Document jm087bxkOGGnX5Ojgp3n5E1bQ

AR226-1891 Final Report Study Title (14C )-8-2 T e lo m e r B A lcohol: Adsorption/D esorption using a Batch Equilibrium M ethod Data Requirem ent Protocol designed to m eet the known requirem ents of OECD Guideline 106 (January 2000) A u th o r S Swales Sponsor Test Facility Katie Smythe, A dm inistrator Telom er Research Program The Rand Corporation 1200 South Hayes Street Arlington, V A 22202 USA C ovance Laboratories Ltd O tley Road, Harrogate North Yorkshire HG3 1PY ENGLAND R E C E IV E D O P P T NCIC 2004 D EC 22 A M 1 1 :20 Covance Study Num ber 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. 2- - Covance Study Number 2304/001 Final Report GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT (14C)-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. S Swales Study Director, Covance Laboratories Ltd Name Submitter Department or Company Name Submitter Department or Company Name Submitter Department or Company Date Date Date Covance Study Number 2304/001 Final Report FLAGGING STATEMENTS (14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium Method 4- - Covance Study Number 2304/001 ___________________ Final Report STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT (14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium Method I, 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 international 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 part of the UK GLP compliance-monitoring program. S Swales BSc MSc PhD Study Director -5- Covance Study Number 2304/001 ___________________ Final Report QUALITY ASSURANCE STATEMENT (14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium Method This study has been reviewed by the Quality Assurance Unit of 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 of a "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 D ales From To 17 Mar 2003 17 Mar 2003 26 Mar 2003 26 Mar 2003 03 Apr 2003 03 Apr 2003 13 A ug 2003 13 A ug 2003 27 N ov 2003 27 N ov 2003 23 D ec 2003 18 Feb 2004 23 Dec 2003 09 Mar 2004 Phase Protocol Review Protocol Amendment Review Test Article Receipt Protocol Amendment R eview Protocol Amendment Review D ose Administration Draft Report and Data R eview Date Reported to SD and SD M anagem ent 17 Mar 2003 26 Mar 2003 03 Apr 2003 13 A ug 2003 27 N ov 2003 23 D ec 2003 09 Mar 2004 Inspection Dates From To 13 Mat 2003 13 M ay 2003 30 May 2003 30 May 2003 24 Jun2003 24 Jun 2003 30 Jun2003 30 Jun2003 Process Phase Data Processing High Performance Liquid Chromatography Analysis Sample Receipt Sample Preparation Date Reported to SD and SD M anagem ent 13 M ay 2003 30 May 2003 24 Jun 2003 30 Jun 2003 I Moftrisoj SectilfHead, Quality Assurance -6- Covance Study Number 2304/001 ___________________ Final Report RESPONSIBLE SCIENTIST'S STATEMENT (14C)-8-2 Telomer B Alcohol: Adsorption/Desorption using a Batch Equilibrium Method I, 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. A Hill BSc PhD Director, Environmental Sciences 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 KDixon KDixon KDixon KDixon PDavies C Clare 8- - 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. 9- - 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 C O N T E N T S ............................................................................................................................................................10 S U M M A R Y .............................................................................................................................................................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 o f 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 o f R esults....................................................................................................................................30 R E S U L T S ................................................................................................................................................................ 31 Preliminary tests.............................................................................................................................................31 Adsorption and desorption isotherms.........................................................................................................33 SUMMARY AND CONCLUSIONS.................................................................................................................35 TABLES.................................................................................................................................................................. 36 Table 1 1:1 Ratio o f soil to aqueous phase following treatment at 0.025 pg/mL............................... 37 Table 2 1:5 Ratio o f soil to aqueous phase following treatment at 0.025 pg/mL............................... 37 Table 3 1:25 Ratio o f soil to aqueous phase following treatment at 0.025 pg/mL.............................37 Table 4 Recovery o f applied radioactivity from the 24 hour equilibrium time samples at a soil to aqueous phase ratio o f 1:1 and 20 1C...............................................................................................38 Table 5 Stability over a 24 hour period (20C) at a soil to aqueous phase ratio o f 1 :1 .....................38 - 10 - 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 o f 1:1)......................................................................................................................38 Table 7 Stability over a 3 hour period (4C) at a soil to aqueous phase ratio o f 1 :1 ........................ 39 Table 8 Percent applied radioactivity recovered in the supernatants from sterile soils at 4C and stability o f 8-2 TBA in the soil extract o f sterile soil...................................................................... 39 Table 9 Percent applied radioactivity recovered in the desorption supernatants from sterile soils at 4C following a 3 h equilibrium tim e...........................................................................................39 Table 10 Values for Ce, X/m, Q and Xi/m for 8-2 TBA on soil SK961089.....................................40 Table 11 Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on soil SK566696.....................................40 Table 12 Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on Emperor Son's Lake sediment............41 Table 13 Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on soil SK179618.....................................41 Table 14 Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on Eurosoil 5 ............................................. 42 Table 15 Quantities o f 8-2 TBA adsorbed to and desorbed from soil SK 961089............................ 42 Table 16 Quantities o f 8-2 TBA adsorbed to and desorbed from soil SK 566696............................ 43 Table 17 Quantities o f 8-2 TBA adsorbed to and desorbed from Emperor Son's Lake sediment...43 Table 18 Quantities o f 8-2 TBA adsorbed to and desorbed from soil SK 179618........................... 44 Table 19 Quantities o f 8-2 TBA adsorbed to and desorbed from Eurosoil 5..................................... 44 Table 20 Adsorption coefficients for 8-2 TBA on soil SK 961089........................................................45 Table 21 Adsorption coefficients for 8-2 TBA on soil SK 566696........................................................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 SK 179618........................................................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 SK 961089.......................................................................................................................................... 47 Table 26 Freundlich adsorption and desorption constants and regression analysis for 8-2 TBA on soil SK 566696.......................................................................................................................................... 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 SK 179618.......................................................................................................................................... 49 Table 29 Freundlich adsorption and desorption constants and regression analysis for 8-2 TBA on Eurosoil 5................................................................................................................................................... 49 Table 30 Recovery o f radioactivity from all five soils treated with 8-2 TBA at 0.125 pg/mL after adsorption, desorption, organic wash and combustion...................................................................50 Table 31 pH o f the blank solutions used for the main isotherms test.................................................. 50 Table 32 pH o f 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 8 2 T B A ...............................................................................................................................................................53 Figure 2 Adsorption equilibrium time data for soil SK566696 following treatment with 8 2 T B A ...............................................................................................................................................................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 8 2 T B A ...............................................................................................................................................................56 Figure 5 Stability o f 8-2 TBA in the soil extract o f non-sterile SK961089 soil following adsorption for 24 h at 20C determined by TLC..................................................................................... 57 Figure 6 Stability o f 8-2 TBA in the soil extract o f non-sterile SK566696 soil following adsorption for 24 h at 20C determined by TLC......................................................................................58 Figure 7 Stability o f 8-2 TBA in the soil extract o f non-sterile Emperor Son's Lake sediment following adsorption for 24 h at 20C determined by TLC....................................................................59 Figure 8 Stability o f 8-2 TBA in the soil extract o f non-sterile SK179618 soil following adsorption for 24 h at 20C determined by TLC..................................................................................... 60 Figure 9 Stability o f 8-2 TBA in the soil extract o f non-sterile SK961089 soil following adsorption for 3 h at 4C determined by T LC .......................................................................................... 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 T LC ..........................................................................................62 - 11 - Covance Study Number 2304/001 Final Report Figure 11 Stability o f 8-2 TBA in the soil extract o f 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 T LC ..........................................................................................64 Figure 13 Stability o f 8-2 TBA in the soil extract o f sterile SK179618 soil following adsorption for 3 h at 4C determined by T LC ..........................................................................................65 Figure 14 Stability o f 8-2 TBA in the soil extract o f sterile Eurosoil 5 soil following adsorption for 3 h at 4C determined by T LC ..........................................................................................66 Figure 15 Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK961089at 4C......67 Figure 16 Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK566696at 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 SK179618at 4C......70 Figure 19 Adsorption and desorption isotherms for 8-2 TBA on sterile Eurosoil 5 soilat 4 C ..... 71 A P P E N D IC E S ........................................................................................................................................................ 72 Appendix 1 Certificates o f analysis............................................................................................................ 73 Appendix 2 Soil certificates o f analysis.................................................................................................... 75 Appendix 3 Radiochemical purity o f (14C)-8-2 TBA and formulation stability................................ 81 Appendix 4 Chromatography o f reference standard................................................................................ 85 Appendix 5 Minor deviations from protocol.............................................................................................86 Appendix 6 Example calculations.............................................................................................................. 87 - 12 - SUMMARY Covance Study Number 2304/001 Final Report 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 pg/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 Organic carbon (0.01M CaCl2) content (%) 7.6 4.6 4.2 0.8 6.0 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 - 13 - 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 pL) 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 pg/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 pg/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 pg/mL) were air-dried then ground prior to combustion in oxygen. - 14 - Covance Study N um ber 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%. aHollis, 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 - 15 - Covance Study Number 2304/001 Final Report INTRODUCTION (14C)-8-2 TBA (2-perfluorooctylethanol) used in this study had the following structure: C 8 F 17 *Position o f the 14C radiolabel The objective of the study was to: OH 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). - 16 - 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 pg/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 - 17 - 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) aInformation on the sampling site, sampling method and pesticide history was not supplied with Eurosoil 5 and, therefore, this information has not been reported. - 18 -  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 Organic carbon (0.01M CaCl2) Content (%) Clay content (%) Soil texture (USDA) SK961089 7.6 4.6 34 Clay loam SK566696 4.2 0.8 11 Loamy sand Emperor Son's Lake 6.0 1.2 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 (1T4C)-F8,2 t7Ba--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 - 19 - 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 pm 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 pL) 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 - 20 - 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 (pg) SSR EQT EQT-Rpt Isotherms Isotherms SSR 2 3 4 5 1stock solution 1, 2 stock solution 2 SSR = soil:solution ratio EQT = equilibrium time EQT-Rpt = repeat equilibrium time 0.3572 0.1471 0.1911 0.1301 0.0451 25 100 130 88 30 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 pg/mL to 0.1 pg/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 - 21 - 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 gg/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 - 22 - 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 qg/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 qg/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. - 23 - 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 gg/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: - 24 - 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 pg/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 42 42 42 52 52 Formulation conc.1 (mg/mL) 0.131 0.131 0.131 0.053 0.053 Volume formulation (ML) 8.6 6.9 5.2 8.6 2.2 Weight of 8-2 TBA (Pg) 1.127 0.904 0.681 0.456 0.117 'concentration 2refer to table on page 21 for preparation of the formulations. Volume of 0.01 MCaCl2 (mL) 9 9 9 9 9 Nominal conc.1 (pg/mL) 0.125 0.1 0.075 0.05 0.0125 Actual conc.1 (Mg/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 - 25 - 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 pg/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 pg/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 pg/mL) and lowest (0.0125 pg/mL) concentrations of 8-2 TBA was measured. The pH of each adsorption supernatant from the adsorption isotherm test was also measured. - 26 - Analytical Methods and Measurements Covance Study Number 2304/001 Final Report HPLC Radiochemical purity determinations were performed using the following high performance liquid chromatography (HPLC) method: Column: Gradient: Genesis C18, 150 x 4.6 mm, 4pm A linear binary gradient system was used consisting of Solvent A: 2mM ammonium acetate in water Solvent B: Methanol Time %A 0 97 15 0 35 0 35.1 97 40 97 %B 3 100 100 3 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 P-ram flow through radioactivity monitor (LabLogic, Sheffield, South Yorkshire) equipped with a liquid mixing (500 pL) 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 - 27 - 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 (pg) = G Dry weight of soil (g) = m Volume of solution associated with soil (mL) = V0 Concentration of test article in adsorption supernatant (pg/mL) = Ce Concentration of test article in desorption supernatant (pg/mL) = Ci Density of 0.01 M calcium chloride solutions and supernatants (g/mL) = p Adsorption Weight of stock solution initially added (g) = W1 Volume of stock solution initially added (mL) = V1= W1/ p Weight of supernatant after adsorption step (g) = W2 Volume of supernatant after adsorption step (mL) = V2= W2/ p Quantity of test substance adsorbed (pg) = X = G - (Cex (V0+ V1)) Percent of test substance adsorbed = A = X x 100 / G - 28 - 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/ p Quantity of test substance adsorbed following desorption (pg) = Xi = X + (Ce x (Vo + Vi - V2)) - (Cl x (Vo + Vi - 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 = Kdx 100 / % organic carbon Adsorption coefficient as a function of the organic matter content of the soil = Kdom= Kdx 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: - 29 - 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. - 30 - 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 gg/mL 0.05 gg/mL 0.025 gg/mL 0.01 gg/mL 0.1 gg/mL1 5.166 2.583 1.291 0.553 5.166 2.933 2.031 0.999 0.467 5.206 1Control 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 gg/mL 0.05 gg/mL 0.025 gg/mL 0.01 gg/mL 0.1 gg/mL1 5.166 2.583 1.291 0.553 5.166 1.838 1.164 0.600 0.345 4.848 1Control 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. - 31 - 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 qg/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 - 32 - 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. - 33 - 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 gg/mL) and lowest (0.0125 gg/mL) concentrations of 8-2 TBA are presented in Table 31. The pH values of all the adsorption supernatants are presented in Table 32. - 34 - 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 aHollis, 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 - 35 - TABLES Covance Study Number 2304/001 Final Report - 36 - Table 1 Covance Study Number 2304/001 Final Report 1:1 Ratio of soil to aqueous phase following treatment at 0.025 pg/mL Unit Soil type Supernatant A2 SK961089 B2 SK566696 C2 Emperor Son's Lake D1 SK179618 1.2 1.5 1.1 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 pg/mL Unit Soil type Supernatant A3 SK961089 A4 SK961089 B3 SK566696 B4 SK566696 C3 Emperor Son's Lake D3 SK179618 D4 SK179618 1.6 1.6 2.6 2.5 2.6 6.3 5.8 Ratio Test not performed for Eurosoil 5. % of applied radioactivity Soil extract Residue Lid Wash 71.5 19.5 71.3 19.4 76.0 4.8 77.7 4.1 78.9 4.0 35.9 45.4 37.6 43.7 2.6 3.3 4.8 5.5 9.6 1.3 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 pg/mL Unit Soil type Supernatant A5 SK961089 A6 SK961089 B5 SK566696 C5 Emperor Son's Lake C6 Emperor Son's Lake D5 SK179618 4.8 5.1 8 10 11.3 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 - 37 - Table 4 Covance Study Number 2304/001 Final Report 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 SK566696 Emperor Son's Lake SK179618 EQ A5 EQ A6 EQ B5 EQ B6 EQ C5 EQ C6 EQ D5 EQ D6 1.0 0.4 1.3 1.2 0.7 0.9 0.6 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 - Table 7 Covance Study Number 2304/001 Final Report 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 o f applied radioactivity 3 h @ 4C Amount in soil 8-2 TBA 1.9 92.0 90.3 3.0 82.6 81.1 1.1 92.9 91.1 1.2 94.1 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 1.5 0.8 3 0.7 6 1.0 24 1.4 0.0 1.2 1.2 1.3 1.8 0.0 0.0 1.0 1.0 1.5 1.3 2.0 1.4 1.3 1.7 Values presented are mean values o f 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 X 1/m for 8-2 TBA on soil SK961089 Nominal Dose level ( bg/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 Ce X/m ( bg/mL) (bg/g) 0.0021 0.0021 0.0021 0.1223 0.1224 0.1223 0.0013 0.0012 0.0012 0.0985 0.0989 0.0987 0.0010 0.0016 0.0013 0.0741 0.0744 0.0743 0.0009 0.0007 0.0008 0.0496 0.0497 0.0497 0.0002 0.0001 0.0002 0.0126 0.0127 0.0127 Desorption C1 X^m ( bg/mL) (bg/g) 0.0010 0.0010 0.0010 0.1225 0.1225 0.1225 0.0008 0.0009 0.0009 0.0984 0.0986 0.0985 0.0005 0.0017 0.0011 0.0742 0.0736 0.0739 0.0004 0.0004 0.0004 0.0497 0.0497 0.0497 0.0001 0.0001 0.0001 0.0126 0.0127 0.0127 Ce and Ci are the aqueous phase concentrations; X/m and X ^m are the concentrations in the soil Table 11 Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on soil SK566696 Nominal Dose level (bg/mL) Replicate Adsorption Ce X/m ( bg/mL) (bg/g) Desorption C1 X!/m (bg/mL) (bg/g) 0.125 1 2 Mean 0.0023 0.0024 0.0023 0.1220 0.1221 0.1221 0.0020 0.0025 0.0023 0.1207 0.1204 0.1205 0.1 1 0.0020 2 0.0017 Mean 0.0018 0.0978 0.0981 0.0979 0.0015 0.0014 0.0014 0.0970 0.0972 0.0971 0.075 1 2 Mean 0.0012 0.0013 0.0013 0.0741 0.0740 0.0740 0.0010 0.0010 0.0010 0.0735 0.0735 0.0735 0.05 1 0.0010 2 0.0009 Mean 0.0010 0.0495 0.0495 0.0495 0.0010 0.0008 0.0009 0.0488 0.0491 0.0489 0.0125 1 2 Mean 0.0002 0.0002 0.0002 0.0126 0.0127 0.0127 0.0002 0.0002 0.0002 0.0124 0.0125 0.0125 Ce and C1are the aqueous phase concentrations; X/m and X 1/m are the concentrations in the soil - 40 - Table 12 Covance Study Number 2304/001 Final Report Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on Emperor Son's Lake sediment Nominal Dose level ( hg/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 Ce X/m (hg/mL) (hg/g) 0.0028 0.0027 0.0027 0.1212 0.1225 0.1218 0.0020 0.0021 0.0021 0.0977 0.0979 0.0978 0.0014 0.0026 0.0020 0.0735 0.0726 0.0731 0.0011 0.0009 0.0010 0.0492 0.0493 0.0492 0.0002 0.0002 0.0002 0.0127 0.0127 0.0127 Desorption C1 X^m (pg/mL) ( hg/g ) 0.0019 0.0018 0.0019 0.1205 0.1217 0.1211 0.0013 0.0013 0.0013 0.0975 0.0974 0.0974 0.0010 0.0009 0.0009 0.0730 0.0729 0.0729 0.0007 0.0005 0.0006 0.0491 0.0492 0.0491 0.0001 0.0001 0.0001 0.0126 0.0126 0.0126 Ce and Ci are the aqueous phase concentrations; X/m and X ^m are the concentrations in the soil Table 13 Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on soil SK179618 Nominal Dose level (pg/mL) Replicate Adsorption Ce (pg/mL) X/m (hg/g) Desorption C1 (pg/mL) X!/m ( hg/g ) 0.125 0.1 0.075 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 0.1218 0.1220 0.1219 0.0976 0.0980 0.0978 0.0735 0.0736 0.0735 0.0015 0.0015 0.0015 0.0013 0.0014 0.0013 0.0009 0.0010 0.0010 0.1212 0.1215 0.1214 0.0971 0.0974 0.0972 0.0732 0.0731 0.0731 0.05 1 2 Mean 0.0010 0.0008 0.0009 0.0492 0.0495 0.0493 0.0006 0.0006 0.0006 0.0492 0.0492 0.0492 0.0125 1 2 Mean 0.0003 0.0002 0.0003 0.0126 0.0126 0.0126 0.0002 0.0002 0.0002 0.0125 0.0125 0.0125 Ce and C1are the aqueous phase concentrations; X/m and X 1/m are the concentrations in the soil - 41 - Covance Study Number 2304/001 Final Report Table 14 Values for Ce, X/m, C1 and X 1/m for 8-2 TBA on Eurosoil 5 Nominal Dose level (bg/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 Ce X/m ( bg/mL) (bg/g) 0.0020 0.0016 0.0018 0.1225 0.1219 0.1222 0.0012 0.0011 0.0012 0.0990 0.0988 0.0989 0.0012 0.0013 0.0012 0.0737 0.0734 0.0735 0.0006 0.0006 0.0006 0.0499 0.0498 0.0499 0.0002 0.0002 0.0002 0.0127 0.0127 0.0127 Desorption C1 X ^m ( bg/mL) ( bg/g ) 0.0010 0.0010 0.0010 0.1223 0.1215 0.1219 0.0009 0.0009 0.0009 0.0985 0.0983 0.0984 0.0006 0.0008 0.0007 0.0736 0.0731 0.0733 0.0003 0.0003 0.0003 0.0497 0.0497 0.0497 0.0001 0.0001 0.0001 0.0127 0.0127 0.0127 Ce and Ci are the aqueous phase concentrations; X/m and X ^m are the concentrations in the soil Table 15 Quantities of 8-2 TBA adsorbed to and desorbed from soil SK961089 Nominal Dose level ( bg/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 (pg) 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 - Table 16 Covance Study Number 2304/001 Final Report Quantities of 8-2 TBA adsorbed to and desorbed from soil SK566696 Nominal Dose level (pg/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 (pg) 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 ( pg/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 (pg) 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 - Table 18 Covance Study Number 2304/001 Final Report Quantities of 8-2 TBA adsorbed to and desorbed from soil SK 179618 Nominal Dose level (pg/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 (pg) 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 ( pg/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 (pg) 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 - Q- QOO Covance Study Number 2304/001 Final Report Table 20 Adsorption coefficients for 8-2 TBA on soil SK961089 Nominal Dose level (pg/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 Kdom 58.12 58.91 58.51 1263 1281 1272 736 746 741 78.71 84.09 81.40 1711 1828 1770 996 1064 1030 74.16 46.60 60.38 1612 1013 1313 939 590 764 57.49 68.03 62.76 1250 1479 1364 728 861 794 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 ( pg/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 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 - Q- QOO Covance Study Number 2304/001 Final Report Table 22 Adsorption coefficients for 8-2 TBA on Emperor Son's Lake Nominal Dose level (pg/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 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 ( pg/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 Kdom 57.96 61.66 59.81 1525 1623 1574 878 934 906 62.73 63.00 62.87 1651 1658 1654 950 955 953 58.06 66.71 62.38 1528 1755 1642 880 1011 945 48.21 59.43 53.82 1269 1564 1416 730 900 815 43.44 52.86 48.15 1143 1391 1267 658 801 729 - 46 - Covance Study Number 2304/001 Final Report Table 24 Adsorption coefficients for 8-2 TBA on Eurosoil 5 Nominal Dose level <Rg/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 Kdom QO 62.58 75.24 68.91 934 1123 1028 539 649 594 83.18 88.44 85.81 1242 1320 1281 717 762 740 61.56 57.33 59.44 919 856 887 531 494 512 85.77 88.83 87.30 1280 1326 1303 739 766 753 82.96 84.55 83.75 1238 1262 1250 715 729 722 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 4941 2823 77.35 9669 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 3414 1966 103.92 2735 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 47.85 572 714 331 412 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 pg/mL after adsorption, desorption, organic wash and combustion Sample A1 A2 B1 B2 C1 C2 D1 D2 E1 E2 % of Applied Radioactivity Ads Supernatant 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 0.125 pg/mL 0.0125 pg/mL pH 6.05 5.90 Table 32 pH of the adsorption supernatants from the main isotherms test following treatment with 8-2 TBA Dose group Replicate 1 2 3 4 5 6 7 8 9 10 pH 7.82 8.14 7.45 7.65 7.88 7.87 7.93 7.94 7.86 8.35 - 50 - Table 32 continued Covance Study Number 2304/001 Final Report pH of the adsorption supernatants from the main isotherms test following treatment with 8-2 TBA Dose group B Replicate 1 2 3 4 5 6 7 8 9 10 C1 2 3 4 5 6 7 8 9 10 D1 2 3 4 5 6 7 8 9 10 E1 2 3 4 5 6 7 8 9 10 pH 4.26 4.22 4.11 4.18 4.14 4.10 4.17 4.14 4.14 4.11 6.21 6.17 6.20 6.24 6.25 6.16 6.23 6.25 6.26 6.31 5.74 5.73 5.43 5.49 5.47 5.58 5.54 5.52 5.58 5.54 3.05 3.00 3.04 3.02 3.02 2.99 2.99 2.99 2.96 2.99 - 51 - FIGURES Covance Study Number 2304/001 Final Report - 52 - Figure 1 Covance Study Number 2304/001 Final Report Adsorption equilibrium time data for soil SK961089 following treatment with 8-2 TBA Adsorption Time (hours) Percent adsorbed to soil 00 3 99 6 98.2 24 98.3 48 98.4 - 53 - Figure 2 Covance Study Number 2304/001 Final Report Adsorption equilibrium time data for soil SK566696 following treatment with 8-2 TBA Adsorption Time (hours) Percent adsorbed to soil 00 3 98.3 6 98.3 24 98.1 48 98.1 - 54 - Figure 3 Covance Study Number 2304/001 Final Report Adsorption equilibrium time data for Emperor Son's Lake sediment following treatment with 8-2 TBA Adsorption Time (hours) Percent adsorbed to soil 00 3 97.8 6 98.2 24 98.4 48 98.8 - 55 - Figure 4 Covance Study Number 2304/001 Final Report Adsorption equilibrium time data for soil SK179618 following treatment with 8-2 TBA Adsorption Time (hours) Percent adsorbed to soil 00 3 97.5 6 97.9 24 97.6 48 96.9 - 56 - Figure 5 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of non-sterile SK961089 soil following adsorption for 24 h at 20C determined by TLC Plate 15, track 1 No Name Type Width [mm] 1 8-2 TBA 15.00 2 10.40 3 Bkg 3.60 4 Bkg 4.20 - Sum 25.40 - Rem 150.20 - Ttl 175.60 ^normalised to this 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 - Figure 6 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of non-sterile SK566696 soil following adsorption for 24 h at 20C determined by TLC Plate 15, track 3 No Name Type Width [mm] 1 8-2 TBA 12.60 2 10.80 3 9.60 4 Bkg 2.40 5 Bkg 2.00 - Sum 33.00 - Rem 142.60 - Ttl 175.60 ^normalised to this 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 Plate 16, track 2 No Name Type Width [mm] 1 8-2 TBA 13.80 2 Bkg 3.40 3 Bkg 2.80 - Sum 13.80 - Rem 161.80 - Ttl 175.60 ^normalised to 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 - Figure 8 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of non-sterile SK179618 soil following adsorption for 24 h at 20C determined by TLC Plate 16, track 3 No Name Type Width [mm] 1 Origin 14.40 2 8-2 TBA 10.40 3 7.40 4 Unk 13.80 5 Bkg 2.80 6 Bkg 4.20 - Sum 46.00 - Rem 129.60 - Ttl 175.60 ^normalised to 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 - Figure 9 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of non-sterile SK961089 soil following adsorption for 3 h at 4C determined by TLC Plate 18, track 1 No Name Type Width [mm] 1 8-2 TBA 13.40 2 Bkg 2.80 3 Bkg 5.20 - Sum 13.40 - Rem 162.20 - Ttl 175.60 ^normalised to 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 - Figure 10 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of non-sterile SK566696 soil following adsorption for 3 h at 4C determined by TLC Plate 18, track 3 No Name Type Width [mm] 1 8-2 TBA 12.00 2 Bkg 4.80 3 Bkg 4.00 - Sum 12.00 - Rem 163.60 - Ttl 175.60 ^normalised to 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 Plate 19, track 1 No Name Type Width [mm] 1 8-2 TBA 13.60 2 Bkg 1.60 3 Bkg 5.00 - Sum 13.60 - Rem 162.00 - Ttl 175.60 *normalised to this value 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 - Figure 12 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of non-sterile SK179618 soil following adsorption for 3 h at 4C determined by TLC Plate 19, track 3 No Name Type Width [mm] 1 8-2 TBA 11.80 2 6.80 3 11.40 4 Bkg 3.80 5 Bkg 4.00 - Sum 30.00 - Rem 145.60 - Ttl 175.60 ^normalised to 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 - Figure 13 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of sterile SK179618 soil following adsorption for 3 h at 4C determined by TLC Plate 21, track 1 No Name Type Width [mm] 1 Origin 7.80 2 8-2 TBA 12.60 3 Bkg 2.60 4 Bkg 3.20 - Sum 20.40 - Rem 155.20 - Ttl 175.60 ^normalised to 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 - Figure 14 Covance Study Number 2304/001 Final Report Stability of 8-2 TBA in the soil extract of sterile Eurosoil 5 soil following adsorption for 3 h at 4C determined by TLC Plate 21, track 3 No Name Type Width [mm] 1 Origin 7.80 2 8-2 TBA 14.60 3 Bkg 3.40 4 Bkg 4.20 - Sum 22.40 - Rem 153.20 - Ttl 175.60 ^normalised to 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 - Figure 15 Covance Study Number 2304/001 Final Report Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK961089 at 4C Log Concentration in Soil (pg/g) - 67 - Figure 16 Covance Study Number 2304/001 Final Report Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK566696 at 4C Log Concentration in Soil (pg/g) - 68 - Figure 17 Covance Study Number 2304/001 Final Report Adsorption and desorption isotherms for 8-2 TBA on sterile Emperor Son's Lake sediment at 4C Log Concentration in Soil (|jg/g) - 69 - Figure 18 Covance Study Number 2304/001 Final Report Adsorption and desorption isotherms for 8-2 TBA on sterile soil SK179618 at 4C Log Concentration in Soil (pg/g) - 70 - Figure 19 Covance Study Number 2304/001 Final Report Adsorption and desorption isotherms for 8-2 TBA on sterile Eurosoil 5 soil at 4C Log Concentration in Soil (pg/g) - 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 !> PerkinElmer life sciences. PerkinElmerLifeSciences, Inc. y 549AlbanyStreet Boston, MA 02118 C USTO M S ER VIC ES Caution: For Laboratory Use. Aresearch chemical for research purposes only. CERTIFICATE OF ANALYSIS Name: Lot Number: Molecular Formula: Formula Weight: Date of Synthesis: Date of Purification: Last Solvent: Physical Appearance: Structure: 1H, 1H, 2H, 2H-Perfluoro-1-decanol, [1,2-14C]- DuPont Sample #H-25628 3489-169 C10H5F17O 464 September 17,2002 (Lot number 3489-165) January 15,2003 Dichloromethane Ethanolic solution CaF1714CH214CH2OH ANALYTICAL METHOD Specific Activity (mass spectrometry on Lot No. 3489-165) Radiochemical purity (HPLC; January 29,2003) RESULT 33 mCi/mmol (0.071 mCi/mg) >99%; Column: Vydac Protein C4; Mobile Phase: 0.1% TFA in Water/ Acetonitrile (50/50); Flow rate: 1 mL/min. Detector: Radioactivity - 73 - Certificate of analysis for non-radiolabelled TBA Covance Study Number 2304/001 Final Report Clariam GmbH, Werk Gendorf CQG / Qualiismanagemem D-84504 Burgkirchen Fax: + + 49 8679 / 2676 Clari ant Inspection certificate according to EN10204-3.1B Date: 13.11.2001 Page: 1 / 1 Our consignment Material Material-no. Batch No. : 1-Decano1, 3,3,4,4/5,5,6,6,7,7,8,8,9,9,10,10, 10-heptadecafluoro : 104705 : P00/001 On the baton, of whicr. the consignment is a part, the following values were determined. They conform to the agreed product specification. Inspection characteri c l u j /-methoriSpecification Result C3 -Per fxuor alkylethar.c 1 Capillary GC Sum Perfluoroaikylethanol Capillary GC Sum Perfiuoralkylbutanol Capillary GC ' Sum Perfluoralkylethene Capillary GC Water content Karl Fischer DIN 51777 N-Methylpyrrolidone content Capillary GC >= 97,5 <-l, 0 <=0, 5 <=0,2 <=0, 2 o cn A 99,3%[a/a] 99,9%[a/a] < 0,l%[a/a] < 0,1%[a/a] < 0, 1% < 0, 1% The above particulars do not release the customer from the obligation to carry out an inspection of goods received. ' Ms. Baumgartner - Worksinspector - 74 - Soil SK961089 Covance Study Number 2304/001 Final Report Appendix 2 Soil certificates of analysis TEST SYSTEM CERTIFICATE OF ANALYSIS Test system code Test system name Collected by Sampling location Date of field sampling Pesticide history Land use Depth o f sampling Method of soil collection Date o f arrival at CLE Handling C S 17/00-628 SK961089 Land Research Associates, Lockington, Derby, DE74 2RH Chapel Hill Farm, Empingham, Rutland. 02-August-2000 No pesticide use in coppice Long-established deciduous coppice, used for pheasant-rearing Topsoil from 15 to 30 cm In accordance with ISO10381-6 04-August-2000 The soil was given a unique code, thoroughly mixed and passed through a 2mm mesh sieve. Prior to use the soil was stored air-dry, in the dark, at room temperature, in loosely tied plastic bags. UK & BBA Particle Size Distribution Sand % (2000 - 63 pm) Silt % (63 - 2 pm) Clay % (< 2 pm) USDA Particle Size Distribution Sand % (2 0 0 0 -5 0 pm) Silt % ( 5 0 - 2 pm) Clay % (< 2 pm) UK Textural Class BBA Textural Class USDA Textural Class 37 29 34 38 28 34 Clay loam sandiger toniger Lehm Clay loam pH in H20 (Commenced: 16-8-01) pH inlM K Cl (Commenced: 16-8-01) pH in 0.01M CaCl2 (Commenced: 16-8-01) Cation Exchange Capacity mEq/lOOg mmol/Kg (Commenced: 22-8-01) 8.0 7.6 7.6 38.2 382 Water Holding Capacity at pF 0 (0.001 bar) % 65.4 Nitrogen Content % * (Commenced: 28-3-01) 0.59 Water Holding Capacity at pF 2.5 (0.33 bar) % 29.4 Carbon/Nitrogen Ratio (Commenced: 28-3-01) 1:0.13 Organic Carbon % (Commenced: 15-8-01) Organic Matter % (Commenced: 15-8-01) Exchangeable acidity * mEq/lOOg (Commenced: 28-03-01) 4.6 7.9 NA Exchangeable Cations * mEq/lOOg Exchangeable Ca Exchangeable Mg Exchangeable Na Exchangeable K Exchangeable Mn (Commenced: 28-03-01) CaC03eq. mg/Kg * (Commenced: 28-03-01) NA NA NA NA NA 187.6 The analytical procedures* undertaken at Covance Laboratories Ltd., Harrogate were performed in accord with the current national UK GLP Regulations and the current OECD Principles of GLP. All data is retained in the GLP archive o f Covance Laboratories Ltd., Harrogate. * Analysis performed by National Soil Resource Institute, Silsoe, Bedford (formally known as the Soil Survey and Land Research Centre) (Laboratory Analysis Plan No. SSHQ/034/01 & SSHQ/035/01). No claim is being made for the compliance status o f this work. Analysed by: - _^ Approved by: JLc o b Quality Assurance: If M C A .L ) 2 0 C 6 - 75 - Soil SK566696 Covance Study Number 2304/001 Final Report TEST SYSTEM CERTIFICATE OF ANALYSIS Test system code Test system name Collected by Sampling location Date o f field sampling Pesticide history Land use Depth o f sampling Method o f soil collection Date o f arrival at CLE Handling CS01/03 SK566696 Land Research Associates, Lockington, Derby, DE74 2RH Grid Reference: SK566696 Warsop, Nottinghamshire 14-January-2003 No pesticide use in coppice Grassy clearing at edge of established wood o f silver birch, oak etc Topsoil from 12 to 20 cm In accordance with ISO 10381-6 16-January-20Q3 The soil was given a unique code, thoroughly mixed and passed through a 2mm mesh sieve. Prior to use the soil was stored air-dry, in die dark, at room temperature, in loosely tied plastic bags. UK & BBA Particle Size Distribution Sand % (2000 - 63 pm) Silt % (63 - 2 pm) Clay % (< 2 pm) USDA Particle Size Distribution Sand % (2 0 0 0 -5 0 pm) Silt % ( 5 0 - 2 pm) Clay % (< 2 pm) UK Textural Class BBA Textural Class USDA Textural Class 84 5 11 85 4 11 Loamy sand schwach toniger Sand Loamy sand pH in H20 (Commenced: 18-1-03) pH inlM K Cl (Commenced: 18-1-03) pH in 0.01M CaCl2 (Commenced: 18-1-03) Cation Exchange Capacity mEq/lOOg mmol/Kg (Commenced: 06-2-03) 5.1 4.2 4.2 13.4 134 Water Holding Capacity at pF 0 (0.001 bar) % 29.0 Nitrogen Content % * (Commenced: 07-02-03) 0.8 Water Holding Capacity at pF 2.5 (0.33 bar) % 7.1 Carbon/Nitrogen Ratio (Commenced: 07-02-03) 1: 1 Organic Carbon % (Commenced: 17-1-03) 0.8 Organic Matter % (Commenced: 17-1-03) 1.4 The analytical procedures'" undertaken at Covance Laboratories Ltd., Harrogate were performed in accord with the current national UK GLP Regulations and the current OECD Principles of GLP. All data is retained in the GLP archive o f Covance Laboratories Ltd., Harrogate. * Analysis performed by National Soil Resource Institute, Silsoe, Bedford (formally known as the Soil Survey and Land Research Centre) (Laboratory Analysis Plan No. SSHQ/011/03). No claim is being made for the compliance status o f this work. Analysed by: Approved by: (v^scp-j Quality Assurance: ' lju/iAilcoa il mxL) zoos - 76 - Soil SK 179618 Covance Study Number 2304/001 Final Report TEST SYSTEM CERTIFICATE OF ANALYSIS Test system code Test system name Collected by Sampling location Date o f field sampling Pesticide history Land use Depth o f sampling Method o f soil collection Date o f arrival at CLE Handling CS37/00-628 SK179618 Land Research Associates, Lockington, Derby, DE74 2RH Kenslow Farm, Middleton, Derbyshire 09-November-2000 No pesticide use Permanent grass field Topsoil from 5 to 20 cm In accordance with ISO10381-6 01-December-2000 The soil was given a unique code, thoroughly mixed and passed through a 2mm mesh sieve. Prior to use the soil was stored air-dry, in the dark, at room temperature, in loosely tied plastic bags. UK & BBA Particle Size Distribution Sand % (2000 - 63 pm) Silt % ( 6 3 - 2 pm) Clay % (< 2 pm) USDA Particle Size Distribution Sand % (2 0 0 0 -5 0 pm) Silt % ( 5 0 - 2 pm) Clay % (< 2 pm) UK Textural Class BBA Textural Class USDA Textural Class 33 47 20 34 46 20 Clay loam schwach sandiger Lehm Loam pH in H20 (Commenced: 16-8-01) pH inlM K Cl (Commenced: 16-8-01) pH in 0.01M CaCl2 (Commenced: 16-8-01) Cation Exchange Capacity mEq/lOOg mmol/Kg (Commenced: 22-8-01) 6.0 5.8 5.6 24.9 249 Water Holding Capacity at pF 0 (0.001 bar) % 75.9 Nitrogen Content % * (Commenced: 28-03-01) 0.33 Water Holding Capacity at pF 2.5 (0.33 bar) % 27.3 Carbon/Nitrogen Ratio (Commenced: 28-03-01) 1:0.09 Organic Carbon % (Commenced: 15-8-01) 3.8 Organic Matter % (Commenced: 15-8-01) 6.6 The analytical procedures'" undertaken at Covance Laboratories Ltd., Harrogate were performed in accord with the current national UK GLP Regulations and the current OECD Principles of GLP. All data is retained in the GLP archive o f Covance Laboratories Ltd., Harrogate. * Analysis performed by National Soil Resource Institute, Silsoe, Bedford (formally known as the Soil Survey and Land Research Centre) (Laboratory Analysis Plan No. SSHQ/034/01). No claim is being made for the compliance status o f this work. Analysed by: .^ A/\pppprruovceud boy: Quality Assurance: L^Oym&CUJJJ 27 W[ajLj 2CA3 - 77 - Emperor Son's Lake Covance Study Number 2304/001 Final Report Page 1 o f 2 TEST SYSTEM CERTIFICATE OF ANALYSIS Test system code Test system name Collected by Sampling location Date o f field sampling Pesticide history Method o f soil collection CS04/03 Site Cl (Emperor's Son Lake) Land Research Associates, Lockington Hall, Lockington, Derby SK 268699 Emperor's Son Lake, Chatsworth, Derbyshire 10-February-2003 No pesticides used in the catchment Water: Collected into container. Sediment: Collected by hand into container. Water Temperature (C) just below the water surface Oxygen Content (%) just below the water surface Water pH Depth o f water (cm) above sediment Depth o f sediment sampled (cm) 4.2 99.0 6.34 15-20cm 0-10cm Water Temperature (C) 5cm above the sediment Oxygen Content (%) 5cm above the sediment Redox potential (mV) of water Redox potential (mV) o f sediment Storage prior to despatch Date o f arrival at CLE Handling ., Stored at 3 - 5C 12-February-2003 The water-sediment system was given a unique code. Prior to 4.3 98.8 416.0 225 Sediment Parameters: UK & BBA Particle Size Distribution Sand % (2000 - 63 pm) Silt % (63 - 2 pm) Clay % (< 2 pm) USDA Particle Size Distribution Sand % (2000 - 50 pm) Silt % (50 - 2 pm) Clay % (< 2 pm) USDA Textural Class UK Textural Class BBA Textural Class Organic Carbon % (Commenced: 19.02.03) incubator routinely maintained at 4 2C. 74 9 17 75 8 17 Sandy loam Sandy loam mittel toniger Lehm or schwach toniger Sand 1.2 pH in 1MKC1 (Commenced: 24.02.03) pH in 0.01M CaCl2 (Commenced: 24.02.03) Cation Exchange Capacity mEq/lOOg mmol/Kg (Commenced: 25.03.03) Microbial Biomass pg C/g (Commenced: 27.03.03) Total Nitrogen mg/kg * % (Commenced: 04.04.03) 6.1 6.0 13.6 136 192.99 882.0 0.09 Organic Matter % (Commenced: 19.02.03) Water Holding Capacity at pF 0 (0.001 bar) % Water Holding Capacity at pF 2.5 (0.33 bar) % 2.1 42.6 8.1 Total Phosophorus mg/kg * (Commenced: 04.04.03) Carbon/Nitrogen Ratio (Commenced: 19.02.03) pH in H20 (Commenced: 24.02.03) 382.9 1: 0.08 6.8 78 - Covance Study Number 2304/001 Final Report Test system code Test system name TEST SYSTEM CERTIFICATE OF ANALYSIS CS04/03 Site Cl (Emperor's Son Lake) Page 2 of 2 Associated Water Parameters: Total Nitrogen mg/L * (Commenced: 04.04.03) 7.0 Total Phosophorus mg/L * (Commenced: 04.04.03) 0.6 Dissolved Organic Carbon (DOC) mg/L * (Commenced: 04.04.03) 35.9 Water Hardness mg/L as CaC03 * (Commenced: 04.04.03) 41.0 Suspended Solids mg/L * (Commenced: 04.04.03) 210.0 The analytical procedures* undertaken at Covance Laboratories Ltd., Harrogate were performed in accord with the current national UK GLP Regulations and the current OECD Principles of GLP. All data is retained in the GLP archive of Covance Laboratories Ltd., Harrogate. * Analysis performed by National Soil Resource Institute, Silsoe, Bedford (formally known as the Soil Survey and Land Research Centre) (Laboratory Analysis Plan No. SSHQ 025/03). No claim is being made for the compliance status o f this work. NSRI is a member of the UK GLP compliance programme and the work supplied to Covance complies with the UK GLP regulations. Analysed by: Approved by: Quality Assurance: - 79 - Eurosoil 5 Covance Study Number 2304/001 Final Report Test system code Test system name Collected by Date o f arrival at CLE Handling TEST SYSTEM CERTIFICATE OF ANALYSIS CS55/03 and CS57/03 Eurosoil 5 (IRMM-443-5) IRRM (Institute for Reference Materials and Measurements) 22/10/03 and 30/10/03 The soil was given a unique code on arrival. The soil was supplied as an air dried fine soil <2mm in batches o f 200g. Each batch supplied by IRMM is from the same bulk sample collected in 1991. The two batches received have been treated as being the same despite having different Covance batch numbers. UK & BBA Particle Size Distribution Sand % (2000 - 63 pm) Silt % (63 - 2 pm) Clay % (< 2 pm) USDA Particle Size Distribution Sand % (2000 - 50 pm) Silt % ( 5 0 - 2 pm) Clay % (< 2 pm) UK Textural Class BBA Textural Class USDA Textural Class Water Holding Capacity at pF 0 (0.001 bar) % Water Holding Capacity at pF 2.5 (0.33 bar) % Organic Carbon % (Commenced: 16/12/03) Organic Matter % (Commenced: 16/12/03) 86 9 5 87 8 5 Loamy sand schwach toniger Sand or reiner Sand Loamy sand 46.9 10.3 6.7 11.6 pH in H20 + (Commenced: Dec 2001) pH in 0.01M CaCl2+ (Commenced: Dec 2001) Cation Exchange Capacity mEq/lOOg (Commenced: 18/12/03) Nitrogen Content % * (Commenced: 13-01-04) Carbon/Nitrogen Ratio (Commenced: 16/12/03) 4.1 3.1 26.8 0.2 1 : 0.03 The analytical procedures*+undertaken at Covance Laboratories Ltd., Harrogate were performed in accord with the current national UK GLP Regulations and the current OECD Principles o f GLP. All data is retained in the GLP archive o f Covance Laboratories Ltd., Harrogate. * Analysis performed by National Soil Resource Institute, Silsoe, Bedford (Laboratory Analysis Plan No. NSR1/001/04). No claim is being made for the compliance status of this work. NSRI is a member of the UK GLP compliance programme and the work supplied to Covance complies with the UK GLP regulations. Analysis supplied by IRMM. No claim is being made for the compliance status o f this work. VV02.-04- Quality Assurance ^ l/l&LUUJj U-l-lOGLf - 80 - Appendix 3 Covance Study Number 2304/001 Final Report Radiochemical purity of (14C)-8-2 TBA and formulation stability Radiochemical Purity by HPLC (stock solution 1) 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min Method: METHOD1 Filename : 4CHR001.R01 Evaluation: A User : S.Swales Channel: C- 14 Detector: Name Start - End RT Height Area %Total (m) (m) (cps) (Counts) (%) Bkg 1 0.7- 2.3 1.5 0.3 TBA 16.6- 17.4 17.0 854.8 10931.4 98.03 17.5- 17.8 17.6 7.8 55.6 0.50 co co i--i CM CO i--1 18.4 10.8 155.4 1.39 Bkg 2 33.7- 34.6 34.1 0.1 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) 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0min Method: METHOD1 Filename : CHR014.R01 Channel: C- 14 Detector: Name Start - End RT (m) (m) Bkg 1 1.6 - 2.3 1.9 8-2 TBA 17.4 - 18.5 17.7 18.5 - 18.9 18.5 18.9 - 19.8 19.5 Bkg 2 33.4 - 33.9 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 Covance Study Number 2304/001 Final Report No Name Type Pos [mm] RF PSL PSL-Bkg %(PSL-Bkg) 1 16.00 -- 174.46 167.34 1.07 2 8-2 TBA 87.80 0.48 15044.37 15014.08 96.14 3 107.80 0.61 94.37 84.95 0.54 4 123.40 0.71 227.07 210.27 1.35 5 Bkg 6.00 -- 8.45 0.00 0.00 6 Bkg 169.60 -- 6.57 0.00 0.00 - Sum -- -- 15540.27 15476.64 99.10 - Rem -- -- 300.72 140.87 0.90 - Ttl -- -- 15840.99 15617.51 100.00 Date of analysis: 7 April 2003 - 83 Radiochemical Purity by TLC (stock solution 1) Plate 23, track 3 Covance Study Number 2304/001 Final Report C O U_ 200 w CL 100 o 0 0 50 100 [m m ] 150 No Name Type Pos [mm] RF PSL PSL-Bkg %(PSL-Bkg) 1 Origin 20.20 0.02 104.36 97.58 1.47 2 8-2 TBA 93.40 0.51 6424.60 6399.84 96.33 3 112.20 0.63 46.38 36.44 0.55 4 127.60 0.73 102.61 88.89 1.34 5 Bkg 5.80 -- 5.25 0.00 0.00 6 Bkg 169.20 -- 4.05 0.00 0.00 - Sum -- -- 6677.96 6622.76 99.69 - Rem -- -- 104.00 20.73 0.31 - Ttl -- -- 6781.95 6643.49 100.00 Date of analysis: 19 December 2003 Prior to formulation of dose formulations for the isotherms test. - 84 - Appendix 4 Covance Study Number 2304/001 Final Report 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) TBA 18.7 1HPLC Chromatogram used is 03CHR002.R01(UV) 2 TLC Plate used is Plate 7 (non-radiolabelled values quoted) Rf 0.49 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: - 85 - Appendix 5 Covance Study Number 2304/001 Final Report 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: 2.63 V V Volume of LSC aliquots (mL): 3 Number of flasks: 0.25 6 Flask number (identity): Volume of formulation aliquot (mL): Diluted to volume (mL): Spac 1 0.05 5 Spac 2 0.05 5 Spac 3 0.05 5 Spac 4 0.05 5 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): 0.0086 Radioactivity applied (pCi): 0.079715 Radioactivity applied (kBq): 2.949446 Weight of test article applied (mg): 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: 2304/1 Sample description: Adsorb supernatant DG B Number of LSC replicates: 2 Flag variability at x% of mean: 10% LOD (times background): 1.5 LOD based on mean counts Number of samples (10 max) 10 LSC DATA INPUT (dpm) Subject code: B1 B2 B3 B4 B5 SDATA file input 100 00 0 200000 Background 00 00 0 Manual input 1 397 393 336 279 214 2 384 403 356 313 205 Background WFIGHT 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): RESUI TS 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 (pg/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 (pg/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 Vo 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 Ci 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 .i7 6 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 Xi 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 N um ber 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 Batch 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 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