxygen and quantification by LSC. The PTFE-lined lids used for each sample were soaked in acetone prior to quantification by LSC.
Mass balance For each of the samples, the mass balance was determined from the sum of the radioactivity present in the adsorption supernatant, the desorption supernatant, the soil extract, the lid wash and the combusted residue.
pH measurements The pH of 0.01 M calcium chloride containing the highest (0.125 g/mL) and lowest (0.0125 g/mL) concentrations of 8-2 TBA was measured. The pH of each adsorption supernatant from the adsorption isotherm test was also measured.
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Covance Study Number 2304/001 Final Report
Analytical Methods and Measurements
HPLC Radiochemical purity determinations were performed using the following high performance liquid chromatography (HPLC) method:
Column: Gradient:
Genesis C18, 150 x 4.6 mm, 4m
A linear binary gradient system was used consisting of
Solvent A:
2mM ammonium acetate in water
Solvent B:
Methanol
Time
% A
% B
0
97
3
15
0
100
35
0
100
35.1
97
3
40
97
3
Flow rate: Detection:
1 mL/min UV absorbance of the eluent was monitored at 215 nm using an Agilent G1314A UV detector or a Jasco UV-975 detector. Radioactivity in the eluent was
monitored using a -ram flow through radioactivity monitor (LabLogic, Sheffield, South Yorkshire) equipped with a liquid mixing (500 L) cell.
Samples were co-injected with non-radiolabelled 8-2 TBA. Chromatograms were evaluated using Laura (version 1.4a) software (LabLogic).
TLC The following TLC system was used for determination of radiochemical purity and analysis of selected sample extracts:
Plates: Solvent:
Whatman silica gel K6F Toluene : Ethyl acetate (1:1)
Following chromatography, non-radiolabelled 8-2 TBA was visualised by spraying the plate with a 2% potassium permanganate solution. (14C)-8-2 TBA was detected by preparation of a radioluminogram of the TLC plate using a Fuji BAS 1500 Bio-image analyser. Chromatograms were evaluated from the radioluminograms using the associated Tina software (version 2.09g).
Radioactivity Radioactivity was measured using a Packard Tricarb model 1900TR liquid scintillation counter with facilities for computing quench corrected disintegrations per minute (dpm). Efficiency correlation curves were routinely checked by the use of commercially available quenched standards (Perkin Elmer) quenched over the range observed in test samples. The limit of detection was taken as 1.5 times the background
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Covance Study Number 2304/001 Final Report
radioactivity, determined by counting vials containing scintillant only in the same batch as the samples.
The radioactivity in liquid samples was determined by calculating the total sample weight and assaying duplicate weighed aliquots by LSC, after mixing with liquid scintillant (ca 5 mL). The density of calcium chloride solutions and supernatants was assumed to be 1.00 g/mL so concentrations per g are equivalent to concentrations per mL.
The radioactivity in solid residues was determined, after air-drying and grinding using a pestle and mortar, by combustion of triplicate weighed aliquots in oxygen using a Harvey OX-500 biological oxidiser. The combusted products were absorbed in scintillation cocktail and the radioactivity absorbed assayed by LSC. Carbon-14 standards were combusted at the beginning and at regular intervals throughout the day to check the carry-over between samples and the efficiency of the combustion process. Combustion efficiencies were 99 4% and therefore no correction factors were applied to the data.
Calculations
Definitions Mass of test article available for adsorption (g) = G Dry weight of soil (g) = m Volume of solution associated with soil (mL) = V0 Concentration of test article in adsorption supernatant (g/mL) = Ce Concentration of test article in desorption supernatant (g/mL) = C1 Density of 0.01 M calcium chloride solutions and supernatants (g/mL) =
Adsorption Weight of stock solution initially added (g) = W1 Volume of stock solution initially added (mL) = V1 = W1 / Weight of supernatant after adsorption step (g) = W2 Volume of supernatant after adsorption step (mL) = V2 = W2 / Quantity of test substance adsorbed (g) = X = G - (Ce x (V0 + V1)) Percent of test substance adsorbed = A = X x 100 / G
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Desorption
Covance Study Number 2304/001 Final Report
Weight of supernatant after desorption step (g) = W3
Volume of supernatant after desorption step (mL) = V3 = W3 /
Quantity of test substance adsorbed following desorption (g) = X1 = X + (Ce x (V0 + V1 - V2)) - (C1 x (V0 + V1 - V2 + V3))
Percent of adsorbed compound desorbed in desorption step = (X - X1) x 100 / X
Adsorption coefficients The following equations were used to calculate adsorption coefficients (mL/g) for individual units:
Adsorption coefficient = Kd = (X/m) / Ce Adsorption coefficient as a function of the organic carbon content of the soil = Kdoc = Kd x 100 / % organic carbon Adsorption coefficient as a function of the organic matter content of the soil = Kdom = Kd x 100 / % organic matter
Adsorption constants The Freundlich constants, Kf, and slope of Freundlich isotherm, 1/n, for each adsorption and desorption isotherm were calculated from the following relationship:
X/m = K x C1/n Where X/m is the concentration of test article in the soil and C is the concentration of test article in the supernatant.
Taking Logs of each side gives: Log (X/m) = Log (Kf) + (1/n) Log (C)
Hence an isotherm of Log (X/m) against Log (C) was plotted for each adsorption and desorption phase. The intercept and the slope of the line of best fit gave Log (K) and (1/n) respectively.
The Freundlich adsorption constant was calculated as a function of the organic carbon content and organic matter content of the soil to give the values for Koc and Kom respectively by using the following equations:
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Koc = K x 100 / % organic carbon Kom = K x 100 / % organic matter
Covance Study Number 2304/001 Final Report
Determination of percent of applied radioactivity in samples The individual estimates of concentration derived from the duplicate or triplicate aliquots were averaged to provide a mean concentration of radioactivity in the sample. Total radioactivity applied to incubation unit (kBq) = T Initial sample weight (g) = I Sub-sample weight (g) = SS Total weight of sample for LSC assay (g) = A Weight of aliquots assayed by LSC (g) = W Background corrected radioactivity in aliquots assayed by LSC (dpm) = D Concentration of radioactivity in sample (dpm/g) = C = D / W Radioactivity in assayed sample (kBq) = B = C x A / 60,000 (1 kBq = 60,000 dpm) Percent of applied radioactivity in sample = (B / T) x (I / SS) x 100
Expression of Results Tables presented in the report have been computer generated and data values displayed may be rounded values of those held in memory. As a result of this, calculation of mean and total values from individual data presented in the report will, in some instances, yield a minor variation to the value shown. Calculations were performed using Microsoft Excel software.
Volumes referred to in the methods section are approximate volumes only. When samples were quantified for radioactive content, liquid samples were accurately weighed and accurately weighed aliquots were removed for LSC.
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RESULTS
Covance Study Number 2304/001 Final Report
Preliminary tests
Solubility and Adsorption to containers The table below shows the results of the test solutions before and after centrifugation.
Test concentration
Dose applied kBq
Radioactivity in solution kBq
0.1 g/mL 0.05 g/mL 0.025 g/mL 0.01 g/mL 0.1 g/mL1
5.166 2.583 1.291 0.553 5.166
2.933 2.031 0.999 0.467 5.206
1 Control vial containing ethanol only
% ARR in solution
56.8 78.6 77.4 84.4 100.8
Radioactivity in solution after centrifugation kBq
2.293 1.502 0.765 0.351 5.180
% ARR in solution after centrifugation
44.5 58.1 59.3 63.5 100.3
The results show that in a sealed vial with minimal headspace, the test article may not exist fully in solution. This may account for the loss of radioactivity after centrifugation. However, this loss may also be attributed to volatilization of the test article because when the vial lids were removed, there was a further loss of radioactivity, confirming that the test article was volatile. The table below shows the results of the test solutions post-centrifugation and after the removal of the vial lid.
Test concentration
Dose applied kBq
Radioactivity in solution after removal
of lid kBq
0.1 g/mL 0.05 g/mL 0.025 g/mL 0.01 g/mL 0.1 g/mL1
5.166 2.583 1.291 0.553 5.166
1.838 1.164 0.600 0.345 4.848
1 Control vial containing ethanol only
% ARR in solution
35.6 45.1 46.5 62.4 93.8
Radioactivity in methanol wash of vial
kBq
0.1051 0.0258 0.0214 0.0081 0.0244
% ARR in unit wash
2.03 1.00 1.66 1.46 0.47
Solubility tests were inconclusive due to the volatile nature of the compound. Tests showed that there was not much adsorption of the test material to the glass vial.
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Covance Study Number 2304/001 Final Report
Ratio of soil to aqueous phase The percentage adsorption of 8-2 TBA to the test soils was >80% at ratios of soil to solution of 1:1 w/v and 1:5 w/v. For a soil:solution ratio of 1:25 the percentage adsorption of 8-2 TBA was >60% for the test soils. The recovery of applied radioactivity from the samples was dependent on the proportion of soil present with >90% being recovered at a soil:solution ratio of 1:1 w/v. Results are shown in Table 1 to Table 3. The 1:1 ratio was selected for the remainder of the study.
Equilibrium time determination Results from the equilibrium time determination showed that for all four soils with a soil : aqueous phase ratio of 1:1, 8-2 TBA at a concentration of 0.1 g/mL in 0.01M calcium chloride had reached adsorption and desorption equilibriums by 3 hours. After this time, the percent change in the quantity of 8-2 TBA in the supernatant was <10%. Results are shown in Figure 1 to Figure 4.
Results from the stability test (conducted using the 24 hour samples only) showed that the overall recovery of applied radioactivity extracted with acetone was 83% to 88% for soils SK961089, SK566696 and Emperor Son's Lake, but only 39% for soil SK179618. Thin layer chromatography of the soil extracts showed that the purity of 8-2 TBA was 72% to 87% in extracts from SK961089, SK566696 and Emperor Son's Lake soils. The test article was degraded to a greater extent in soil SK179618 with 5% of the applied radioactivity being present as 8-2 TBA in the acetone extract of the soil. Results are presented in Table 4 and Table 5. TLC chromatograms of the soil extracts are shown in Figure 5 to Figure 8.
A repeat test was performed by shaking duplicate samples for 3 hours, the proposed equilibrium time, at a reduced temperature of 4 1C. The level of radioactivity remaining in the supernatants after 3 hours was similar to that previously obtained at 20C (i.e. <3% of applied radioactivity). This, therefore, demonstrated that equilibrium had been established at 3 hours. The stability was checked for all soils and, with the exception of SK179618 soil, (14C)-8-2 TBA was the only component present in the soil extracts. Results are presented in Table 6 and Table 7. TLC chromatograms of the soil extracts are shown in Figure 9 to Figure 12.
Due to the ongoing stability problem with soil SK179618, an additional soil (Eurosoil 5) was obtained. The previous test was repeated using both SK179618 soil and Eurosoil 5 under sterile conditions and at 4 1C.
The level of radioactivity remaining in the supernatants after 3 hours at 4C was again similar to that previously obtained for SK179618 (i.e. 3% of applied
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Covance Study Number 2304/001 Final Report
radioactivity). This indicated that equilibrium had been established at 3 hours. The stability was checked for both soils and (14C)-8-2 TBA was the only component present in the soil extracts. The results are shown in Table 8, Figure 13 and Figure 14.
Under sterile conditions the results for SK179618 soil and Eurosoil 5 confirm that the (14C)-8-2 TBA was stable over the 3 hour equilibrium time at 4 1C.
The determination of the desorption equilibrium time demonstrated that 1% to 2% of the adsorbed radioactivity was desorbed from the soils at 1.5, 3, 6 and 24 hours. Therefore, the adsorbed 8-2 TBA is not desorbed from the soils even after 24 h. Therefore, 3 hours was a selected as the desorption equilibrium time. The results are shown in Table 9.
Adsorption and desorption isotherms Values for the concentrations of 8-2 TBA in solution and on the soil at the adsorption and desorption equilibrium times are presented in Table 10 to Table 14.
Quantities of 8-2 TBA adsorbed to and desorbed from soil are presented in Table 15 to Table 19.
Adsorption coefficients (Kd, Kdoc and Kdom) determined at each concentration for the soil used for the adsorption/desorption test are presented in Table 20 to Table 24.
Freundlich adsorption and desorption constants (Kf, Koc and Kom) are presented in Table 25 to Table 29.
The Freundlich adsorption constants (Kf) calculated by linear regression analysis were 35, 40, 20, 130 and 38 for the SK961089, SK566696, Emperor Son's Lake, SK179618 and Eurosoil 5 respectively.
The Freundlich adsorption constants related to organic carbon content (Koc) were 765, 4941, 1667, 3414 and 572 for the SK961089, SK566696, Emperor Son's Lake, SK179618 and Eurosoil 5 respectively.
The adsorption and desorption isotherms, presented in Figure 15 to Figure 19, showed a strong positive monotonic relationship between log Ce and log X/m, indicated by the correlation coefficients being close to 1.
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Covance Study Number 2304/001 Final Report
Mass balance results are presented in Table 30. Mean recoveries of applied radioactivity were 86 to 96%. The pH values of 0.01M calcium chloride solutions containing the highest (0.125 g/mL) and lowest (0.0125 g/mL) concentrations of 8-2 TBA are presented in Table 31. The pH values of all the adsorption supernatants are presented in Table 32.
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Covance Study Number 2304/001 Final Report
SUMMARY AND CONCLUSIONS
The Freundlich adsorption constants (Kf) were calculated by linear regression analysis and the results were as follows:
Soil
SK961089 (clay loam) SK566696 (loamy sand) Emperor Son's Lake sediment (sandy loam)
SK 179618 (loam) Eurosoil 5 (loamy sand)
Freundlich constant (Kf)
35 40 20 130 38
The Freundlich adsorption constants related to organic carbon content (Koc) as shown below:
Soil
SK961089 (clay loam) SK566696 (loamy sand) Emperor Son's Lake sediment (sandy loam)
SK 179618 (loam) Eurosoil 5 (loamy sand)
(Koc)
765 4941 1667 3414 572
These values characterise 8-2 TBA as slightly mobile (Koc 500 to 4000) to non-mobile (Koc>4000) in the four soils and one sediment used in this study (Hollis, 1991)a
a Hollis, J M (1991). Mapping the vulnerability of aquifers and surface water to pesticide contamination at the national/regional scale. Pesticides in Soil and Water: Current Perspectives (A Walker Ed), BCPC Monograph 47, pp 169 to 174
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TABLES
Covance Study Number 2304/001 Final Report
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Covance Study Number 2304/001 Final Report
Table 1
1:1 Ratio of soil to aqueous phase following treatment at 0.025 g/mL
Unit
Soil type
Supernatant
A2
SK961089
1.2
B2
SK566696
1.5
C2
Emperor Son's Lake
1.1
D1
SK179618
2.5
Ratio Test not performed for Eurosoil 5.
% of applied radioactivity
Soil extract
Residue
Lid Wash
74.6
18.1
3.9
86.6
5.6
6.1
80.4
6.0
10.6
39.1
51.3
1.4
Total
97.8 99.8 98.1 94.3
Table 2 1:5 Ratio of soil to aqueous phase following treatment at 0.025 g/mL
Unit
Soil type
Supernatant
A3
SK961089
1.6
A4
SK961089
1.6
B3
SK566696
2.6
B4
SK566696
2.5
C3
Emperor Son's Lake
2.6
D3
SK179618
6.3
D4
SK179618
5.8
Ratio Test not performed for Eurosoil 5.
% of applied radioactivity
Soil extract
Residue
Lid Wash
71.5
19.5
2.6
71.3
19.4
3.3
76.0
4.8
4.8
77.7
4.1
5.5
78.9
4.0
9.6
35.9
45.4
1.3
37.6
43.7
1.9
Total
95.2 95.6 88.2 89.8 95.1 88.9 89.0
Table 3 1:25 Ratio of soil to aqueous phase following treatment at 0.025 g/mL
Unit
Soil type
Supernatant
A5
SK961089
4.8
A6
SK961089
5.1
B5
SK566696
8
C5
Emperor Son's Lake
10
C6
Emperor Son's Lake
11.3
D5
SK179618
8.4
Ratio Test not performed for Eurosoil 5.
% of applied radioactivity
Soil extract
Residue
Lid Wash
61.1
20.8
7.9
59.9
19.4
7.7
67.0
5.2
9.3
60.8
3.4
12.0
56.6
3.8
15.8
30.0
45.1
2.9
Total
94.6 92.1 89.5 86.2 87.5 86.4
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Covance Study Number 2304/001 Final Report
Table 4
Recovery of applied radioactivity from the 24 hour equilibrium time samples at a
soil to aqueous phase ratio of 1:1 and 20 1C
Soil
Sample Supernatant
SK961089
EQ A5
1.0
EQ A6
0.4
SK566696
EQ B5
1.3
EQ B6
1.2
Emperor Son's Lake EQ C5
0.7
EQ C6
0.9
SK179618
EQ D5
0.6
EQ D6
1.6
Stability Test not performed for Eurosoil 5.
Percent of applied radioactivity
Soil Extract
Residue
Lid wash
84.1
13.9
1.5
83.0
13.2
1.2
87.4
3.9
2.1
84.9
3.7
2.3
87.9
5.5
3.3
86.2
5.6
4.8
38.9
52.7
0.0
37.0
53.7
0.9
Total
100.5 97.8 94.7 92.1 97.4 97.5 92.2 93.2
Table 5 Stability over a 24 hour period (20C) at a soil to aqueous phase ratio of 1:1
Soil
Sample
SK961089 SK566696 Emperor Son's Lake SK179618
EQ A5 EQ A6 EQ B5 EQ B6 EQ C5 EQ C6 EQ D5 EQ D6
Stability Test not performed for Eurosoil 5.
Percent of applied radioactivity
Amount in soil extract
8-2 TBA
84.1
72.4
83.0
73.2
87.4
77.0
84.9
76.6
87.9
87.0
86.2
85.7
38.9
5.2
37.0
4.4
Table 6 Comparison of the applied radioactivity recovered in the supernatants from soils
(soil to aqueous phase ratio of 1:1)
Soil
Sample
SK961089 SK566696 Emperor Son's Lake SK179618
EQ A9 EQ A10 EQ B9 EQ B10 EQ C9 EQ C10 EQ D9 EQ D10
Stability Test not performed for Eurosoil 5.
Percent of applied radioactivity
3 h @ 4C
3 h @ 20C
1.9
1.1
1.3
0.9
1.7
2.1
1.8
1.3
1.8
2.0
2.7
2.4
1.3
2.4
1.3
2.6
- 38 -
Covance Study Number 2304/001 Final Report
Table 7
Stability over a 3 hour period (4C) at a soil to aqueous phase ratio of 1:1
Soil
Sample
SK961089 SK566696 Emperor Son's Lake SK179618
EQ A9 EQ A10 EQ B9 EQ B10 EQ C9 EQ C10 EQ D9 EQ D10
Stability Test not performed for Eurosoil 5.
Percent of applied radioactivity
Amount in soil extract
8-2 TBA
93.5 101.6 95.2 95.4 91.9 88.5 86.5 84.4
93.4 100.0 95.0 95.1 90.7 88.0 69.5 66.9
Table 8 Percent applied radioactivity recovered in the supernatants from sterile soils at
4C and stability of 8-2 TBA in the soil extract of sterile soil
Soil SK179618
Eurosoil 5
Sample
EQ D11 EQ D12
EQ E11 EQ E12
Percent of applied radioactivity
3 h @ 4C
Amount in soil
1.9
92.0
3.0
82.6
1.1
92.9
1.2
94.1
8-2 TBA
90.3 81.1
91.1 91.5
Table 9 Percent applied radioactivity recovered in the desorption supernatants from
sterile soils at 4C following a 3 h equilibrium time
Desorption time (h)
SK961089
Percent of applied radioactivity in solution
SK566696
Emperor Son's Lake
SK179618
0
0.0
0.0
0.0
0.0
1.5
0.8
1.2
1.0
1.0
3
0.7
1.2
1.5
1.3
6
1.0
1.3
2.0
1.4
24
1.4
1.8
1.3
1.7
Values presented are mean values of two replicate samples.
Eurosoil 5
0.0 0.6 0.7 0.7 0.8
- 39 -
Covance Study Number 2304/001 Final Report
Table 10
Values for Ce, X/m, C1 and X1/m for 8-2 TBA on soil SK961089
Nominal
Dose level (g/mL)
Replicate
Adsorption
Ce (g/mL)
X/m (g/g)
Desorption
C1 (g/mL)
X1/m (g/g)
0.125 0.1
0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
0.0021 0.0021 0.0021
0.0013 0.0012 0.0012
0.0010 0.0016 0.0013
0.0009 0.0007 0.0008
0.0002 0.0001 0.0002
0.1223 0.1224 0.1223
0.0985 0.0989 0.0987
0.0741 0.0744 0.0743
0.0496 0.0497 0.0497
0.0126 0.0127 0.0127
0.0010 0.0010 0.0010
0.0008 0.0009 0.0009
0.0005 0.0017 0.0011
0.0004 0.0004 0.0004
0.0001 0.0001 0.0001
0.1225 0.1225 0.1225
0.0984 0.0986 0.0985
0.0742 0.0736 0.0739
0.0497 0.0497 0.0497
0.0126 0.0127 0.0127
Ce and C1 are the aqueous phase concentrations; X/m and X1/m are the concentrations in the soil
Table 11 Values for Ce, X/m, C1 and X1/m for 8-2 TBA on soil SK566696
Nominal
Dose level (g/mL)
Replicate
Adsorption
Ce (g/mL)
X/m (g/g)
Desorption
C1 (g/mL)
X1/m (g/g)
0.125 0.1 0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
0.0023 0.0024 0.0023
0.0020 0.0017 0.0018
0.0012 0.0013 0.0013
0.0010 0.0009 0.0010
0.0002 0.0002 0.0002
0.1220 0.1221 0.1221
0.0978 0.0981 0.0979
0.0741 0.0740 0.0740
0.0495 0.0495 0.0495
0.0126 0.0127 0.0127
0.0020 0.0025 0.0023
0.0015 0.0014 0.0014
0.0010 0.0010 0.0010
0.0010 0.0008 0.0009
0.0002 0.0002 0.0002
0.1207 0.1204 0.1205
0.0970 0.0972 0.0971
0.0735 0.0735 0.0735
0.0488 0.0491 0.0489
0.0124 0.0125 0.0125
Ce and C1 are the aqueous phase concentrations; X/m and X1/m are the concentrations in the soil
- 40 -
Covance Study Number 2304/001 Final Report
Table 12
Values for Ce, X/m, C1 and X1/m for 8-2 TBA on Emperor Son's Lake sediment
Nominal
Dose level (g/mL)
Replicate
Adsorption
Ce (g/mL)
X/m (g/g)
Desorption
C1 (g/mL)
X1/m (g/g)
0.125 0.1 0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
0.0028 0.0027 0.0027
0.0020 0.0021 0.0021
0.0014 0.0026 0.0020
0.0011 0.0009 0.0010
0.0002 0.0002 0.0002
0.1212 0.1225 0.1218
0.0977 0.0979 0.0978
0.0735 0.0726 0.0731
0.0492 0.0493 0.0492
0.0127 0.0127 0.0127
0.0019 0.0018 0.0019
0.0013 0.0013 0.0013
0.0010 0.0009 0.0009
0.0007 0.0005 0.0006
0.0001 0.0001 0.0001
0.1205 0.1217 0.1211
0.0975 0.0974 0.0974
0.0730 0.0729 0.0729
0.0491 0.0492 0.0491
0.0126 0.0126 0.0126
Ce and C1 are the aqueous phase concentrations; X/m and X1/m are the concentrations in the soil
Table 13 Values for Ce, X/m, C1 and X1/m for 8-2 TBA on soil SK179618
Nominal
Dose level (g/mL)
Replicate
Adsorption
Ce (g/mL)
X/m (g/g)
Desorption
C1 (g/mL)
X1/m (g/g)
0.125 0.1
0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
0.0021 0.0020 0.0020
0.0016 0.0016 0.0016
0.0013 0.0011 0.0012
0.0010 0.0008 0.0009
0.0003 0.0002 0.0003
0.1218 0.1220 0.1219
0.0976 0.0980 0.0978
0.0735 0.0736 0.0735
0.0492 0.0495 0.0493
0.0126 0.0126 0.0126
0.0015 0.0015 0.0015
0.0013 0.0014 0.0013
0.0009 0.0010 0.0010
0.0006 0.0006 0.0006
0.0002 0.0002 0.0002
0.1212 0.1215 0.1214
0.0971 0.0974 0.0972
0.0732 0.0731 0.0731
0.0492 0.0492 0.0492
0.0125 0.0125 0.0125
Ce and C1 are the aqueous phase concentrations; X/m and X1/m are the concentrations in the soil
- 41 -
Covance Study Number 2304/001 Final Report
Table 14
Values for Ce, X/m, C1 and X1/m for 8-2 TBA on Eurosoil 5
Nominal
Dose level (g/mL)
Replicate
Adsorption
Ce (g/mL)
X/m (g/g)
Desorption
C1 (g/mL)
X1/m (g/g)
0.125 0.1
0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
0.0020 0.0016 0.0018
0.0012 0.0011 0.0012
0.0012 0.0013 0.0012
0.0006 0.0006 0.0006
0.0002 0.0002 0.0002
0.1225 0.1219 0.1222
0.0990 0.0988 0.0989
0.0737 0.0734 0.0735
0.0499 0.0498 0.0499
0.0127 0.0127 0.0127
0.0010 0.0010 0.0010
0.0009 0.0009 0.0009
0.0006 0.0008 0.0007
0.0003 0.0003 0.0003
0.0001 0.0001 0.0001
0.1223 0.1215 0.1219
0.0985 0.0983 0.0984
0.0736 0.0731 0.0733
0.0497 0.0497 0.0497
0.0127 0.0127 0.0127
Ce and C1 are the aqueous phase concentrations; X/m and X1/m are the concentrations in the soil
Table 15 Quantities of 8-2 TBA adsorbed to and desorbed from soil SK961089
Nominal Dose level (g/mL)
Replicate
0.125 0.1 0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
Percent applied chemical adsorbed
98.2 98.2 98.2
98.7 98.8 98.7
98.6 97.8 98.2
98.2 98.5 98.3
98.3 98.8 98.5
Weight (g) of adsorbed chemical following
Adsorption Desorption
1.1010 1.1013 1.1011
0.8882 0.8888 0.8885
0.6684 0.6631 0.6657
0.4457 0.4470 0.4464
0.1140 0.1146 0.1143
1.1026 1.1024 1.1025
0.8872 0.8863 0.8868
0.6687 0.6564 0.6625
0.4461 0.4469 0.4465
0.1142 0.1144 0.1143
Percent of adsorbed chemical desorbed
0.0 0.0 0.0
0.1 0.3 0.2
0.0 1.0 0.5
0.0 0.0 0.0
0.0 0.2 0.0
Percent of adsorbed chemical not desorbed
100.1 100.1 100.1
99.9 99.7 99.8
100.0 99.0 99.5
100.1 100.0 100.0
100.1 99.8 100.0
- 42 -
Covance Study Number 2304/001 Final Report
Table 16
Quantities of 8-2 TBA adsorbed to and desorbed from soil SK566696
Nominal Dose level (g/mL)
Replicate
0.125 0.1 0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
Percent applied chemical adsorbed
98.1 98.1 98.1
98.0 98.3 98.1
98.3 98.2 98.3
98.0 98.1 98.1
98.2 98.3 98.3
Weight (g) of adsorbed chemical following
Adsorption Desorption
1.0996 1.0992 1.0994
0.8818 0.8843 0.8830
0.6668 0.6657 0.6662
0.4448 0.4456 0.4452
0.1139 0.1141 0.1140
1.0879 1.0834 1.0856
0.8747 0.8763 0.8755
0.6617 0.6608 0.6613
0.4391 0.4414 0.4402
0.1124 0.1126 0.1125
Percent of adsorbed chemical desorbed
1.1 1.4 1.2
0.8 0.9 0.9
0.8 0.7 0.8
1.3 0.9 1.1
1.4 1.3 1.3
Percent of adsorbed chemical not desorbed
98.9 98.6 98.8
99.2 99.1 99.1
99.2 99.3 99.2
98.7 99.1 98.9
98.6 98.7 98.7
Table 17 Quantities of 8-2 TBA adsorbed to and desorbed from Emperor Son's Lake sediment
Nominal Dose level (g/mL)
Replicate
0.125 0.1 0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
Percent applied chemical adsorbed
97.7 97.8 97.7
97.9 97.8 97.9
98.1 96.4 97.2
97.7 98.1 97.9
98.4 98.4 98.4
Weight (g) of adsorbed chemical following
Adsorption Desorption
1.0951 1.0958 1.0954
0.8810 0.8804 0.8807
0.6649 0.6536 0.6593
0.4438 0.4454 0.4446
0.1142 0.1142 0.1142
1.0893 1.0889 1.0891
0.8788 0.8754 0.8771
0.6602 0.6561 0.6581
0.4424 0.4445 0.4434
0.1138 0.1135 0.1136
Percent of adsorbed chemical desorbed
0.5 0.6 0.6
0.3 0.6 0.4
0.7 0.0 0.2
0.3 0.2 0.3
0.4 0.6 0.5
Percent of adsorbed chemical not desorbed
99.5 99.4 99.4
99.7 99.4 99.6
99.3 100.4 99.8
99.7 99.8 99.7
99.6 99.4 99.5
- 43 -
Covance Study Number 2304/001 Final Report
Table 18
Quantities of 8-2 TBA adsorbed to and desorbed from soil SK 179618
Nominal Dose level (g/mL)
Replicate
0.125 0.1 0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
Percent applied chemical adsorbed
98.0 98.2 98.1
98.2 98.2 98.2
98.1 98.3 98.2
97.7 98.1 97.9
97.4 97.9 97.6
Weight (g) of adsorbed chemical following
Adsorption Desorption
1.0972 1.0984 1.0978
0.8819 0.8819 0.8819
0.6630 0.6646 0.6638
0.4435 0.4454 0.4444
0.1130 0.1135 0.1132
1.0922 1.0935 1.0928
0.8772 0.8759 0.8765
0.6600 0.6603 0.6601
0.4428 0.4431 0.4430
0.1125 0.1130 0.1128
Percent of adsorbed chemical desorbed
0.5 0.4 0.5
0.5 0.7 0.6
0.4 0.7 0.5
0.1 0.5 0.3
0.4 0.4 0.4
Percent of adsorbed chemical not desorbed
99.5 99.6 99.5
99.5 99.3 99.4
99.6 99.3 99.5
99.9 99.5 99.7
99.6 99.6 99.6
Table 19 Quantities of 8-2 TBA adsorbed to and desorbed from Eurosoil 5
Nominal Dose level (g/mL)
Replicate
0.125 0.1 0.075 0.05 0.0125
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
1 2 Mean
Percent applied chemical adsorbed
98.4 98.7 98.5
98.8 98.8 98.8
98.4 98.2 98.3
98.8 98.8 98.8
98.8 98.8 98.8
Weight (g) of adsorbed chemical following
Adsorption Desorption
1.1009 1.1040 1.1024
0.8868 0.8876 0.8872
0.6649 0.6641 0.6645
0.4486 0.4488 0.4487
0.1146 0.1146 0.1146
1.0989 1.1004 1.0997
0.8828 0.8834 0.8831
0.6639 0.6617 0.6628
0.4475 0.4477 0.4476
0.1144 0.1143 0.1144
Percent of adsorbed chemical desorbed
0.2 0.3 0.3
0.4 0.5 0.5
0.2 0.4 0.3
0.2 0.2 0.2
0.1 0.2 0.2
Percent of adsorbed chemical not desorbed
99.8 99.7 99.7
99.6 99.5 99.5
99.8 99.6 99.7
99.8 99.8 99.8
99.9 99.8 99.8
- 44 -
Covance Study Number 2304/001 Final Report
Table 20
Adsorption coefficients for 8-2 TBA on soil SK961089
Nominal Dose level (g/mL)
0.125
0.1
0.075
0.05
0.0125
Replicate
1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean
Adsorption coefficients
Kd
Kdoc
Kdom
58.12
1263
736
58.91
1281
746
58.51
1272
741
78.71 84.09 81.40
1711 1828 1770
996 1064 1030
74.16
1612
939
46.60
1013
590
60.38
1313
764
57.49
1250
728
68.03
1479
861
62.76
1364
794
60.84 87.74 74.29
1323 1907 1615
770 1111 940
Table 21 Adsorption coefficients for 8-2 TBA on soil SK566696
Nominal Dose level (g/mL)
0.125
0.1
0.075
0.05
0.0125
Replicate
1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean
Adsorption coefficients
Kd
Kdoc
Kdom
52.56 51.80 52.18
6570 6475 6522
3754 3700 3727
48.88 58.20 53.54
6110 7275 6693
3492 4157 3824
61.12 55.70 58.41
7639 6962 7301
4365 3979 4172
49.53 54.79 52.16
6191 6849 6520
3538 3914 3726
57.44 60.71 59.08
7181 7588 7384
4103 4336 4220
- 45 -
Covance Study Number 2304/001 Final Report
Table 22
Adsorption coefficients for 8-2 TBA on Emperor Son's Lake
Nominal Dose level (g/mL)
0.125
0.1
0.075
0.05
0.0125
Replicate
1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean
Adsorption coefficients
Kd
Kdoc
Kdom
43.26 45.82 44.54
3605 3818 3712
2060 2182 2121
48.00 46.97 47.49
4000 3914 3957
2286 2237 2261
51.79 27.54 39.66
4316 2295 3305
2466 1311 1889
44.96 53.66 49.31
3747 4471 4109
2141 2555 2348
64.21 65.03 64.62
5351 5420 5385
3057 3097 3077
Table 23 Adsorption coefficients for 8-2 TBA on soil SK179618
Nominal Dose level (g/mL)
0.125
0.1
0.075
0.05
0.0125
Replicate
1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean
Adsorption coefficients
Kd
Kdoc
Kdom
57.96
1525
878
61.66
1623
934
59.81
1574
906
62.73
1651
950
63.00
1658
955
62.87
1654
953
58.06 66.71 62.38
1528 1755 1642
880 1011 945
48.21
1269
730
59.43
1564
900
53.82
1416
815
43.44
1143
658
52.86
1391
801
48.15
1267
729
- 46 -
Covance Study Number 2304/001 Final Report
Table 24
Adsorption coefficients for 8-2 TBA on Eurosoil 5
Nominal Dose level (g/mL)
0.125
0.1
0.075
0.05
0.0125
Replicate
1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean
Adsorption coefficients
Kd
Kdoc
Kdom
62.58
934
539
75.24
1123
649
68.91
1028
594
83.18
1242
717
88.44
1320
762
85.81
1281
740
61.56
919
531
57.33
856
494
59.44
887
512
85.77
1280
739
88.83
1326
766
87.30
1303
753
82.96
1238
715
84.55
1262
729
83.75
1250
722
Table 25 Freundlich adsorption and desorption constants and regression analysis for
8-2 TBA on soil SK961089
Experimental Stage
Adsorption Step
Desorption Step
Adsorption constant
Kf
Koc
Kom
35.20 91.57
765 1991
446 1159
1/n 0.9105 0.9636
r2 0.9534 0.9922
- 47 -
Covance Study Number 2304/001 Final Report
Table 26
Freundlich adsorption and desorption constants and regression analysis for
8-2 TBA on soil SK566696
Experimental Stage
Adsorption Step
Desorption Step
Adsorption constant
Kf
Koc
Kom
39.53 77.35
4941 9669
2823 5525
1/n 0.9522 1.0340
r2 0.9937 0.9632
Table 27 Freundlich adsorption and desorption constants and regression analysis for
8-2 TBA on Emperor Son's Lake sediment
Experimental Stage
Adsorption Step
Desorption Step
Adsorption constant
Kf
Koc
Kom
20.00 34.25
1667 2854
952 1631
1/n 0.8633 0.8875
r2 0.9967 0.9902
- 48 -
Covance Study Number 2304/001 Final Report
Table 28
Freundlich adsorption and desorption constants and regression analysis for
8-2 TBA on soil SK179618
Experimental Stage
Adsorption Step
Desorption Step
Adsorption constant
Kf
Koc
Kom
129.73 103.92
3414 2735
1966 1575
1/n 1.1188 1.0422
r2 0.9864 0.9927
Table 29 Freundlich adsorption and desorption constants and regression analysis for
8-2 TBA on Eurosoil 5
Experimental Stage
Adsorption Step
Desorption Step
Adsorption constant
Kf
Koc
Kom
38.34
572
331
47.85
714
412
1/n 0.9049 0.8718
r2 0.9719 0.9834
- 49 -
Covance Study Number 2304/001 Final Report
Table 30
Recovery of radioactivity from all five soils treated with 8-2 TBA at 0.125 g/mL
after adsorption, desorption, organic wash and combustion
Sample
A1 A2 B1 B2 C1 C2 D1 D2 E1 E2
Ads Supernatant
% of Applied Radioactivity
Des Supernatant Soil extract
Lid wash
0.8
0.4
85.6
0.9
0.8
0.4
83.8
1.8
1.3
1.1
87.0
1.6
1.3
1.4
82.2
3.2
1.3
0.8
77.5
3.0
1.3
0.9
76.9
2.8
1.0
0.7
86.0
0.8
0.9
0.7
88.4
0.9
1.0
0.5
89.7
0.7
0.8
0.5
90.6
1.0
Residue
4.9 3.7 1.8 2.0 3.8 3.7 4.3 4.7 1.6 1.5
Total
92.6 90.5 92.8 90.1 86.4 85.6 92.8 95.6 93.5 94.4
Table 31 pH of the blank solutions used for the main isotherms test
Nominal Concentration
pH
0.125 g/mL
6.05
0.0125 g/mL
5.90
Table 32 pH of the adsorption supernatants from the main isotherms test following
treatment with 8-2 TBA
Dose group
Replicate
pH
A
1
7.82
2
8.14
3
7.45
4
7.65
5
7.88
6
7.87
7
7.93
8
7.94
9
7.86
10
8.35
- 50 -
Covance Study Number 2304/001 Final Report
Table 32 continued
pH of the adsorption supernatants from the main isotherms test following
treatment with 8-2 TBA
Dose group
Replicate
pH
B
1
4.26
2
4.22
3
4.11
4
4.18
5
4.14
6
4.10
7
4.17
8
4.14
9
4.14
10
4.11
C
1
6.21
2
6.17
3
6.20
4
6.24
5
6.25
6
6.16
7
6.23
8
6.25
9
6.26
10
6.31
D
1
5.74
2
5.73
3
5.43
4
5.49
5
5.47
6
5.58
7
5.54
8
5.52
9
5.58
10
5.54
E
1
3.05
2
3.00
3
3.04
4
3.02
5
3.02
6
2.99
7
2.99
8
2.99
9
2.96
10
2.99
- 51 -
FIGURES
Covance Study Number 2304/001 Final Report
- 52 -
Covance Study Number 2304/001 Final Report
Figure 1
Adsorption equilibrium time data for soil SK961089 following treatment
with 8-2 TBA
Percent adsorption to soil
100 90 80 70 60 50 40 30 20 10 0 0
12
24
36
48
Adsorption Tim e (hours)
Soil A
Adsorption Time (hours) Percent adsorbed to soil
0
0
3
99
6
98.2
24
98.3
48
98.4
- 53 -
Covance Study Number 2304/001 Final Report
Figure 2
Adsorption equilibrium time data for soil SK566696 following treatment
with 8-2 TBA
Percent adsorption to soil
100 90 80 70 60 50 40 30 20 10 0 0
12
24
36
48
Adsorption Tim e (hours)
Soil B
Adsorption Time (hours) Percent adsorbed to soil
0
0
3
98.3
6
98.3
24
98.1
48
98.1
- 54 -
Covance Study Number 2304/001 Final Report
Figure 3
Adsorption equilibrium time data for Emperor Son's Lake sediment
following treatment with 8-2 TBA
Percent adsorption to soil
100 90 80 70 60 50 40 30 20 10 0 0
12
24
36
48
Adsorption Tim e (hours)
Soil C
Adsorption Time (hours) Percent adsorbed to soil
0
0
3
97.8
6
98.2
24
98.4
48
98.8
- 55 -
Covance Study Number 2304/001 Final Report
Figure 4
Adsorption equilibrium time data for soil SK179618 following treatment
with 8-2 TBA
Percent adsorption to soil
100 90 80 70 60 50 40 30 20 10 0 0
12
24
36
48
Adsorption Tim e (hours)
Soil D
Adsorption Time (hours) Percent adsorbed to soil
0
0
3
97.5
6
97.9
24
97.6
48
96.9
- 56 -
Covance Study Number 2304/001 Final Report
Figure 5
Stability of 8-2 TBA in the soil extract of non-sterile SK961089 soil following
adsorption for 24 h at 20C determined by TLC
8-2 TBA Front
Start
[PSL]
70 60 50 40 30 20 10
0 0
50
100
[mm]
150
Plate 15, track 1
No Name
Type
1
8-2 TBA
2
3
Bkg
4
Bkg
-
Sum
-
Rem
-
Ttl
*normalised to this
Width [mm] 15.00 10.40 3.60 4.20 25.40 150.20 175.60 value
Pos [mm] 102.80 143.80 166.20 13.00 ----
RF 0.59 0.86 ------
PSL 2266.23 781.36 191.43 224.04 3047.60 8009.06 11056.66
PSL-Bkg 1467.26 227.41 0.00 0.00 1694.66 8.62 1703.28
%(PSL-Bkg) 86.14 13.35 0.00 0.00 99.49 0.51 100.00*
- 57 -
Covance Study Number 2304/001 Final Report
Figure 6
Stability of 8-2 TBA in the soil extract of non-sterile SK566696 soil following
adsorption for 24 h at 20C determined by TLC
8-2 TBA Front
[PSL]
90 80 70 60 50 40 30 20 10
0 0
Start
50
100
[mm]
150
Plate 15, track 3
No Name
Type
1
8-2 TBA
2
3
4
Bkg
5
Bkg
-
Sum
-
Rem
-
Ttl
*normalised to this
Width [mm] 12.60 10.80 9.60 2.40 2.00 33.00 142.60 175.60 value
Pos [mm] 103.00 147.00 15.00 6.60 170.60 ----
RF 0.59 0.88 0.01 ------
PSL 2644.03 717.25 611.78 127.12 107.67 3973.06 7635.39 11608.44
PSL-Bkg 1971.68 140.95 99.52 0.00 0.00 2212.16 26.16 2238.31
%(PSL-Bkg) 88.09 6.30 4.45 0.00 0.00 98.83 1.17 100.00*
- 58 -
Covance Study Number 2304/001 Final Report
Figure 7
Stability of 8-2 TBA in the soil extract of non-sterile Emperor Son's Lake
sediment following adsorption for 24 h at 20C determined by TLC
8-2 TBA Front
Start
[PSL]
100 90 80 70 60 50 40 30 20 10 0 0
50
100
[mm]
150
Plate 16, track 2
No
Name
1 8-2 TBA 2 3 *normalised to
Type
Width
[mm]
13.80
Bkg
3.40
Bkg
2.80
Sum
13.80
Rem
161.80
Ttl
175.60
this value
Pos [mm]
106.00 12.40 166.60
----
RF
0.60 --
1.00 ----
PSL
3070.13 187.87 128.56 3070.13 8271.88 11342.01
PSL-Bkg
2365.81 0.00 0.00
2365.81 13.94
2379.74
%(PSLBkg) 99.41 0.00 0.00 99.41 0.59
100.00 *
- 59 -
Covance Study Number 2304/001 Final Report
Figure 8 Stability of 8-2 TBA in the soil extract of non-sterile SK179618 soil following
adsorption for 24 h at 20C determined by TLC
30
[PSL] Start
Origin 8-2 TBA
Unk Front
20
10
0 0
50
100
[mm]
150
Plate 16, track 3
No
Name
1
Origin
2 8-2 TBA
3
4
Unk
5
6
-
-
-
*normalised to
Type
Width
[mm]
14.40
10.40
7.40
13.80
Bkg
2.80
Bkg
4.20
Sum
46.00
Rem
129.60
Ttl
175.60
this value
Pos [mm]
17.00 106.20 138.60 153.00
3.80 167.00
----
RF
0.01 0.60 0.81 0.91
------
PSL
1157.72 733.43 505.66 1360.35 141.69 234.14 3757.16 6985.75 10742.91
PSL-Bkg
384.57 175.05 108.35 619.41
0.00 0.00 1287.38 27.43 1314.82
%(PSLBkg) 29.25 13.31 8.24 47.11 0.00 0.00 97.91 2.09
100.00 *
- 60 -
Covance Study Number 2304/001 Final Report
Figure 9
Stability of 8-2 TBA in the soil extract of non-sterile SK961089 soil following
adsorption for 3 h at 4C determined by TLC
8-2 TBA Front
Start
[PSL]
70 60 50 40 30 20 10
0 0
50
100
[mm]
150
Plate 18, track 1
No
Name
1 8-2 TBA 2 3 *normalised to
Type
Width
[mm]
13.40
Bkg
2.80
Bkg
5.20
Sum
13.40
Rem
162.20
Ttl
175.60
this value
Pos [mm]
94.20 9.80 163.00
----
RF
0.54 --
1.00 ----
PSL
2010.28 128.47 229.86 2010.28 7266.32 9276.60
PSL-Bkg
1410.08 0.00 0.00
1410.08 1.19
1411.26
%(PSLBkg) 99.92 0.00 0.00 99.92 0.08
100.00 *
- 61 -
Covance Study Number 2304/001 Final Report
Figure 10
Stability of 8-2 TBA in the soil extract of non-sterile SK566696 soil following
adsorption for 3 h at 4C determined by TLC
[PSL] Start
8-2 TBA Front
80 70 60 50 40 30 20 10
0 0
50
100
[mm]
150
Plate 18, track 3
No
Name
1 8-2 TBA 2 3 *normalised to
Type
Width
[mm]
12.00
Bkg
4.80
Bkg
4.00
Sum
12.00
Rem
163.60
Ttl
175.60
this value
Pos [mm]
103.00 15.40 172.80
----
RF
0.56 ------
PSL
2266.70 203.68 176.52 2266.70 7072.23 9338.93
PSL-Bkg
1748.24 0.00 0.00
1748.24 3.96
1752.21
%(PSLBkg) 99.77 0.00 0.00 99.77 0.23
100.00 *
- 62 -
Covance Study Number 2304/001 Final Report
Figure 11
Stability of 8-2 TBA in the soil extract of non-sterile Emperor Son's Lake
sediment following adsorption for 3 h at 4C determined by TLC
8-2 TBA Front
Start
[PSL]
80 70 60 50 40 30 20 10
0 0
50
100
[mm]
150
Plate 19, track 1
No
Name
1
8-2 TBA
2
3
-
-
-
*normalised to
Type
Bkg Bkg Sum Rem Ttl this value
Width [mm] 13.60 1.60 5.00 13.60 162.00 175.60
Pos [mm]
93.60 4.80 170.00
----
RF
0.57 ------
PSL
2322.16 66.60 224.27
2322.16 7162.19 9484.36
PSL-Bkg
1722.79 0.00 0.00
1722.79 22.64
1745.44
%(PSLBkg) 98.70 0.00 0.00 98.70 1.30
100.00 *
- 63 -
Covance Study Number 2304/001 Final Report
Figure 12
Stability of 8-2 TBA in the soil extract of non-sterile SK179618 soil following
adsorption for 3 h at 4C determined by TLC
8-2 TBA Front
Start
50
40
[PSL]
30
20
10
0 0
50
100
[mm]
150
Plate 19, track 3
No
Name
1
8-2 TBA
2
3
4
5
-
-
-
*normalised to
Type
Width
[mm]
11.80
6.80
11.40
Bkg
3.80
Bkg
4.00
Sum
30.00
Rem
145.60
Ttl
175.60
this value
Pos [mm]
103.60 20.20 148.40 164.40 11.20
----
RF
0.58 0.02 0.87 0.98
-----
PSL
1599.66 350.20 684.24 169.85 169.45 2634.09 6355.74 8989.83
PSL-Bkg
1086.36 54.40 188.34 0.00 0.00
1329.11 22.21
1351.32
%(PSLBkg) 80.39 4.03 13.94 0.00 0.00 98.36 1.64
100.00 *
- 64 -
Covance Study Number 2304/001 Final Report
Figure 13 Stability of 8-2 TBA in the soil extract of sterile SK179618 soil following
adsorption for 3 h at 4C determined by TLC
150
Front
Start
8-2 TBA
100
[PSL]
50
Origin
0 0
50
100
[mm]
150
Plate 21, track 1
No
Name
1
Origin
2 8-2 TBA
3
4
-
-
-
*normalised to
Type
Width
[mm]
7.80
12.60
Bkg
2.60
Bkg
3.20
Sum
20.40
Rem
155.20
Ttl
175.60
this value
Pos [mm]
11.40 87.80 4.40 159.60
----
RF
0.01 0.51
-0.98
----
PSL
169.26 3545.26
42.61 49.30 3714.53 2476.17 6190.70
PSL-Bkg
45.65 3345.58
0.00 0.00 3391.24 16.64 3407.87
%(PSLBkg) 1.34 98.17 0.00 0.00 99.51 0.49
100.00 *
- 65 -
Covance Study Number 2304/001 Final Report
Figure 14 Stability of 8-2 TBA in the soil extract of sterile Eurosoil 5 soil following
adsorption for 3 h at 4C determined by TLC
150
Front
8-2 TBA
Start
100
[PSL] Origin
50
0 0
50
100
[mm]
150
Plate 21, track 3
No
Name
1
Origin
2
8-2 TBA
3
4
-
-
-
*normalised to
Type
Width
[mm]
7.80
14.60
Bkg
3.40
Bkg
4.20
Sum
22.40
Rem
153.20
Ttl
175.60
this value
Pos [mm]
15.40 93.60 8.00 161.40
----
RF
0.00 0.52
-0.96
----
PSL
158.93 3735.09
54.71 61.96 3894.02 2384.03 6278.05
PSL-Bkg
39.19 3510.96
0.00 0.00 3550.15 32.25 3582.40
%(PSLBkg) 1.09 98.01 0.00 0.00 99.10 0.90
100.00 *
- 66 -
Covance Study Number 2304/001 Final Report
Figure 15
Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK961089 at
4C
Log Concentration in Soil (g/g)
-4.5
-4.0
-3.5
-3.0
-2.5
-2.0
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
-2.0 Log Concentration in Solution (g/mL)
Adsorption Desorption
- 67 -
Covance Study Number 2304/001 Final Report
Figure 16
Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK566696 at
4C
Log Concentration in Soil (g/g)
-3.8
-3.6
-3.4
-3.2
-3.0
-2.8
-2.6
-2.4
-2.2
-2.0
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
-2.0 Log Concentration in Solution (g/mL)
Adsorption Desorption
- 68 -
Covance Study Number 2304/001 Final Report
Figure 17
Adsorption and desorption isotherms for 8-2 TBA on sterile Emperor Son's
Lake sediment at 4C
Log Concentration in Soil (g/g)
-4.0
-3.5
-3.0
-2.5
-2.0
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
-2.0 Log Concentration in Solution (g/mL)
Adsorption Desorption
- 69 -
Covance Study Number 2304/001 Final Report
Figure 18
Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK179618 at
4C
Log Concentration in Soil (g/g)
-4.0
-3.5
-3.0
-2.5
-2.0
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
-2.0 Log Concentration in Solution (g/mL)
Adsorption Desorption
- 70 -
Covance Study Number 2304/001 Final Report
Figure 19
Adsorption and desorption isotherms for 8-2 TBA on sterile Eurosoil 5 soil at
4C
Log Concentration in Soil (g/g)
-4.5
-4.0
-3.5
-3.0
-2.5
-2.0
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
-2.0 Log Concentration in Solution (g/mL)
Adsorption Desorption
- 71 -
APPENDICES
Covance Study Number 2304/001 Final Report
- 72 -
Appendix 1 Certificates of analysis
Covance Study Number 2304/001 Final Report
Certificate of analysis for (14C)-8-2 TBA
- 73 -
Certificate of analysis for non-radiolabelled TBA
Covance Study Number 2304/001 Final Report
- 74 -
Soil SK961089
Covance Study Number 2304/001 Final Report
Appendix 2
Soil certificates of analysis
- 75 -
Soil SK566696
Covance Study Number 2304/001 Final Report
- 76 -
Soil SK179618
Covance Study Number 2304/001 Final Report
- 77 -
Emperor Son's Lake
Covance Study Number 2304/001 Final Report
- 78 -
Covance Study Number 2304/001 Final Report
- 79 -
Eurosoil 5
Covance Study Number 2304/001 Final Report
- 80 -
Covance Study Number 2304/001 Final Report
Appendix 3 Radiochemical purity of (14C)-8-2 TBA and formulation stability
Radiochemical Purity by HPLC (stock solution 1)
cps 800 700 600 500 400 300 200 100
0 0.0
Bkg 1
8-2 TBA
5.0 10.0 15.0 20.0 25.0 30.0 35.0 min
Bkg 2
Method: METHOD1 Filename:
Channel: C-14 Detector:
Name
Start - End
(m)
Bkg 1
0.7- 2.3
TBA
16.6- 17.4
17.5- 17.8
18.2- 18.8
Bkg 2
33.7- 34.6
4CHR001.R01
RT (m) 1.5 17.0 17.6 18.4 34.1
Evaluation: A User: S.Swales
Height (cps) 0.3 854.8 7.8 10.8 0.1
Area %Total
(Counts)
(%)
10931.4 55.6
155.4
98.03 0.50 1.39
3 Peaks
11142.4 99.92
Bkg Area Total Area Unallocated
395.5 Counts 11151.5 Counts
9.0 Counts (
0.08 %)
Date of analysis: 4 April 2003
- 81 -
Covance Study Number 2304/001 Final Report
Radiochemical Purity by HPLC (stock solution 1)
cps 700 600
500
400 300
200
100 0 0.0
Bkg 1
8-2 TBA
5.0 10.0 15.0 20.0 25.0 30.0 35.0min
Bkg 2
Method: METHOD1 Filename:
Channel: C-14 Detector:
Name
Start - End
(m)
Bkg 1
1.6- 2.3
8-2 TBA
17.4- 18.5
18.5- 18.9
18.9- 19.8
Bkg 2
33.4- 33.9
CHR014.R01
RT (m) 1.9 17.7 18.5 19.5 33.6
Evaluation: A User: S.Swales
Height (cps) 0.2 718.8 5.8 6.8 0.2
Area %Total
(Counts)
(%)
7338.1 48.9 86.1
98.14 0.65 1.15
3 Peaks
7473.1 99.94
Bkg Area Total Area Unallocated
429.6 Counts 7477.4 Counts
4.4 Counts (
0.06 %)
Date of analysis: 19 December 2003 Prior to formulation of dose formulations for the isotherms test.
- 82 -
Radiochemical Purity by TLC (stock solution 1) Plate 6, track 3
600
Covance Study Number 2304/001 Final Report
Front
Start
8-2 TBA
500
400
[PSL]
300
200
100
0
0
50
100
150
[mm]
No
Name
Type Pos [mm]
1
16.00
2
8-2 TBA
87.80
3
107.80
4
123.40
5
Bkg
6.00
6
Bkg
169.60
-
Sum
--
-
Rem
--
-
Ttl
--
Date of analysis: 7 April 2003
RF -0.48 0.61 0.71 ------
PSL 174.46 15044.37 94.37 227.07
8.45 6.57 15540.27 300.72 15840.99
PSL-Bkg 167.34 15014.08 84.95 210.27
0.00 0.00 15476.64 140.87 15617.51
%(PSL-Bkg) 1.07 96.14 0.54 1.35 0.00 0.00 99.10 0.90
100.00
- 83 -
Radiochemical Purity by TLC (stock solution 1) Plate 23, track 3
Covance Study Number 2304/001 Final Report
Front
8-2 TBA
Start
200
[PSL]
100
Origin
0
0
50
100
150
[mm]
No
Name
Type Pos [mm]
RF
PSL
PSL-Bkg
1
Origin
20.20
0.02 104.36
97.58
2 8-2 TBA
93.40
0.51 6424.60 6399.84
3
112.20
0.63
46.38
36.44
4
127.60
0.73 102.61
88.89
5
Bkg
5.80
--
5.25
0.00
6
Bkg
169.20
--
4.05
0.00
-
Sum
--
--
6677.96 6622.76
-
Rem
--
--
104.00
20.73
-
Ttl
--
--
6781.95 6643.49
Date of analysis: 19 December 2003 Prior to formulation of dose formulations for the isotherms test.
%(PSL-Bkg) 1.47 96.33 0.55 1.34 0.00 0.00 99.69 0.31
100.00
- 84 -
Covance Study Number 2304/001 Final Report
Appendix 4
Chromatography of reference standard
Retention times and Rf values of 8-2 TBA by HPLC and TLC
Reference Standard
Retention time by UV detection (min)
Rf
TBA
18.7
0.49
1 HPLC Chromatogram used is 03CHR002.R01(UV) 2 TLC Plate used is Plate 7 (non-radiolabelled values quoted)
HPLC and TLC chromatographic conditions are described on page 27.
The retention time of the UV peak was ca 0.1 min before the retention time of the corresponding 14C peak due to the positioning of the detectors. The retention time of the reference standard varied slightly depending on the nature of the samples and the amount of material chromatographed. HPLC chromatogram 03CHR002.R01 is shown below:
mV 10
9 8 7 6 5 4 3 2 1 0
0.0
8-2 TBA
5.0 10.0 15.0 20.0 25.0 30.0 35.0 min
- 85 -
Covance Study Number 2304/001 Final Report
Appendix 5 Minor deviations from protocol The study supervisor changed from J Cross to K Dixon and the study co-ordinator changed from S Irlam to C Unsworth. Soil SK179618 was classified as being a clay loam in the protocol. However, this was the UK textural classification not USDA as stated. The USDA textural classification is loam as indicated by the certificate of analysis. The amount of test substance in the supernatant was not determined during the stability test because the radioactivity remaining in the adsorption supernatants was too low to enable any form of analysis to be performed. The amount of test substance was determined in the soil extracts only. The five concentrations selected for the adsorption isotherms test did not cover two orders of magnitude. The test compound had a low water solubility (0.14 mg/L) and was highly adsorbed to the solid phase (<3% remaining in the aqueous phase at the adsorption equilibrium time). These parameters made it impossible to cover two orders of magnitude and still be able to detect radioactivity in the adsorption supernatants. The adsorption isotherms test was , therefore, conducted over one order of magnitude. These deviations do not affect the scientific integrity of the study.
- 86 -
Appendix 6 Example calculations
Covance Study Number 2304/001 Final Report
Dose solution concentration calculations The spreadsheet below shows how the concentration of (14C)-8-2 TBA was determined in the dose solutions applied to soils:
Source file: EF_DOSE.XLS Version 1 File name: I:\D2142\Study Data\2304-001\Data\[SpAc 16.xls]Report
Study details Study number: 2304/1 Experiment type:Adsorption/ Desorption Dosing occasion:Main Isotherms- highest concentration Dosing vehicle:Ethanol Radiolabelled test article HD and Lot number(s):319/03-2304 Lot 1 Analysis date: 22/12/03
Assay details Test article specific radioactivity (MBq/mg): Formulation quantities in weight / volume: LSC aliquot quantities in weight / volume: Number of LSC replicates:
Flask number (identity): Volume of formulation aliquot (mL):
Diluted to volume (mL):
2.63 V V Volume of LSC aliquots (mL): 3 Number of flasks:
Spac 1 0.05 5
Spac 2 0.05 5
Spac 3 0.05 5
Spac 4 0.05 5
0.25 6
Spac 5 0.05 5
Spac 6 0.05 5
Radioactivity in aliquots (dpm) 1: 2: 3:
Background radioactivity (dpm):
51901 51857 51544
32
52575 52454 52393
51561 51393 51527
51438 51221 51440
50954 50857 50827
50836 51035 50752
Background corrected dpm/mL 1: 2: 3:
kBq/mL of formulation: SD: CV:
Flasks omitted from mean marked with X: Values used: n: Sum:
Mean kBq/mL of formulation: SD: CV:
207476 207300 206048
345 1
0.38%
345 6
2058 343 4
1.17%
210172 209688 209444
350 1
0.18%
350
206116 205444 205980
343 1
0.17%
343
205624 204756 205632
342 1
0.25%
342
203688 203300 203180
339 0
0.13%
339
203216 204012 202880
339 1
0.29%
339
Volume of formulation applied (mL): Radioactivity applied (Ci): Radioactivity applied (kBq):
Weight of test article applied (mg):
0.0086 0.079715 2.949446 0.001121
- 87 -
Covance Study Number 2304/001 Final Report
Determination of applied radioactivity The following spreadsheet shows how the percent of applied radioactivity was determined in soil extracts. The same spreadsheet was used for quantification of combusted residues.
Source file: R_RCALC.XLS Version 1 File name: I:\D2142\Study Data\2304-001\Data\Isotherms\[Adsorb supernatant DG B.xls]Sheet1
ASSAY DETAILS Covance study number: Sample description:
Number of LSC replicates: Flag variability at x% of mean:
LOD (times background): Number of samples (10 max)
2304/1 Adsorb 2 10% 1.5 10
supernatant DG B LOD based on
mean
counts
LSC DATA INPUT (dpm)
Subject code:
B1
B2
B3
B4
B5
SDATA file input
1
0
0
0
0
0
2
0
0
0
0
0
Background
0
0
0
0
0
Manual input
1
397
393
336
279
214
2
384
403
356
313
205
Background
WEIGHT DATA INPUT (g) Weight of empty pot
Weight of pot + sample Total assayed sample weight
LSC aliquot weights 1 2
25
6.167 6.167 0.997 0.999
25
6.192 6.192 1.001 1.004
32
6 6 0.994 0.996
32
6.192 6.192 0.993 0.993
20
6.079 6.079 0.995 0.986
B6 0 0
0 223 236
20
6.176 6.176 0.995 1.003
B7 0 0
0 251 256
24
6.047 6.047 1.477 1.437
B8 0 0
0 207 246
24
6.148 6.148 1.367 1.466
B9 0 0
0 72 74
24
6.123 6.123 1.395 1.433
B10 0 0
0 69 71
24
6.036 6.036 1.397 1.393
REFERENCE DATA Dose / Reference (kBq) ... (A) Weight of sub-sample (g) Initial sample weight (g) Equivalent dose (kBq) ... (B) Specific activity (MBq/mg):
RESULTS Background corrected dpm 1 2
2.949
2.63
372 359
2.949
2.63
368 378
2.366
2.63
304 324
2.366
2.63
247 281
1.783
2.63
194 185
1.783
2.63
203 216
1.194
2.63
227 232
1.194
0.3054
0.3054
2.63
2.63
2.63
183
48
45
222
50
47
Dpm per g 1 373.119358 367.632368 305.83501 248.741188 194.974874 204.020101 153.689912 133.869788 34.4086022 32.2118826 2 359.359359 376.494024 325.301205 282.980866 187.626775 215.353938 161.44746 151.432469 34.8918353 33.7401292
Mean dpm per g Concentration (g/g) Variability limit exceeded?
LSC input method Radioactivity recovered
dpm in total sample kBq in total sample dpm in sub-sample kBq in sub-sample
366 2.321E-03
NO Manual
2259 0.0376
NA NA
372 2.358E-03
NO Manual
2304 0.0384
NA NA
316 2.000E-03
NO Manual
1893 0.0316
NA NA
266 1.685E-03
NO Manual
1646 0.0274
NA NA
191 1.212E-03
NO Manual
1163 0.0194
NA NA
210 1.329E-03
NO Manual
1295 0.0216
NA NA
158 9.985E-04
NO Manual
953 0.0159
NA NA
143 9.040E-04
NO Manual
877 0.0146
NA NA
35 2.196E-04
NO Manual
212 0.0035
NA NA
33 2.090E-04
NO Manual
199 0.0033
NA NA
% Recovery of radioactivity LOD (%)
based on reference Mean % recovery
Sample concentration (g/g)
1.3 0 A
1.3 2.321E-03
1.3 0 A
2.358E-03
1.3 0.1
A 1.3 2.000E-03
1.2 0.1
A
1.685E-03
1.1 0.1
A 1.2 1.212E-03
1.2 0.1
A
1.329E-03
1.3 0.1
A 1.3 9.985E-04
1.2 0.1
A
9.040E-04
1.2 0.3
A 1.2 2.196E-04
1.1 0.3
A
2.090E-04
- 88 -
Adsorption coefficient calculations The following spreadsheet shows how the adsorption coefficients were calculated:
AdsorptionDesorption.XLS Version 3.0 (Microsoft Excel 97)
File name:
Study number:
2304-001
Treatment:
Soil B
% Organic carbon:
0.8
% Organic matter:
1.4
Assumed solution density (g/mL): 1
Concentration
0.125 0.125
0.1 0.1 0.075 0.075 0.05 0.05 0.0125 0.0125
Replicate
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10
m
9.014 8.999 9.019 9.017 9.002 8.993 8.993 8.996 9.033 8.991
V0 9.232 9.266 9.12 9.33 9.24 9.256 9.244 9.31 9.337 9.22
G 1.121 1.121
0.9 0.9 0.678 0.678 0.454 0.454 0.116 0.116
W2 6.167 6.192
6 6.192 6.079 6.176 6.047 6.148 6.123 6.036
W3 6.136 6.275 6.028 6.216 6.122 6.165 5.93 6.175 6.092 6.35
Ce 0.002321 0.002358
0.002 0.001685 0.001212 0.001329 0.0009985 0.000904 0.0002196 0.000209
C1 0.002045 0.002457 0.001459 0.001419 0.0009592 0.0009755 0.0009743 0.0007586 0.0002418 0.0002241
V2 6.167 6.192
6 6.192 6.079 6.176 6.047 6.148 6.123 6.036
V3 6.136 6.275 6.028 6.216 6.122 6.165 5.93 6.175 6.092 6.35
X 1.09957253 1.09915077
0.88176 0.88427895 0.66680112 0.66569878 0.44476987 0.44558376 0.11394959 0.11407302
%A 98.08854 98.050916 97.973333 98.253217 98.348248 98.18566 97.966931 98.146203 98.232409 98.33881
X1 1.08787035 1.08342877 0.87465307 0.87629315
0.661728 0.6607736 0.43906963 0.44135916 0.1124052 0.11260191
%D 1.06424812 1.43037711 0.80599392 0.90308562 0.7608148 0.73985077 1.28161371 0.94810462 1.35533295 1.28962431
%ND 98.9357519 98.5696229 99.1940061 99.0969144 99.2391852 99.2601492 98.7183863 99.0518954 98.6446671 98.7103757
X/m 0.12198497 0.12214143 0.09776694 0.09806798 0.07407255 0.0740241 0.04945734 0.04953132 0.01261481 0.01268747
X1/m 0.12068675 0.12039435 0.09697894 0.09718234
0.073509 0.07347644 0.04882349 0.04906171 0.01244384 0.01252385
Kd 52.5570749 51.7987425 48.8834682 58.2005799 61.1159676 55.6991008 49.5316383 54.7912838 57.4444981 60.705586
Kdoc 6569.63437 6474.84281 6110.43353 7275.07249 7639.49595 6962.3876 6191.45478 6848.91048 7180.56226 7588.19825
Covance Study Number 2304/001 Final Report
-89-
-90-
Bookmark code bm01 bm02
bm03 bm04 bm05 bm06
Description Study Number Study Title
Report Status Study Director Company name of sponsor Sponsor address
bm08 bm08a bm09 bm01a bm13
bm14
bm16 bm17 bm18 bm19
Test article test article chemical name OECD Guideline UK GLP Guideline
OECD GLP Guideline
Report issue date Protocol signed date First practical work date Last practical work date
Bookmark
2304/001 (14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Equilibrium Method Draft S Swales Katie Smythe, Administrator Telomers Research Program The Rand Corporation 1200 South Hayes Street Arlington, VA22202 USA 8-2 TBA 8-2 TBA 2-perfluorooctylethanol
Batch
United Kingdom Statutory Instrument 1999 No. 3106, The Good Laboratory Practice Regulations 1999 OECD Principles of Good Laboratory
Practice (revised 1997, Issued Jan 1998) ENV/MC/CHEM(98)17
5 November 2001 23 January 2002
