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Organotin compounds: their analyses and effect on model biomembranes Nwata, Basil Ugwunna 1994

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ORGANOTIN COMPOUNDS:- THEIR ANALYSES AND EFFECT ON MODEL BIOMEMBRANES BY BASIL UGWUNNA NWATA B.Sc (Hons), University of Ilorin, Nigeria, 1981 M.Sc, University of Ibadan, Nigeria 1984 M.Sc, University of British Columbia, Canada 1989  A THESIS SUBMITrID IN PARTIAL FULFILLMENT OF THE REQUIREMENTS  FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY  We accept this thesis as conforming to the required standard  COLUMBIA JUNE 1994 ©  Basil Ugwunna Nwata, 1994  In presenting this thesis in partial fulfilment of the requirements for degree at the University of British Columbia, I agree that the Library freely available for reference and study. I further agree that permission copying of this thesis for scholarly purposes may be granted by the department  or  by  his  or  her  representatives.  It  is  understood  an advanced shall make it for extensive  head of my that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  C/17’1’ i7’  The University of British Columbia Vancouver, Canada  Date ditW4 i’?  DE-6 (2)88)  11  ABSTRACT Studies involving the analyses of organotin compounds in marine organisms of British Columbia, and the effect of organotin compounds on the permeability of model biological membranes are presented in this thesis. Analysis of organotin compounds by  gas chromatography-selected  ion  monitoring mass spectrometry (GC-MS SIM) affords a very specific technique for the identification and quantitation of organotin compounds, by using the peculiar isotope pattern for tin compounds. This methodology is therefore able to distinguish organotin compounds from other compounds that may co-elute with them from the gas chromatograph. Although some British Columbian locations such as Hastings Arm, Alice Arm, etc showed no organotin contamination, the major organotin pollutants found for some coastal areas such as Denman Island, Dundas Island, etc were tributyltin and dibutyltin species. The butyltin body content for Blue mussels in the contaminated areas range from 14.4 to 37.3 ng/g (wet wt as Sn) for tributyltin and 6.7 to 67.3 ng/g (wet wt as Sn) for dibutyltin species. Dicyclohexyltin levels of 3.5 ng/g and 21.3 ng/g (wet wt as Sn) were found only at Wreck Beach Vancouver, and Anyox respectively. The effect of organotin compounds on egg phosphatidyicholine  (EPC)  liposomes or organotin-EPC liposomes, were established by studing the efflux of a probe compound; dimethylarsinic acid (DMA) trapped inside these liposomes, by using 1 H NMR spectroscopy. The probe compound at pH 7.4 exists as two chemical species; DMAH and DMK which are capable of diffusing from these liposomes.  111  When the organotin compounds were added externally to the EPC liposomes, tributyltin chloride caused an increased permeability of the liposomes, which was linearly dependent on the concentration of the externally added tributyltin chloride solution. Monobutyltin trichioride decreased the permeability coefficient of DMAH to the EPC liposomes from 1.7 x 10-8 to 4 x i0 cm/s, while trimethyltin cation facilitated the efflux of DMK from the liposomes. For TBT-EPC liposomes formed by a mixture of tributyltin chloride and EPC, the efflux of DMA from these liposomes was facilitated by the tributyltin cation only if the liposomes were not in contact with externally added tributyltin chloride solution. When in contact with externally added tributyltin chloride, the ability of the tributyltin cation to act as carrier for DMK was lost. The activation energy for the passive efflux of DMAH from TBT-EPC liposomes varied from 52.3 to 64.4 kJ/mol depending on the tributyltin content of the liposome. For  the  monobutyltin  trichloride-EPC  liposomes  (MBT-EPC),  the  monobutyltin cation did not exhibit any ability to act as carrier for DMA irrespective of whether it was externally added to the liposomes or not. The DMAH species permeate by passive diffusion with activation energy of 106.8 to 121.5 kJ/mol. A modified  batch hydride generation-graphite  furnace atomic absorption  spectrophotometric method (HG-GFAAS) is described for total tin determination. In this method, tin hydride was adsorbed and pre-concentrated on graphite furnace tubes pre-coated with palladium or sodium tungstate matrix modifiers, prior to their atomization in the graphite furnace.  iv TABLE OF CONTENTS  Abstract Table of contents  iv  List of Tables  xiv  List of Figures  xix  List of Abbreviations  xxiii  Acknowledgements  xxvi  Dedication  xxvii  Chapter 1 General Introduction  1  1.1  Historical background of the organotin compounds  1  1.2  Industrial applications and use of organotin compounds  1  1.3  Need for a chemical antifouling agent  3  -  1.3.1 Contact leaching antifouling paints  5  1.3.2 Ablative formulation  5  1.3.3 Self polishing co-polymer paints  6  1.4  Toxicity of organotin compounds  6  1.5  Metabolism and behaviour of tributyltin compounds in the environment  1.6  Analytical methods for organotin compounds  9 11  1.6.1 Molecular spectrophotometry and spectrofluorimetric techniques  12  1.6.2 Electrochemical techniques  13  V  1.6.3 Atomic spectrometry  14  1.6.4 Gas chromatography  17  1.6.4.1  Hydride generation gas-chromatography (HG-GC)  17  1.6.4.2  Conversion to tetraalkyltin compounds  19  1.6.4.2(a)  Gas chromatography with flame photometric detection (GC-FPD)  1.6.4.2(b)  Gas chromatography with atomic absorption spectrometric detection (GC-AAS)  1.6.4.2(c)  20  21  Gas chromatography with mass spectrometric detection (GC-MS)  1.6.5 Liquid chromatography (LC)  21 21  1.6.6 Thin layer chromatography (TLC) and high performance thin layer chromatography  24  1.7  Tributyltin and government regulations  25  1.8  Objectives and scope of the present study  26  Chapter 2  Speciation and quantitation of butyltin and cyclohexyltin compounds in marine organisms by using capillary column GC-MS SIM  29  2.1  Introduction  29  2.2  Experimental  30  2.2.1 Instrumentation 2.2.1.1  Gas chromatography (GC)  30 30  vi 2.2.1.2  NMR and mass spectrometry  30  2.2.1.3  Gas chromatography-mass spectrometry (GC-MS)  31  2.2.1.4  Mechanical shaker and blender  31  2.2.2 Materials and reagents  32  2.2.3 Synthesis of standard organotin compounds  33  2.2.3.1  Synthesis of tributylmethyltin  33  2.2.3.2  Synthesis of dibutyldimethyltin  33  2.2.3.3  Synthesis of tricyclohexylmethyltin  34  2.2.3.4  Synthesis of dicyclohexyldimethyltin  34  2.2.3.5  Synthesis of the internal standards  34  2.2.3.5(a)  Synthesis of tetrapropyltin  34  2.2.3.5(b)  Direct synthesis of deuterated internal standards  35  2.2.3.6  Synthesis of methylmagnesium iodide  36  Analytical procedure  36  2.3.1 Gas chromatography  36  2.3  2.3.1.1  Establishment of elution profile and retention data  36  2.3.1.2  Suitability of tetrapropyltin as internal standard  37  2.3.1.3  n(CH as 3 S 9 H 4 (C 2 SnCH and ) 3 ) 9 H 2 4 Suitability of (C internal standards  37  2.3.2 Low resolution mass spectrometry  38  2.3.3 GC-MS retention data, calibration curves and precision  38  2.3.4 Recovery studies  39  vi’ 2.3.5 Extraction of the organotin compounds from marine animals  41  2.4  43  Results and discussion  2.4.1 Characterization of the standard tetraorganotin compounds  43  2.4.2 Fragment ions and intensities of the standard tetraorganotin compounds  48  2.4.3 GC-MS elution profile and masses of selected fragment ions used for selected ion monitoring  51  2.4.4 Suitability of tetrapropyltin as internal standard as studied by gas chromatography  53  2.4.5 Detection limit, calibration curves, and precision for the GC-MS SIM analysis 2.4.5.1  57  Detection limit and calibration curves obtained  2.4.5.2  by GC-MS SIM  57  Precision of the GC-MS SIM method  62  2.4.6 Recovery studies on the extraction procedure  63  2.4.7 Organotin concentrations in some marine organisms of British Columbia, Canada 2.4.7.1  Organotin concentrations in oysters  2.4.8 Spread of organotin compounds in the Canadian environment  67 67 71  2.4.9 Organotin concentrations in various organisms from the same locations  79  vii’ 2.4.10 Distribution of organotin compounds in marine animals studied over a period of three years  Chapter 3  81  Effect of tributyltin chloride, monobutyltin trichioride and trimethyltin hydroxide on the permeability of egg phosphatidyicholine liposomes  3.2  83  Dimethylarsinic acid (DMA) as a probe for studying the effect of organotin compounds on the membranes of liposomes  84  3.3  Liposomes as models for biological membranes  85  3.4  Types of liposomes and methods of preparation  87  3.4.1 Multilamellar vesicles (MLVs)  88  3.4.2 Small unilamellar vesicles (SUVs)  88  3.4.3 Large unilamellar vesicles (LUV5)  89  Transport processes in membranes  90  3.5  3.5.1 Simple or passive diffusion  90  3.5.2 Facilitated diffusion  91  3.5.2.1  Solute translocation through channels  92  3.5.2.2  Translocation through carriers  92  3.5.3 Active transport 3.6  Solute transport across liposomal membranes  93 94  3.6.1 Transport of non-ionic solutes  94  3.6.2 Transport of ions  97  ix 3.7  Properties of liposomes capable of yielding investigative information  99  3.8  Butyltin compounds: the need for the present study  99  3.9  Theoretical description of the diffusion experiment applicable to NMR spectrometry  101  3.9.1 Passive diffusion  101  3.9.2 Facilitated diffusion  101  3.10  111  Experimental  3.10.1 Instrumentation  111  3.10.1.1  Nuclear magnetic resonance spectrometry (NMR)  111  3.10.1.2  Lipid extruder and membrane filters  111  3.10.1.3  UV-Visible spectrophotometry  111  3.10.2 Chemicals and reagents  112  3.10.3 Preparation of large unilamellar vesicles (LUVs) from egg phosphatidyicholine (EPC) and the encapsulation of dimethylarsinic acid  112  3.10.4 Preparation of butyltin-EPC LUVs and the encapsulation of DMA  114  3.10.5 The NMR water suppression and spectral acquisition conditions for DMA efflux from EPC and butyltin-EPC liposomes  116  x 3.10.6 Determination of phospholipid concentrations by phosphorus assay 3.10.6.1  3.10.6.2  117  Extraction of phospholipid from liposomes prior to phosphorus determination  117  Lipid concentration determination  117  3.10.7 Processing of the NMR spectra  118  3.10.8 Analysis and treatment of data  120  3.10.8.1  Determination of rate constants and mode of permeation 120  3.10.8.2  Determination of permeability coefficients  3.11  Results and Discussion  124 125  3.11.1 The use of DMA as a probe in permeability studies of EPC liposomes in the presence and absence of organotin compounds in the extraliposomal aqueous compartment  125  3.11.2 Effect of organotin concentration on the efflux of DMA  135  3.11.3 Efflux of DMA from tributyltin chloride-EPC liposomes (with tributyltin chloride absent in the extraliposomal compartment) 3.11.4 Efflux of DMA from monobutyltin trichloride-EPC liposomes  139 145  3.11.5 Effect of the butyltin chloride concentrations of the liposome on permeability properties of TBT-EPC and MBT-EPC liposomes  149  xi 3.11.6 Effect of temperature on the permeability of organotin-EPC liposomes  151  3.11.7 Activation energies for the permeation of butyltin chloride-EPC liposomes  153  3.11.8 Relevance of this NMR study to the enviromnental toxicity of. butyltin compounds Chapter 4  158  Hydride generation methods of atomic absorption spectrophotometry for total tin determination  160  4.1  Introduction  160  4.2  Experimental  162  4.2.1 Instrumentation 4.2.1.1  Continuous hydride generation atomic absorption spectrophotometry (HG-AAS)  4.2.1.2  162  162  Batch hydride generation-graphite furnace atomic absorption spectrophotometry (HG-GFAAS)  164  4.2.2 Materials and Reagents  165  4.2.3 Methodology for HG-AAS  166  4.2.3.1  Continuous hydride generation method (HG-AAS)  4.2.3.2  Batch hydride generation-graphite  furnace method (HG-GFAAS) 4.2.4 Preparation of matrix modifiers and standard tin solutions 4.2.4.1  Preparation of palladium modifier  166  166 168 168  XII  4.2.4.2  Preparation of sodium tungstate modifier  165  4.2.4.3  Preparation of standard tin solutions  169  4.2.5 Optimum concentration of reagents used in HG-AAS  169  4.2.6 Use of L-cysteine to remove interferences  170  Optimum concentration of L-cysteine required to  4.2.6.1  remove interferences 4;2.7 Optimum conditions for the batch HG-GFAAS 4.2.7.1  Optimization of reagent concentrations for HG-GFAAS  4.2.7.2  Optimization of the trapping temperatures and trapping time for tin hydride in the graphite furnace tube  171 171 172  172  4.2.8 Treated graphite furnace tubes:- coating the graphite  furnace tubes with solutions of sodium tungstate and palladium modifiers  172  4.2.8.1  Optimum modifier treatment of graphite furnace tubes  173  4.2.8.2  Calibration curves for the HG-GFAAS method  173  4.3  Sample digestion and preparation  174  4.4  Results and Discussion  175  4.4.1 Optimum concentrations of sodium borohydride and HC1  necessary for the production of stannane in the continuous hydride generator  175  4.4.2 Optimum concentration of L—cysteine required to eliminate inteferences in HG-AAS  177  XIII  4.4.3 HG-AAS determination of total tin in oysters and standard reference material (Tort 1)  179  4.4.4 Batch hydride generation-graphite furnace atomic absorption spectrophotometry (HG-GFAAS) 4.4.4.1  Optimum concentrations of reagents needed for tin  hydride production in the HG-GFAAS method 4.4.4.2  184  Optimum flow rate of sodium borohydride into the batch hydride generator  4.4.4.3  182  185  Optimum temperature for trapping tin hydride in the pre-treated graphite furnace tubes  186  4.4.4.4  Optimum trapping time  187  4.4.4.5  Pre-treatment of graphite furnace tubes with modifiers  188  4.4.4.6  Determination of total tin content of a standard reference material by the HG-GFAAS method  Chapter 5  Summary and conclusions  192 195  References  202  Appendix A Map of locations sampled for organotin pollution  220  Appendix B The NMR spectral acquisition and water suppression parameters for the efflux of dimethylarsinic acid from liposomes Appendix C Michael is-Mentons equations for enzyme kinetics  221 223  Appendix D Wet ashing apparatus with air cooled reflux condenser used for digestion of marine animals  224  xiv LIST OF TABLES 2.1  PAGE  Sn NMR chemical shifts for the standard 119  organotin compounds  44  2.2  Major fragment ions of tributylmethyltin  48  2.3  Major fragment ions for dibutyldimethyltin and tricyclohexylmethyltin  2.4  Major fragment ions for dicyclohexyldimethyltin and tetrapropyltin  2.5  51  Fragment ions and masses used to detect and quantitate each organotin compound in GC-MS SIM  2.7  50  Retention time and retention time window used for GC-MS SIM analysis  2.6  49  53  Regression data for graphs obtained with various concentrations of the internal standard tetrapropyltin  54  2.8  Detection limits for organotin compounds by GC-MS SIM  60  2.9  Calibration equations used for the quantitation of environmental samples by GC-MS SIM  61  2.10  Precision of the GC-MS SIM  63  2.11  Recovery of organotin compounds spiked into Shrimp  64  2.12  Organotin concentrations in the oyster Crassostrea gigas from some coastal areas of British Columbia  2.13  Organotin concentration, spread and speciation in some  68  xv locations of British Columbia 2.14  Some organotin concentrations reported for the Blue mussels Mytilus edulis  2.15  72  77  Organotin concentrations in the Blue mussel Mytilus edulis converted to tg/g wet wt as organotin cation  2.16  Organotin distribution in marine animals from Camano Sound British Columbia  2.17  79  Organotin distribution in marine animals from Tasu Sound British Columbia  2.18  78  80  Organotin body burden for soft shell clams from Quatsino Sound, British Columbia studied over a period of three years  3.1  Efflux data for the diffusion of DMAH from EPC liposomes in the absence of organotin compounds  3.2  131  Data for the efflux of DMA from EPC liposomes in the presence of 33.2 M trimethyltin hydroxide  3.5  127  Effect of 33.2 iM monobutyltin trichloride on the efflux of DMAH from EPC liposomes  3.4  126  Effect of 33.2 .LM tributyltin chloride on the efflux of DMAH from EPC liposomes  3.3  81  Effect of tributyltin chloride concentration on  135  xvi DMAH efflux 3.6  Effect of monobutyltin trichloride concentration on the efflux of DMAH  3.7  136  138  Diffusion parameters for the efflux of DMA from tributyltin chloride-EPC liposomes by a mixture of passive and facilitated diffusion  3.9  141  Parameters for the efflux of DMAH from TBT-EPC B liposomes in the presence of externally added tributyltin chloride (16 iiM)  3.9  145  Permeability data for efflux of DMAH from MBT-EPC B liposomes (with monobutyltin trichioride absent in the extraliposomal compartment)  3.10  148  Permeability data for efflux of DMAH from MBT-EPC B liposomes (with monobutyltin trichioride present in the extraliposomal compartment)  3.11  148  Effect of tributyltin chloride concentration on the permeability of tributyltin chloride-EPC liposomes with tributyltin chloride also present in the extraliposomal volume  3.12  Effect of monobutyltin trichloride concentration on the permeability of monobutyltin trichloride-EPC liposomes with monobutyltin trichioride also present in the extraliposomal  150  xvii volume 3.13  Effect of temperature on the permeability properties of TBT-EPC A liposomes  3.14  152  Effect of temperature on the permeability properties of MBT-EPC B liposomes  3.17  151  Effect of temperature on the permeability properties of MBT-EPC A liposomes  3.16  151  Effect of temperature on the permeability properties of TBT-EPC B liposomes  3.15  150  152  Effect of tributyltin chloride content of liposome on the activation energy for efflux of DMAH from TBT-EPC liposomes  3.18  155  Effect of monobutyltin trichioride content of liposomes on the activation energy for efflux of DMAH from MBT-EPC liposomes  3.19  Arrhneius pre-exponential factor for DMAH efflux from TBT-EPC liposomes  3.20  157  Graphite furnace atomization program for tin determination by HG-GFAAS  4.2  157  Arrhneius pre-exponential factor for DMAH efflux from MBT-EPC liposomes  4.1  155  Operating conditions for the continuous hydride generation  167  xviii -atomic absorption spectrophotometry (HG-AAS) 4.3  170  Total tin content of samples analyzed by the HG-AAS method  181  4.4  Reagent ratios needed to maximize tin hydride generation  184  4.5  Comparison of palladium and sodium tungstate-treated graphite furnace tubes showing the atomic absorbance of tin hydride generated from 14 g/mL tin solution  4.6  Total tin content of a standard reference material Tort 1 obtained by different authors  4.7  192  193  Comparison of figures of merit obtained with the two atomic absorption spectrophotometric methods used in this study  194  xix LIST OF FIGURES Figure 2.1  PAGE  Flow diagram for the extraction of organotin from marine animals  42  Figure 2.2  Mass spectra (El) of tricyclohexylmethyltin  45  Figure 2.3  H NMR spectra of dicyclohexyldimethyltin 1  46  Figure 2.4  Mass spectra (El) of dicyclohexyldimethyltin  47  Figure 2.5  GC-MS elution profile of the standard tetraorganotin compounds  Figure 2.6  Effect of internal standard concentration on the linearity of calibration curves for tributylmethyltin  Figure 2.7  58  GC-MS calibration curves for (a) dicyclohexyldimethyltin and (b) tricyclohexylmethyltin  Figure 2.10  56  GC-MS calibration curves for (a) dibutyldimethyltin and (b) tributylmethyltin  Figure 2.9  55  Effect of internal standard concentration on the linearity of calibration curves for tributylmethyltin  Figure 2.8  52  59  Selected ion current chromatogram of standard organotin compounds spiked into shrimp  65  Figure 2.11  Mass spectra of peak A in Figure 2.10  65  Figure 2.12  Mass spectra of (i) peaks C and (ii) peak D in Figure 2.10  66  Figure 2.13  (a) Selected ion current chromatogram of extract from Blue mussel from Wreck Beach, Vancouver. (b) Mass spectra of  xx peak D in Figure 2.13(a)  76  Figure 3.1  Structure of a phospholipid (phosphatidyicholine)  86  Figure 3.2  Liposome  87  Figure 3.3  Schematic diagram of facilitated diffusion (efflux) mediated by a carrier  Figure 3.4  Passive diffusion of a permeant HA across a liposomal membrane  Figure 3.5  93  96  Proposed mechanism of tributyltin mediated efflux of dimethylarsinate (DMK) from a liposome and the equilibria of the carrier-permeant interactions  103  Figure 3.6  H NMR spectra of DMA as it diffuses out of EPC liposomes 1  119  Figure 3.7  Log plot for the efflux of DMA from EPC liposomes  121  Figure 3.8  Chemical species of dimethylarsinic acid present at pH 7.4  123  Figure 3.9  Efflux of DMA from EPC liposomes (organotin compounds are absent in the extraliposomal comparment)  Figure 3.10  Time course for the efflux of DMA from EPC liposomes (tributyltin chloride present in the extraliposomal compartment)  Figure 3.11  126  128  Time course for the efflux of DMA from EPC liposomes (monobutyltin trichloride present in the extraliposomal compartment)  Figure 3.12  128  Log plot of DMA efflux from EPC liposomes (33.2 M  trimethyltin hydroxide present in extraliposomal volume)  133  xxi Figure 3.13  Time course for DMA efflux from EPC liposomes (33.2 jM trimethyltin hydroxide present in extraliposomal volume)  Figure 3.14  133  Effect of tributyltin chloride concentration on the permeability of EPC liposomes (tributyltin chloride was added into the extraliposomal compartment)  Figure 3.15  137  Effect of monobutyltin trichioride on the permeability of EPC liposomes (monobutyltin trichioride was added into the extraliposomal compartment)  137  Figure 3.16  Log plot of DMA efflux from TBT-EPC C liposomes  140  Figure 3.17  Time course for DMA efflux from TBT-EPC C liposomes  140  Figure 3.18  Contribution of passive and facilitated diffusion to the efflux of DMA from TBT-EPC liposomes of different tributyltin chloride composition  Figure 3.19  142  Log plot for efflux of DMA from TBT-EPC C liposomes when 16.7 iM tributyltin chloride is present in the extraliposomal compartment  Figure 3.20  143  Time course for efflux of DMA from TBT-EPC C liposomes when 16.7 iM tributyltin chloride is present in the extraliposomal compartment  144  Figure 3.21  Log plot of DMA efflux from MBT-EPC B liposomes  147  Figure 3.22  Time course for DMA efflux from MBT-EPC B liposomes  147  xxii Figure 3.23  Arrhenius plot for DMAH efflux from TBT-EPC B liposomes  154  Figure 3.24  Arrhenius plot for DMAH efflux from MBT-EPC B liposomes  154  Figure 4.1  Schematic diagram of the apparatus used for the HG-AAS method  Figure 4.2  Schematic diagram of the hydride generator used for the HG-GFAAS method  Figure 4.3  163  163  Effect of sodium borohydride and HC1 concentrations on the absorbance of tin hydride produced from 4 JLg/mL tin solution  Figure 4.4  176  Effect of the concentration of L-cysteine on the absorbance of tin hydride  Figure 4.5  Effect of sodium borohydride flow rate on absorbance  Figure 4.6  Effect of trapping temperature on the atomic absorbance  178 185  of tin hydride  187  Figure 4.7  Effect of trapping time on absorbance  188  Figure 4.8  Effect of sodium tungstate concentration on absorbance  191  Figure 4.9  Effect of palladium on absorbance  191  xxiii LIST OF ABBREVIATIONS b.p  Boiling point  Calcd  Calculated value  cm  Centimeter  Conc  Concentration  Contd  Continued  DMA  Dimethylarsinic acid or equilibrium mixture of DMA and DMAH Dimethylarsinate; negatively charged species of  DMK  dimethylarsinic acid present in solution at pH 7.4 Undissociated dimethylarsinic acid present in solution at  DMAH  pH 7.4 El  Electron ionization  EPC  Egg phosphatidyicholine  GC  Gas chromatograph/chromatography  GC-MS  Gas chromatography with mass spectrometric detection or  Gas chromatograph coupled to a mass spectrometer GC-MS SIM  Gas chromatography with mass spectrometric detection in the selected ion monitoring mode.  GFAAS  Graphite furnace atomic absorption spectrophotometry  h  hour  xxiv acid  HEPES  N-2-Hydroxyethylpiperazine-N’-2-ethanesulphonic  HG-AAS  Hydride generation atomic absorption spectrophotometry  HG-GFAAS  Hydride generation-graphite furnace atomic absorption spectrophotometry  Hz  Hertz  i.d  internal diameter  LUVs  Large unilamellar vesicles  M  ) 1 Molar (mol L  MBT  Monóbutyltin trichioride  MBT-EPC liposome  Liposome  formed  by a mixture  of monobutyltin  trichioride and egg phosphatidyicholine mm  minutes  MS  Mass spectrometer/spectrometry  ND  Not detected  NMR  -  Nuclear magnetic resonance spectrometer/spectrometry  ppb  Parts per billion  ppm  Parts per million  psi  Pounds per square inch  RSD  Relative standard deviation  SIM  Selected ion monitoring  TBT  Tributyltin chloride  TBT-EPC liposome  Liposome formed by a mixture of tributyltin chloride and  xxv egg phosphatidyicholine iris  Tris(hydroxymethyl)aminomethane  TSP  Deuterated 3-(trimethylsilyl) propionic acid sodium salt  v/v  Volume to volume ratio  w/v  Weight to volume ratio  xxvi  ACKNOWLEDGEMENTS. I wish to express my gratitude to my research supervisor Professor W.R. Cullen for his guidance and interest in this research, and for my financial support. I would like to thank Dr Gunther  Eigendorf for his advice on mass  spectrometry, and for his kindness towards me. I am also grateful to Madiba Saidy, Ryan Males, and the following people: Dr. ‘s Christopher Harrington, Bruce Todd, Roshan Cader, Kian Pang for their help in the various stages of this thesis, and to the past and present members of Professor Cullen’s research group for many helpful discussions. My gratitude also goes to the following people; Ms Lina Madilao, Mr Steve Rak, Kim Wong and Gary Hewitt for their help in the various technical aspects of this work, and to Dr P.R. Cullis and Professor F.G. Herring for the use of their facilities. I am thankful to the Department of Chemistry, University of British Columbia, Canada for financial support and provision of research facilities. I also wish to express my sincere gratitude to my mother, brothers and sisters, and brother-Inlaw Dr A.N. Ewunonu, for their encouragement to me. To my wife Joyce, and children; Edoziem and Chima, I owe you much gratitude for the encouragement and moral support you gave me.  xxvii DEDICATION This thesis is dedicated to the memory of my beloved father Chief Nelson Ukachi Nwata. Your departure before the task could be completed was very painful, but the daily remembrance of you is my source of inspiration. Rest in perfect peace.  1 CHAPTER 1 GENERAL INTRODUCTION.  1.1.  HISTORICAL BACKGROUND OF THE ORGANOT1N COMPOUNDS. The synthesis of diethykin diiodide by Frankland’ in 1849, marked the  introduction of a new class of compounds that were later to occupy a significant position in industry and agriculture. By definition, organotin compounds are those compounds that have a carbon-tin covalent bond in the molecule. Progress in the chemistry of organotin compounds was enhanced by the discovery of Grignard reagents which made possible the production of a variety of organotin compounds n, from which lower alkyl- or aryl- tin compounds could easily be R S of formula 4 made. The first industrial application of organotin compounds was made in 1936, when Yngve of the Carbide and Carbon Chemical Company, U.S.A., discovered the heat stabilizing effect of organotin compounds on polyvinyl chloride (PVC) and other . The organotin compounds that have been found 2 chlorinated hydrocarbon polymers useful in this application are the mono and dibutyltin compounds and the dioctyltin compounds.  1.2  INDUSTRIAL  APPLICATIONS  AND  USE  OF  ORGANOTEN  COMPOUNDS. The organotin compounds find a wide range of use in the manufacturing  2 industry, agriculture and medicine. Because of their very low toxicity, the dioctyltin derivatives are used as stabilizers for food packaging polymers. Organotin compounds  are also used for cold curing of silicone rubber and as polymerisation catalysts; for example, butyichiorotin dihydroxide . Dibutyltin diacetate is a catalyst for flexible 3 foams. Some organotin dihalides having the formula 2 SnX (R=ethyl or phenyl, R L X=chloride or bromide, L = o-phenanthroline or 2-(2-pyridyl) benzimidazole exhibit 2 anti-tumOur and anti-herpes activity in vitro . Dibutyltin dilaurate is also effective in 4 the removal of intestinal worms in poultry . Dicyclohexyltin derivatives of dipeptides 3 having the formula Cy SnL (L=glycylglycine, glycylalanine, glycyiphenylalanine and 2 . 5 glycyltyrosine) exhibit high cytotoxicity in vitro to breast cancer cells The triorganotin compounds are the most important of the organotin compounds in agricultural applications. For example, tricyclohexyltin compounds are effective as miticides and and possess marked acaricidal action against plant-feeding mites, but have very little effect on predacious mites and insects . They also have 3 been reported as antifeedants for the Gypsy moth Porthetria dispar . The pesticide 6 Plictran® marketed by Dow Chemical Company has tricyclohexyltin hydroxide as the active ingredient , and has been used in Canada for crop protection on apples and 7 . Peropal®, a pesticide marketed by Bayer AG has 1-tricyclohexyltin-1,2,48 pears triazole as the active ingredient. The triphenyltin compounds show antifungal activity. The fungicide Brestan® marketed by Hoechst A.G, Germany, contains triphenyltin acetate and has been used . Du-Ter®, a 3 against a broad range of fungal organisms in sugar beet and potatoes  3 fungicide marketed by Philips-Duphar, Holland contains triphenyltin hydroxide as the active ingredient . The use of triphenyltin hydroxide as an antifeedant for the 3 Colorado beetle Leptinotarsa decemlineata has also been reported . 9 The tributyltin compounds are fungicides, algaecides and slimicides, and have been widely used as wood preservatives. Presently, their major use is in marine antifouling paint formulations for protecting the hulls of ships and boats from algae, fungi, sponges, molluscs, barnacles, diatoms, shipworms etc. Such fouling has the effect of increasing weight and drag, causing the ship to consume more fuel to maintain speed. The tributyltin compounds  used in marine paint formulations are  bis(tributyltin) oxide, bis(tributyltin) dodecenylsuccinate, bis(tributyltin) suiphide, tributyltin fluoride, tributyltin resinate, tributyltin methacrylate,  bis(tributyltin)  adipate. Unfortunately the tributyltin compounds are the major organotin compounds of concern from the point of view of environmental marine pollution. When used as antifouling agents, they pollute the marine environment. They do not remain localized but spread throughout the marine environment causing considerable problems such as imposex in marine gastropods’° and the deformation and decline of oyster stock . 11  1.3  NEED FOR A CHEMICAL ANTIFOULING AGENT. The nuisance caused by the growth of unwanted marine organisms is a major  problem in the maritime industry. One of the early approaches taken to prevent fouling in wooden ships, was the use of copper metal sheathing 12 on the ship’s hulls.  4 This achieved moderate success in the control of fouling. In steel ships, the use of copper metal sheathing is not appropriate because of the severe galvanic corrosion of steel when in contact with copper and sea water . The usual method of fouling 12 prevention in steel ships is to use chemical agents which are usually incorporated in the paints used to paint the hulls. These antifouling paints act by releasing biocides, which kill the larvae and spores of any marine animals and plants attempting to settle on the ship’s hull. . 12 Among the early biocides employed for this purpose was cuprous oxide Cuprous oxide exhibits a wide spectrum of toxicity to animals, but many plants are resistant to it. Continuous use of the copper oxide results in the formation of insoluble greenish salts within the surface layers of the paint film. The build up of these salts on the surface prevents further controlled release of fresh biocide. This process limits the life time, and the efficiency of the paint. The search for biocides to boost the performance of cuprous oxide led to the screening of the organotin compounds for biocidal activity. Tributyltin compounds were found to be suitable biocides because they exhibit low mammalian toxicity, but high toxicity to fungi and algae at low concentrations. They are also typically colorless and can therefore be incorporated into brightly colored paints. In the course of searching for efficient ways to deliver tributyltin compounds to the target organisms, the following antifouling paint formulations have become available.  5 1.3.1 Contact leaching antifouling paints. In this design, the antifouling system is composed of a tough insoluble filmforming resin such as a chlorinated rubber within which tributyltin fluoride is physically dispersed . When immersed in water, the freely dispersed molecules of 12 tributyltin fluoride near the surface of the paint are able to diffuse out of the matrix of the paint film. As the biocide leaches out of the film, it leaves behind microscopic pores within the paint matrix. The inflow of sea water into these tiny pores causes the release of fresh tributyltin biocide from beneath the surface layers of the film. A major disadvantage of this paint design is that with the passage of time, the microscopic pores become clogged with insoluble materials thereby making it difficult for biocide in the deeper strata of the paint matrix to be released. As a result, contact leaching antifouling paints work best only during the early part of their life span. When the antifouling action of the paint fails, a large amount of the biocide is still trapped in the inner matrix thereby creating a severe problem in the proper disposal of spent antifouling paints . 12  1.3.2 Ablative formulation. In this design, the tributyltin compound is dispersed into a film matrix composed of a mixture of soluble polymeric materials which are designed to break down over time . As the film matrix breaks down, the biocide is released. A 12 disadvantage of this design is that it is difficult to control the actual breakdown of the paint film and the release of the biocide because the rate of paint film  6 breakdown is affected by water conditions and vessel speed . 4  1.3.3 Self polishing copolymer paints. In self polishing copolymer paints, the paint film is composed of a copolymer of methylmethacrylate and tributyltin methacrylate which also is the source of the  biocide. At the surface of the paint, sea water interacts with the hydrophobic co polymer and initiates a saponification reaction which cleaves tributyltin cation from the co-polymer backbone, releasing it into the sea. The release rate of the tributyltin cation is gradual thereby enabling the biocidal action of the antifouling paint to last a long time.  1.4  TOXICiTY OF ORGANOTIN COMPOUNDS. In general, the toxicity of the organotin compounds RSnX .. increases with 4  the increase in the number of alkyl or aryl substituents bonded directly to the tin . Maximum toxicity is obtained when n=3. On increasing the number of alkyl 15 atom or aryl substituents compounds  above n=3, the toxicity drops. Therefore, tetraorganotin  on their own have no toxicity. The toxicity observed with the  tetraorganotin compounds is believed to be due to their in vivo metabolism to triorganotin compounds . As the number of carbon atoms in the alkyl chain is 13 increased above three, mammalian toxicity decreases. The type of R- group on the tin atom determines the level of toxicity to specific organisms . The trimethyltin compounds are the most toxic to insects while 8  7 the triethyltin compounds are the most toxic to mammals . For gram-negative 8 bacteria,  the tripropyltin compounds  are the most toxic . The tri-n-butyltin 8  compounds are the most toxic to gram-positive bacteria and fungi . 8  The toxic effect of the trialkyltin compounds is attributed to the inhibition of mitochondrial oxidative phosphorylation, and subsequent disruption of a fundamental energy process . The trialkyltin compounds bind to a number of proteins, and 13 mortality may arise from direct reaction of the organotin species with proteins . 13 Differences in protein binding sites among groups of organisms would result in the varying spectrum of effectiveness of the triorganotin compounds to different organisms’ 14 ’ 3 Another mechanism by which trialkyltin compounds may derive their toxicity has been described by Selwyn  . According to these 6 and Tosteson and Weith’  authors tributyltin’ , tripropyltin and triphenyltin’ 6 ” 5 5 cations mediate chloridehydroxide exchange in the mitochondria and smectic mesophases (liposomes) , and 15 in planar lipid 16 bilayers Also, the tripropyltin cation has been reported to mediate . chloride-chloride exchange across a lipid bilayer . The ability of the trialkyltin 17 cations to mediate anion transport is the subject of study in chapter three of this thesis. The dialkyltin compounds 2 SnX also show a similar trend of decreasing R . The mode of toxicity of the 8 toxicity with increasing length of the alkyl chain dialkyltin compounds has been shown to be different from that of trialkyltin . The toxic action of the lower dialkyltin compounds is due to their 18 compounds  8 ability to combine with enzymes possessing two thiol groups in the correct 18 The biochemical effect of this is an interference with a-keto acid ’ 8 conformation 18 ’ 8 oxidation The mono-organotin compounds RSnX , do not show any important toxic 3 . 8 effect The organotin compounds do not appear to show any carcinogenic or  teratogenic effect . However, di- and tn- alkyl and aryl tin compounds have been 8 shown to induce chromosomal contraction in human lymphocytes . Alterations in the 19 spermatocyte chromosomes of the mesogastropod Truncatella subcvlindrica induced by dibutyltin dichloride and tributyltin chloride have also been reported by Vitturi et a1 , thus demonstrating the genotoxicity of these compounds. 20 Of particular  interest  in the marine  environment  are the tributyltin  compounds. Tributyltin compounds are very toxic to marine life at very low concentrations, and are suspected of inducing imposex in the female dogwhelk Nucella . 22 Tributyltin compounds have also been reported to induce shell ’ 21 lapillus malformations in the oyster Crassostrea gigas at very low concentrations’ , and 24 have also been reported to have caused high mortality in the larvae of the common mussel Mytilus edulis . The toxicity of tributyltin species to the following non-target 25 , lobster 26 marine organisms at the ppb level has been reported:- amphipod larvae larvae and zoeal shore crab , and the sheepshead minnow Cyprinodon variegatus 27 . 28 At the low ppb levels, tributyltin species cause sublethal effects in the zoeal mud crab . 30 Rhithropanopeus harnisii 29 and copepods Acartia tonsa  9 1.5  METABOLISM AN]) BEHAVIOUR OF TRIBUTYLTIN COMPOUNDS  IN THE ENVIRONMENT. On introduction into the marine environment, tributyltin compounds are removed from the water column by photolysis and assimilation by plants and , and by adsorption to the sediment, and particulate matter. Hydrolysis and 21 animals volatilization do not appear to be major degradative pathways . The affinity of 22 tributyltin compounds for sediments and particulate matter makes them far less bioavailable to organisms in the upper water layer, but bottom feeding organisms are exposed to higher concentrations of tributyltin compounds. Therefore, the feeding habit of a marine organism is an important factor in determining its tributyltin body burden. According to Maguire ’, tributyltin species adsorb so firmly to particles that 3 under abiotic conditions, there was no desorption of tributyltin oxide from harbour sediments over a period of ten months. However, under biotic conditions there was microbial degradation resulting in the liberation of butylated and methylated products. Tributyltin cation is hydrophobic, and therefore has a high tendency to preferentially accumulate in the surface microlayer of natural waters . Its octanol 32 water  partition  0 coefficient(K  =  coefficient  =55OO-7OO) 0 (K 33  and  sediment-water  partition  16OO) values favour accumulation in the surface microlayer. The  surface microlayer attracts and sequesters hydrophobic species such as tributyltin species. The preferential accumulation of tributyltin compounds in the surface microlayer is expected to render tributyltin species unavailable to most organisms. However, bioaccumulation has been observed for a variety of organisms. Bacteria  10 and phytoplankton accumulate tributyltin species to concentrations 600 times and 33 Also, a ’ 32 concentrations 30,000 times respectively, greater than their exposure . 36 for the bioaccumulation factor of 4400 has been reported by Evans and Laughlin hepatopancreas  of the mud crab Rhithropanopeus  harrisii. Accumulation  of  tributyltin species up to a concentration factor of 12,000 by the plant Eelgrass Zostera marina has been reported . 37 Tributyltin compounds have been found to exhibit preferential accumulation in certain tissues. Ward  .j38  observed that the viscera of the sheepshead minnow  contained higher concentrations of tributyltin oxide than the cranial or muscle tissues. The reported bioaccumulation factors for tributyltin compounds are high enough to warrant concern with regard to their persistence and accumulation in food chains. However, they are degraded in vivo by bacteria , fungi 39 , algae 60 35 and fish . The 34 detoxification route for tributyltin compounds involves their conversion to the less toxic dibutyltin, monobutyltin, and inorganic tin species. Getzendaner and Corbin 41 have also reported similar detoxification pattern for tricyclohexyltin species. Barug ° 4 has observed the degradation of tributyltin oxide to monobutyltin derivatives by the bacteria Pseudomonas aeruginosa. However, tributyltin chloride was not degraded under anaerobic conditions by the same bacteria. Maguire in vivo  degradation  have observed the  of tributyltin species by a green algae with the major  degradation product being dibutyltin species. Tributyltin compounds do not appear to be amenable to biomagnification. Macek  ti42  have presented data indicating that chemicals with short or moderate  11 half-lives in vivo do not pose a biomagnification problem. Tributyltin species have . 43 a half-life considerably shorter than forty days by aerobic metabolism  1.6  ANALYTICAL METHODS FOR ORGANOTIN COMPOUNDS.  The early analytical methods available for the determination of tin were classical gravimetric or volumetric procedures that gave only total inorganic tin concentrations. Beginning in the early 1930s, and extending into the early 1960s, optical spectrographic methods for total tin determination were extensively used for the determination of geological samples. With the passage of time, the more sensitive colorimetric, fluorimetric, neutron activation and flame atomic absorption techniques were introduced. The wide use of flame atomic absorption spectrometry was hampered by the low sensitivity of the tin absorption lines . 44 For the organotin compounds, early analytical methods relied on the conversion of the organotin compounds to inorganic tin, usually by digestion with mineral acids followed by ignition. L.ater, more diversified techniques such as electrochemical,  chromatographic, and mass spectrometric techniques capable of  providing speciation  information  were introduced.  Recently, a tandem  mass  45 has been applied to a mixture of standard speetrometry (MS-MS) technique organotin compounds with a view to analyzing them without prior derivatization to volatile species or prior chromatographic separation. This method relies on the fixed relationship between parent and daughter ions of any compound under fixed experimental conditions. The applicability of this technique to the analysis of  12 environmental samples has not yet been demonstrated. Not much attention has been given to the qualitative analysis of organotin compounds. However, infrared spectroscopy 47 nuclear ’ 46 spectrometry , Mossbauer , 46 51 magnetic resonance ’ 49 and electron spin resonance spectrometry 48 spectrometry ° 5 have been applied to provide information on molecular structure and speciation. The various quantitative analytical methods applied over the years are described in the following sections.  1.6.1 Molecular spectrophotometry and spectrofluorimetric techniques. These methods rely on the attachment of a chromophoric ligand to the organotin compound. This makes it possible to analyze the organotin compounds by using uv-visible, or fluorescence spectroscopy, since the alkyl groups of the organotin compounds are not of much spectroscopic importance. A variety of ligands have been employed for this purpose. Aidridge and Cremer 52 were the first to use dithizone for the spectrophotometric determination of diethyltin and triethyltin species. Diethyltin and triethyltin chlorides react with dithizone to form colored complexes. Analysis of the complexes  is effected following partitioning  between aqueous  potassium  hydroxide and chloroform. The diethyltin species partition into the alkali layer, while the triethyltin species migrate to the chloroform layer. The separated organotin compounds can then be determined by uv-spectrophotometry. A modified method for the reaction between dithizone and the organotin compounds has been reported by 54 53 for the determination of bis(tributyltin) oxide. Skeel and Bricker Havir and Vretal  13 developed  a spectrophotometric  method  for the determination  of dibutyltin  dichioride by using diphenyl carbazone. This method achieved a sensitivity in the microgram range. Other colorimetric reagents that have been used in the analyses of the organotin compounds are dithiol , 57 , 8-hydroxyquinoline 56 , haematoxylin 55 , pyrocathechol , 58 phenylfluorone 61 (3-hydroxyflavone). 60 flavinol ’ 59 violet For the  fluorimetric  determination  of the  organotin  compounds,  3-  62 and morin hydroxyflavone 63 (2’ ,3 ,4,4’ ,5 ,7,-pentahydroxyflavone) have been applied to the determination of phenyltin, and alkyltin compounds respectively. A major disadvantage of the spectrophotometric  and spectrofluorimetric  techniques is the lack of specificity of these organic ligands to organotin compounds. However, limited selectivity can be achieved by employing various extraction techniques prior to spectrophotometry or spectrofluorimetry.  1.6.2 Electrochemical techniques. Electrochemical techniques rely on the difference in the redox potentials of the various organotin compounds for speciation. Polarography, in the various modes , and potentiometric titrations’ 65 such as anodic stripping voltammetry’ 67 have been used for the determination of organotin compounds in aqueous and nonaqueous media. Diethyltin dichioride was the first organotin compound whose polarographic reduction was recorded . The polarographic behavior of other organotin compounds 68 has also been described . The ease of the polarographic reduction of the organotin 69  14 . 70 compounds has been found to be a function of the alkyl moiety on the tin atom The ease of reduction follows the order ° ethyl > propyl > butyl. Polarography, in 7 the differential pulse mode has also been applied to the determination of organotin 273 7 ’ 71 compounds Potentiometric titration of organotin compounds in dimethylsulfoxide (DMSO) has been reported to show well defined differential pulse polarographic peaks whose heights are linearly dependent  on concentration . 74  Quantitation  of organotin  compounds based on this observation has also been accomplished . 74 The half wave potentials of some organotin compounds have been determined by Abeed et alTh, by using voltammetry, and cyclic voltammetry at rotating disc electrodes (gold and glassy carbon electrodes) in non aqueous solvents. Their results showed that the reduction becomes more difficult as the electron donating ability of the alkyl or aryl groups attached to the tin atom increases (phenyl- methyl-buty1). The variation of the anion attached to the tin atom had little effect on the half wave potentials. The application of electrochemical techniques to organotin analysis is restricted by the sample matrix. Organic matter present in environmental samples coats the electrode surface causing broadening of peaks, and shifts in peak potentials.  1.6.3 Atomic spectrometry. Atomic absorption spectrometry has been used extensively in the analysis of organotin compounds. Atomic absorption spectrometry is not capable of speciation unless a prior separation of the organotin compounds is achieved. The methods  15 usually employed to separate organotin compounds prior to their determination by atomic spectrometry include solvent-solvent extractions or conversion to organotin hydrides. The first analytical speciation and quantitation of organotin compounds by atomic absorption spectrometry after their derivatization to organotin hydrides was . The method involved the reaction of organotin 76 developed by Hedge et a1 compounds in natural water, acid digest of sediment or macroalgae with sodium borohydride. The organotin hydrides produced were collected in a hydride trap which was cooled in liquid nitrogen. The hydride trap was warmed to release the organotin hydrides into a quartz tube furnace according to their boiling points. Since the , numerous atomic absorption 76 publication of this analytical method by the authors methods based on hydride generation and the boiling point differences of the organotin hydrides have been reported  77,78,79,80,81,82,83,84  Many gas chromatographic techniques based on the hydride generation method of Hedge  are presently in use (Section 1.6.4.1).  The speciation of butyltin compounds by liquid-liquid extraction prior to 85 . This method 85 atomic absorption spectrometry has been described by Mckie involved the extraction of butyltin species into acidified hexane and the subsequent removal of dibutyltin and monobutyltin species by washing with 3 % sodium hydroxide solution. The hexane layer was evaporated and the solution of the residue in  nitric  acid was analyzed for tributyltin species by using graphite furnace-Atomic absorption spectrometry  (GFAAS). The matrix modifier used in this determination  was  85 has PO An analytical method similar to the technique reported by McKie 2 H NH . 4  16 been applied to the determination of tributyltin species in shellfish and sediments by Cr as a matrix modifier. K 7 O Stewart and de Mora. These authors employed 2 The use of atomic absorption spectrometry for the determination of organotin compounds is usually affected by severe matrix interferences. To overcome this problem, various matrix modifiers have been introduced. One of such matrix modification methods was the coating of the interior surfaces of a graphite furnace tube with zirconyl acetate . This has been shown to increase the atomization 87 efficiency of tin . Peetre and Smith have reported that there is a relationship 87 between the atomic absorption sensitivity and the structure of an organotin compound. According to this report, the atomic absorption sensitivity decreased as the energy of the alkyl-tin bond decreased. Atomic emission spectrometry of the organotin compounds has not been popularly used except in plasma ’ 89 emission 9 0 detection or flame photometric 1 2 detection in gas chromatography (Section 1.6.4.2a). Atomic emission spectrographic . Prior 93 method has been described for the determination of bis(tributyltin) oxide separation of other organotin compounds present in the sample is necessary if speciation is desired because emission spectrography on itself, is not capable of distinguishing between the different organotin compounds.  1.6.4 Gas chromatography. Organotin  compounds  are  usually converted  to volatile  hydrides or  tetraalkyltin compounds prior to gas chromatographic separations. In a few reports,  17 the analysis of organotin compounds without prior derivatization to hydrides or tetraalkyltin compounds has been . 94 accomplished 9 9 ’ 798 5 In such methods, the 6 chromatograms are usually characterized by peak broadening and tailing. The column efficiency is also decreased. Both packed and capillary gas chromatographic columns have been used with various detection techniques. The chromatographic techniques employed in the analyses of the organotin compounds are discussed below.  Hydride generation gas chromatography (HG-GC).  1.6.4.1  This method involves the conversion of the organotin compounds to volatile hydrides by the use of excess borohydride to produce alkyltin hydrides of the formula ..,. The generated hydrides are purged from solution with the help of an inert 4 RSnH gas, and can be cryoscopically trapped. The cold trap is subsequently warmed, to release the organotin hydrides into the column of the gas chromatograph. Detection of the separated organotin hydrides is achieved by using various gas chromatographic detectors. Woollins and Cullen 99 have described a hydride generation-GC  flame  ionization detection technique based on the method previously developed by Hodge 76  for the analysis of organotin compounds. Hattori LV°° have also described  the determination of organotin compounds in environmental water and sediments, on a packed column by using HG-GC electron capture detection. An ultratrace method for the analysis of aquatic butyltin by HG-GC with flame photometric  18 . A novel on-column hydride 101 detection has been described by Matthias et a1 generation  analysis of organotin  compounds  by gas chromatography-atomic  absorption spectrometry (GC-AAS) has been described by Clark 102 Li and Takami , 103 In the method described by Takaini t.i J . , fish samples were extracted with 103 hydrochloric acid-ethanol mixture. The extracted organotin compounds in the fish were transferred to ethylacetate/hexane  by using liquid-liquid extraction, and then  applied onto a sulfonated cation exchange column where the organotin compounds were trapped. On-column hydride generation was effected by passage of an ethanolic sodium borohydride solution through the cation exchange resin. The generated hydrides were extracted into hexane, and analyzed by GC-MS. The GC-hydride generation method of organotin determination has been very 05 07 08 ”° 104 authors 6 ’’° 1 ’ . extensively used by numerous 9 Two detection methods have widely been used in gas chromatography for the analyses of organotin compounds after their conversion to volatile derivatives. These are flame photometric detection (FPD) and atomic absorption spectrometry (AAS). Coupling the gas chromatograph directly to an atomic absorption spectrometer (GC AAS) appears to be the most popular technique for element specific speciation of the organotin compounds after hydridization. Methyltin species in natural water have been determined  after hydride  derivatization by using GC-graphite furnace atomic absorption spectrometry (GC . Butyltin species in natural water and sediments have been analyzed by 10 GFAAS)’ , by using GC-HG-quartz Donard’ ’ Quevauviller and 1  tube atomic absorption  19 spectrometry. In enviromnental samples, production of volatile hydrides may be inhibited by severe matrix 11 interferences 2• Such matrix interferences can be eliminated by the use of L-Cysteine” . 3  1.6.4.2  Conversion to telraalkyltin compounds. Conversion of organotin compounds to tetraalkyltin derivatives is usually  accomplished by reacting the organotin compounds with a Grignard reagent, or sodium tetraethylborate.  The reaction of monoalkyltin, dialkyltin, and trialkyltin  species with the Grignard reagent proceeds to completion at very low concentrations, and no rearrangement of the original alkyl groups attached to the tin atom is usually 14 rved  The tetraalkyltin derivatives formed are usually stable in organic  0 and , 2 ” 19 , pentyV 8 15 Various alkyl groups such as methyl . 1 ’ 114 solvents , ethyV’ 7 ’ 116 n-hexyV .122, have been attached to the butyltin compounds to facilitate their 21 analyses.  Detection  of the  derivatized  tetraalkyltin  compounds  is usually  accomplished by the use of various gas chromatographic detectors. The GC-detector systems that have been used for tetraalkyltin analyses are described below.  1.6.4.2(a)  Gas chromatography with flame photometric detection (GC-FPD).  The flame photometric detector has been used extensively for the detection and quantitation  of organotin compounds  in the environment  following their  derivatization to tetraalkyltin compounds. Developed by Aue and Flinn’, flame  20 photometric detection has been used for tin-specific detection in gas chromatographic , l O 2 infishUS,lZO,waterl 2 l 4 t 120 analyses of butyltin species 22 sediments and , 25 22 ’’ for the determination of methyltin species in water . 126 A disadvantage of the flame photometric detector is the decrease in sensitivity which may be caused by the accumulation of Sn0 2 on the internal surfaces of the detector, and by tropolone, a ligand sometimes used in the extraction of organotin compounds 27• 1 Flame photometric detection of the organotin compounds relies on the conversion of tin to Snil in air/hydrogen flame. SnH yields a red emission line in the gas phase at about 610 nm and almost all analyses for tin have been carried out at jjflfl had described another emission line for this wavelength. Earlier, Aue and 23 tin at 390 nm in the flame photometer.This emission line was unstable, irreproducible and easily quenched , and was later attributed to a quartz surface induced tin 123 . Jang 28 luminescence’  Ii29  have described a sensitive flame photometric analysis  involving the 390 nm emission line. According to the authors, a clean quartz surface is required in the vicinity of the flame to achieve stability of the emission line, and only the Shimadzu flame photometric detector has this feature. The detection limit was claimed to be about thirty times better than that at the 610 nm emission line.  1.6.4.2(b)  Gas chromatography with atomic absorption spectromeiric detection  (GC-AAS). Analysis of organotin compounds by GC-AAS after conversion to tetraalkyltin  21 ° have 13 compounds is not popular and has only been seldomly used. Forsyth et a1 applied this method to the determination of organotin compounds in fruit juices.  1.6.4.2(c)  Gas chromatography with mass spectromeiric detection (GC-MS).  GC-MS affords a very reliable method in the analysis of the organotin compounds, since detection is based both on retention data and fragmentation pattern. The first application of GC-MS to the analysis of organotin compounds was described by Meinema compounds  were  In this procedure, benzene extracts of pure butyltin  converted  their  to  butylmethyltin  derivatives  by using  methylmagnesium bromide. The derivatized butylmethyltin compounds were analyzed by using GC-MS with dibutyihexylmethyltin as the internal standard. The application of GC-MS to the determination of organotin compounds in environmental samples , Unger t.i 131 has been reported by Cullen etalm, Forsyth .Lai’ , Muller 30 . 132  1.6.5 Liquid chromatography (LC). Liquid chromatography  of the organotin compounds does not require  derivatization to volatile species, and hence could be useful for the analysis of non volatile organotin compounds. The alkyltin species are difficult to detect by uv-visible spectroscopy therefore, derivatization might be necessary to enhance their detection. Various liquid chromatographic  detectors have been used for the analysis of  organotin compounds. An indirect photometric method for the determination of alkyltin compounds has been described by Whang t.ilV 33 for tributyltin, tripropyltin,  22 triethyltin, and trimethyltin species after their separation on a strong cation exchange column. For high sensitivity, inductively coupled plasma-mass spectrometers (ICP 6 inductively coupled plasma-atomic emission spectrometers , 1 ’ 34 MS)’ 3 ” 35  (ICP  36 and atomic absorption ’ ” 135 AE) 38 have been used to 39 40 41 137 spectrometers 1 detect organotin compounds in high performance liquid chromatography (HPLC). Direct coupling of the liquid chromatograph to a mass spectrometer or atomic absorption spectrometer is associated with problems such as solvent interferences. The large amount of solvent and sometimes non-volatile buffers that go into the detector systems are also a major concern. This concern can be addressed by interfacing the LC and the detector systems, or by the use of a microbore column. The small solvent flow rate (10-100 iL/min) in microbore HPLC has been shown to be compatible with direct effluent introduction to a flame atomic absorption . 42 spectrophotometer’ A coupled LC-AAS technique with enhanced laser ionization detection has , for the analysis of tributyltin species in sediment. 43 been described by Epler et al’ In their technique, tributyltin species in a sediment sample were extracted into 1butanol, separated on a strong cation exchange column and detected by using a premixed air-acetylene flame which was irradiated with two pulsed lasers. The 143 was attributed to a rapid enhanced ionization of the tin atom in their technique collisional ionization of the excited tin atoms in the flame. Another method of solving the problem of excessive solvent introduction into  23 the detector system, is by the post column derivatization of the organotin compounds to volatile hydrides which can then be introduced into an atomic absorption . This method has been applied to the determination of methyltin 144 spectrometer . 144 species Organotin compounds adsorb strongly onto unmodified silica gel columns therefore, most liquid chromatographic separations are performed on reversed phase columns, size exclusion columns, and other modified columns such as cyano bonded silica gel columns. Jessen et al 145 have studied the adsorption behavior of alkyltin halides on various chromatographic  columns, and concluded that silica based  octadecyl (ODS) and cyano columns are not sufficiently inert to alkyltin halides while silica gel columns pyrolytically coated with carbon black are inert. However, the organotin halides have been reported to adsorb on a commercially available graphitized carbon black . The ability of organotin halides to adsorb on graphitized 146 carbon black was the basis for a selective determination of the organotin compounds . 46 by Fern et al’ The separation of the alkyltin halides on a cyanopropyl bonded silica gel column  and  their  detection  by fluorescence  spectrometry  after  on-column  . It has been reported by 47 complexation with morin has been described by Langseth’ Jessen et al’ , that rearrangement of the alkyl groups on the tin atom can occur 45 especially if tetraalkyltin compounds are co-injected with other organotin compounds into the LC column.  24 1.6.6 Thin layer chromatography  (TLC) and high performance  thin layer  chromatography (HPTLC). Thin layer chromatography is not widely used for the quantitative analysis of the organotin compounds. However, it has been applied to qualitatively identify organotin compounds. The separation of butyltin compounds and their colorimetric detection after complexation with pyrocatechol violet has been reported by Laughlin et a1 . 148 Speciation of the mammalian organotin metabolic products has been reported . Their method involved both normal and two dimensional TLC 149 by Kimmel et a1 techniques, and visual detection of the organotin species after complexation with colorimetric reagents such as pyrocathechol violet or dithizone. Vasundhara Li 56 have demonstrated the good resolving power of the TLC for a series of tn- and di organotin compounds. Detection of the separated organotin compounds was by treatment of the TLC plate with haematoxylin. High performance  thin layer chromatography  (HPTLC)  has not been  popularly used for qualitative analyses of the organotin compounds. However the quantitation of butyltin compounds in a wood matrix, by using HPTLC has been reported by Ohlsson ct.i . Their method involved the post column development 150 photolysis and complexation of the butyltin species with pyrocatechol violet, followed by colorimetry of the tin-pyrocatechol violet complex. A HPTLC method of quantitation has also been described by Tomboulian 151 j 2  for phenyltin  compounds.  Quantitation  was by fluorescence  scanning  25  S  densitometry, following in situ complexation of the phenyltin compounds with morin.  1.7  TRIBUTYLTIN AND GOVERNMENT REGULATIONS. Tributyltin compounds dissolved in marine waters exhibit acute toxicity to a  variety of aquatic life. Available data in the literature tend to suggest that fish and larger crustacea are less sensitive to tributyltin compounds than bivalves, molluscs, phytoplankton and small crustaceans. It has been established that molluscs are . Following the establishment of 152 generally very sensitive to organotin compounds a correlation between tributyltin compounds, shell malformation and abnormal growth in oysters, the French Government in 1982 banned the use of antifouling paints containing more than 3 % by weight of tributyltin compound on boats less than 25 tons. In 1987, a total ban on the use of organotin paints on vessels less than 25 . 52 metres came into effect in France’ In 1986, the Government of England prohibited the retail sale of some antifouling paints containing tributyltin compounds. In 1987, the use of tributyltin . 53 containing paints on small boats and mariculture equipment was banne& The use of organotin compounds in fresh water antifouling paints is prohibited . 54 in Germany and Switzerland’ In Canada, tributyltin compounds are registered under the Pest Control Products Act for use as a slimicide and for general lumber preservation. Its use as a preservative for nets is not allowed”. In 1989, the Canadian Government prohibited the use of tributyltin compounds on vessels less than 25 metres in length,  26 and also stipulated a maximum release rate of 4 j.tg tributyltin compound per square centimetre of ship’s hull . 155  1.8  OBJECTIVES AND SCOPE OF THE PRESENT STUDY. Tributyltin compounds have been shown to cause shell malformation in oyster,  and other , 11 molluscs 2 ’ 3 and have also been linked to imposex in the marine 4 gastropods such as the female dogwhelk . Cullen Li” 10 7 have reported the presence of butyltin and cyclohexyltin species in some coastal areas of British Columbia, Canada. The toxic effect of the cyclohexyltin species is not known with certainty. Therefore, it is necessary to provide data on the extent of organotin pollution in the coastal areas of British Columbia, Canada. Chapter 2 of this thesis provides information on the levels of butyltin and dicyclohexyltin species in some marine locations of British Columbia. Although the trialkyltin compounds are thought to exert their toxicity by the inhibition of mitochondrial oxidative phosphorylation, little attention has been given , 15 to the effect of the alkyltin compounds on biomembranes. Early studies by Selwyn and Tosteson and Wieth’ 6 showed that tributyltin, triphenyltin cations act as carriers for CF and OW, and therefore mediate CF/OW, while the propyltin cation mediates CF/CF exchange across artificial biomembranes called liposomes. Later, 156 showed that tributyltin chloride affects the dipole potential Tosteson and Weith of phosphatidylethanolamine  lipid bilayer, and lowers it by about 70 millivolts. The  effect of organotin compounds on the other properties of membranes such as  27 permeability, elasticity, etc has not been reported. A knowledge of the effect of the organotin compounds on the other properties of the membrane is important for a complete understanding of the mechanisms of their toxicity. 157 have reported the inhibition of intracellular calcium Arakawa et a1 58 mobilization by tributyltin chloride and dibutyltin dichioride. Arakawa and Wada’ 2 mobilization may be due to changes in the then surmised that the inhibition of Ca 158 had membrane structure caused by the butyltin compounds. Also, the authors suggested that the toxicity of the alkyltin compounds should depend on their solubility in biological fluids, and their extent of incorporation into cells. Therefore, the experiments described in Chapter 3 of this thesis are concerned with providing information on the permeability changes of model biomembranes known as liposomes, and the effect of organotin incorporation into these liposomes on membrane permeability. It is necessary to use liposomes as models for biological membranes because  of their similarity with true biomembranes (Chapter 3 Section 3.3). Also, the use of liposomes eliminates complications that may arise in interpreting experimental data if true biomembranes are used for permeability studies. The ease or difficulty of efflux of an encapsulated  probe permeant;  dimethylarsinic acid (DMA) from these liposomes in the presence and absence of the organotin compounds, is an indication of how the membrane permeability responds to the presence of the organotin compounds. Since the advent of organotin pollution in the marine environment, every  28 effort by workers in the field of environmental pollution has been directed towards providing data on the level and speciation of the organotin compounds. The total tin content of marine animals has been largely neglected. Therefore, Chapter 4 of this thesis is concerned with total tin determination in oysters, and the analytical method development for total tin determination by hydride generation-atomic absorption spectrophotometry.  29 CHAPTER 2 SPECIATION AND QUANT1TATION OF BUTYLTIN AND CYCLOHEXYLTIN COMPOUNDS IN MARINE ORGANISMS BY USING CAPILLARY COLUMN GC-MS SIM.  2.1  INTRODUCTION. The work reported in this section involved the analysis of marine animals for  organotin compounds, and the synthesis of standard tetraorganotin compounds which were used as calibration standards for quantitation. The speciation and quantitation procedure involved a prior extraction of the organotin compounds from the marine organisms by the use of methylene chloride. The extracted organotin compounds were reconstituted in n-hexane and subjected to Grignard methylation, a well known chemical reaction to yield tetraorganotin compounds. Speciation and quantitation were by capillary column GC-MS SIM. The high sensitivity and specificity of the mass spectrometric detector especially for tin compounds makes it the detector of choice. Tin has thirteen stable isotopes which in mass spectrometry give rise to a very characteristic isotope pattern. The isotope pattern for tin is very diagnostic for distinguishing tin compounds from other compounds that may co-elute with them during chromatographic separations. The use of the mass spectrometer as a detector for gas chromatographic separations is accomplished by the direct coupling of the GC capillary column to the ion source  of the mass spectrometer.  30 2.2  EXPERIMENTAL  2.2.1 Instrumentation.  2.2.1.1  Gas chromatography (GC). The gas chromatograph  used to establish the retention times, and the  optimum chromatographic conditions for organotin separation was a Hewlett Packard Model 5890 instrument equipped with a flame ionization detector (FID). Data acquisition from the gas chromatograph was achieved by using a Hewlett Packard 3393A integrator. The GC column was a DB-1 polysiloxane stationary phase wall coated open tubular (WCOT) capillary column (15 m x 0.25 mm i.d) purchased from J & W Scientific, Folsom, California. The carrier gas was helium at a linear velocity of 30 cm/s. The column temperature was held at 50°C for 10 minutes, and then increased at the rate of 20°C per minute to a final temperature of 240°C until complete elution was obtained.  2.2.1.2  NMR and mass spectrometry. H NMR spectra were run at 300 MHz by using a Varian XL 300 1  spectrometer. Chemical shifts are quoted relative to tetramethylsilane as external Sn chemical reference. 119 Sn NMR spectra were obtained on the same instrument. 119 shifts are quoted relative to tetramethyltin as external reference. Low resolution mass spectra (using electron ionization, El) for characterizing  31 the calibration standards  were obtained on a Kratos MS 50 mass spectrometer.  Gas chromatography-mass speciromeiry (GC-MS).  2.2.1.3  The GC-MS system consisted of a Carlo-Erba Fractovap series 4160 gas  interfaced  chromatograph  to a Kratos MS 80 RFA double focusing mass  spectrometer equipped with a Kratos DS55 data system. The mass spectrometer was operated in El under selected ion monitoring mode (SIM), and was calibrated by introducing perfluorokerosine into the ion source. The operating conditions for the mass spectrometer were; calibration range =  1 second, filament current  temperature  =  =  =  118-331, sweep  1-2 Amp., electron voltage  =  1500 ppm, cycle time  =  70 eV., ion source  180 °C.  The gas chromatograph was operated in the temperature programming mode. The temperature of the column was kept constant at 50 °C for 10 minutes, and then increased to 240 °C at a rate of 20 °C per minute until complete elution was obtained. The injector temperature was maintained at 250 °C. The GC column is as described in section 2.2.1.1, and was connected to the mass spectrometer via a capillary interface. The carrier gas was helium at a linear velocity of 30 cm/s.  2.2.1.4  Mechanical shaker and blender.  The mechanical shaker employed during the extraction step of the organotin compounds from environmental samples was a Magniwhirl reciprocating shaker Blue M Electric Company, Blue Island, Illinois, U.S.A.).  32 The blender used to homogenize the biological samples was obtained commercially.  2.2.2 Materials and reagents. Dibutyltin dichioride and tributyltin chloride were purchased from M & T Chemicals Inc., Rahway, New Jersey, and Ventron (Alfa Inorganics) Beverly Massachusetts,  U.S.A. respectively. Tin metal (20 mesh) was obtained  from  Mallinckrodt Chemical Works, St Louis, Missouri, U.S.A.. Tricyclohexyltin chloride (Technical Grade) and methylmagnesium bromide (3M in diethyl ether) were obtained from Aldrich Chemical Company, Milwaukee, U.S.A.. Dicyclohexyltin dibromide  was purchased  from Johnson  Matthey  (Alfa products),  Danvers,  Massachusetts, U.S.A. .The following chemicals silica gel (230-400 mesh) and sodium chloride (Reagent Grade) were purchased from BDH, Poole, England. lodobutane d9 and 2-ethoxyethanol were supplied by Merck Frosst Canada Inc. (MSD isotope division) Montreal, Canada and Matheson, Coleman and Bell. Manufacturing Chemists, Norwood, Ohio, U.S.A. respectively. The following reagents and solvents were procured from Fisher Scientific, Fair Lawn, New Jersey, U.S.A :- methyl iodide (Certified Grade), anhydrous magnesium sulfate (Certified Grade), anhydrous diethyl ether (Reagent Grade), n-hexane (HPLC Grade), n-pentane (Spectrograde), n heptane (HPLC Grade).  33 2.2.3 Synthesis of standard organotin compounds.  2.2.3.1  SnCH ) 9 H 4 (C . Synthesis of tributylmethyltin, 3 Tributyltin chloride (2.99 g, 0.0092 mol) was dissolved in n-hexane (100 mL)  in a 250 mL Erlenmeyer flask. Excess methylmagnesium bromide (6 mL of 3M, 0.0 180 mol) in ether was added to the reaction mixture and stirred for six hours at room temperature, after which the excess methylmagnesium bromide was destroyed by the gradual addition of sulphuric acid (1 M) while the reaction flask was cooled in an ice bath. The reaction mixture was transferred to a separatory funnel where the aqueous layer was removed, and the hexane layer was washed five times with hydrochloric acid (10 mL of 10% HC1), dried with anhydrous sodium sulphate and filtered into a round bottom flask (250 mL). The hexane was removed on a rotary evaporator to yield the crude product which was distilled twice at reduced pressure to obtain 1.70 g (61% yield) tributylmethyltin, b.p 58°C I 10 mm Hg. Analysis: % Found: C, 51.40; H, 9.80. % Calcd: C, 51.18; H, 9.91.  2.2.3.2  Sn(CH 9 H 4 (C . 2 ) Synthesis of dibutyldimethyltin 3  Dibutyltin dichloride (1.46g, 0.0048 mol) and excess methylmagnesium bromide (15 mL of 0.045 mol) were reacted for six hours as described in Section 2.2.3.1 to yield a crude product which was distilled twice to obtain 0.9 g of dibutyldimethyltin (71% yield), b.p 33°C I 4 mm Hg. Analysis: % Found: C, 45.83; H, 9.30. % Calcd: C, 45.67; H, 9.20.  34 2.2.3.3  SnCH ) 1 . 13 H 6 Synthesis of tricyclohexyimethyltin (C  Tricyclohexyltin chloride (2.02g. 0.0050 mol) and excess methylmagnesium bromide (10 mL of 3M, 0.030 mol) were reacted for 12 hours as described in Section 2.2.3.1. The crude product was distilled to obtain 1 g tricyclohexylmethyltin (52% yield), b.p 128°C / 0.6 mm Hg. Analysis: % Found: C, 59.93; H, 9.47. % Calcd: C, 59.56; H, 9.47.  13 H 6 Sn(CH 1 . 2 ) Synthesis of dicyclohexyldimethyltin (C  2.2.3.4  Dicyclohexyltin dibromide (2.00g. 0.0045 mol) and excess methylmagnesium bromide (10 mL of 3M, 0.030 mol) were reacted for 12 hours as described in Section 2.2.3.1. The  obtained  crude  product  was  distilled  to  obtain  0.87g  dicyclohexyldimethyltin (61 % yield), b.p 88°C / 0.06mm Hg. which was identified by mass spectrometry and NMR spectrometry (Section 2.4.1).  2.2.3.5  Synthesis of the internal standards.  2.2.3.5(a)  n. (C S 4 ) 7 H Synthesis of tetrapropyltin 3  Tetrapropyltin  was synthesized according to the method described for  , by reacting magnesium turnings (8.26 g, 0.34 mol), n-propyl bromide 159 tetraethyltin (46.74 g, 0.38 mol) and tin(IV) chloride (13.81 g, 0.053 mol) in anhydrous diethyl ether. The obtained crude product was distilled twice to yield 8.5 g tetrapropyltin, b.p 108°C / 11 mm Hg. Analysis: % Found: C, 49.61; H, 9.76. % Calcd: C, 49.52; H,  35 9.70.  2.2.3.5(b)  Direct synthesis of 2 SnI and the subsequent 3 ) 9 H 2 4 SnI and (C ) 9 H 4 (C  synthesis of deuteriated  internal  standards  1 2 4 (C S 3 nCH L)  and  Sn(CH 9 H 4 (C . 2 ) 3 This synthesis was carried out according to the method reported by Oakes and ° for dibutyltin diiodide. Deuteriated butyliodide (3. 13g, 0.017 mol) and 216 Hutton ethoxyethanol (1 mL) were mixed together in a round bottom flask (50 mL). Lithium (0.097g, 0.014 mol) and tin metal (0.89g, 0.0075 mol) were also added to the round bottom flask. The contents of the flask were refluxed for two hours with stirring, and SnI ) 9 H 4 (C vacuum distilled to obtain 0.77 g of a mixture of the crude products 2 (71% yield) and (C SnI (29% yield) as identified by GC-MS. The crude 3 ) 9 H 2 4 products in pentane were treated with aqueous potassium hydroxide (2% w/v) to precipitate the deuteriated dibutyltin dihydroxide (mlz=286). The pentane solution  was filtered and evaporated under reduced pressure to give deuteriated tributyltin hydroxide as identified by mass spectrometry (mlz=335). Aliquots (0.18 g) of the deuteriated  tributyltin. hydroxide and dibutyltin  dihydroxide were each reacted with methylmagnesium bromide (0.3 mL, 0.0009 mol) in hexane  as  described  in Section  2.2.3.1  SnCH ) 9 H 2 4 (C to give 3  Sn(CH respectively as identified by GC-MS. 9 H 4 (C 2 ) 3  and  36 2.2.3.6  Synthesis of methylmagnesium iodide.  Magnesium turnings in slight excess (O.062g, 1.50 mol) and methyl iodide 10.07g, 1.48 mol) were reacted in anhydrous diethyl ether according to standard 2 ( 161 to obtain the Grignard reagent, methylmagnesium iodide. procedure  2.3  ANALYTICAL PROCEDURE.  2.3.1 Gas chromatography.  2.3.1.1  Establishment of elution profile and retention data.  Appropriate  amounts of the well characterized  standard tetraorganotin  13 H 6 SnCH (C ) 1 , Sn(CH and 1 2 ) 13 H 6 n(CH (C 3 S 9 H 4 (C , 2 SnCH ) 3 ) 9 H 4 (C compounds , n were each dissolved in n-heptane in different volumetric flasks (25 mL) (C S 4 ) 7 H 3 to form the stock solutions (50 tg/mL as Sn). A working solution (5 ig/mL as Sn) of each tetraorganotin compound was prepared in n-heptane from the stock solutions. Aliquots of each working solution (1 J.LL) of each standard tetraorganotin solution (5 gImL as Sn) were injected into the capillary column of the gas chromatograph by using splitless injection.  The retention time of each standard  organotin compound was noted. Aliquots of each stock solution were pipetted into the same volumetric flask (5 mL) to form a mixture of all the standard tetraorganotin compounds (5 jgImL as Sn). This mixture (1 L) was separated on the capillary column of the gas  37 chromatograph. Each of the tetraorganotin compounds was detected and identified on the basis of the earlier established retention times.  2.3.1.2  Suitability of tetrapropyltin as internal standard.  The suitability of tetrapropyltin as internal standard was verified on the tributylmethyltin solutions according to the following procedure:-four sets of tributylmethyltin solutions in heptane were prepared. Each set of solutions contained 2, 4, 6, 8, 10 g/mL (as Sn) tributylmethyltin, and also contained the internal standard tetrapropyltin at only one of the following concentration levels:- 2 ,4,6,50 g/mL as Sn. Each mixture of tributylmethyltin and the internal standard (1 L) was injected into the capillary column of the gas chromatograph and separated. Three replicate injections of each sample solution were made. The peak area ratios of the tributylmethyltin  to the internal  standard  were plotted  against the various  concentrations of the tributylmethyltin solutions (Section 2.4.4).  2.3.1.3  Suitability of 3 SnCH ) 9 H 2 4 (C  and 3 H 4 (C S 2 ) n(CH )  as internal  standards. SnCH and tributylmethyltin ) 9 H 2 4 (C , A mixture of the deuterated compound 3 in heptane was injected into the capillary column of the gas chromatograph. No separation was obtained. Similarly, no separation was obtained for a mixture of Sn(CH and dibutyldimethyltin. Although no separation was obtained on 9 H 4 (C 2 ) 3 the gas chromatographic column, the deuterated butyltin compounds can still be used  38 as internal standards if high instrument sensitivity is not desired, because under conditions of GC-MS SIM, the butyltin compounds and their analogous deuterated compounds co-elute, and are simultaneously detected by the mass spectrometer.  Therefore at a given retention time, only a few number of scans can be obtained for each fragment ion, thereby leading to decreased sensitivity.  2.3.2 Low resolution mass spectrometry: selection of fragment ions used for selected ion monitoring CC-MS. The tetraorganotin  compounds, as  neat liquids were subjected to low  resolution mass spectrometry. The fragment ions, and their intensities were recorded (Section 2.4.2 Tables 2.2 to 2.4). Fragment ions possessing the highest intensities were selected to be monitored in the GC-MS SIM analysis.  2.3.3 GC-MS retention data, calibration curves, and precision. Appropriate  amounts of the well characterized  standard tetraorganotin  SnCH ) 1 13 H 6 Sn(CH and (C 1 2 ) 13 H 6 n(CH (C 3 S 9 H 4 (C , 2 SnCH ) 3 ) 9 H 4 (C compounds, , were each dissolved in n-heptane in separate volumetric flasks (25 mL) to form standard solutions. Appropriate amounts of each standard solution were pipetted into the same volumetric flask (50 mL), and made up to the mark with n-heptane to form the stock solution (5 tgImL as Sn, of each of the standard tetraorganotin compounds). Appropriate amounts of the stock solution were placed into six different volumetric flasks (10 mL) together with aliquots of the tetrapropyltin solution in  39 heptane, so that each volumetric flask contained 0.2 g/mL (as Sn) of the internal standard, and 0.2 to 1.2 tg/mL of a mixture of all the tetraorganotin standards. Each mixture of the standard organotin compounds and the internal standard (1 jL) was analyzed by GC-MS SIM. Only the fragment ions shown in Table 2.7 (Section 2.4.3) were monitored at the retention times shown in Table 2.6. The peak area ratios of the standard organotin compounds to the internal standard were plotted against the standard tetraorganotin concentrations (Section 2.4.5, Figures 2.8 and 2.9) to afford the calibration curves.  2.3.4 Recovery studies. Recovery studies were performed in duplicate at one level of organotin concentration (1.5 tg as Sn). Shrimp (Pandalus tridens) (40 g, wet wt) from a batch whose prior analysis by GC-MS revealed the absence of any organotin compound was homogenized in a blender, and transferred to an Erlenmeyer flask (1 L). The shrimp homogenate was spiked with 1.5 g (as Sn) of each of the following compounds: tributyltin chloride, dibutyltin dichioride, Iricyclohexyltin chloride, and dicyclohexyltin dibromide all dissolved in n-heptane. The solution was vortex mixed and sodium chloride (20 g) was added together with concentrated hydrochloric acid (50 mL) and methylene chloride (100 mL). The resulting slurry was shaken on a mechanical shaker for one hour and filtered through a pyrex glass wool into a separatory funnel (1 L) where the lower methylene chloride layer was removed. The shrimp residue, and the glass wool were returned to the Erlenmeyer flask (1 L). The aqueous layer  40 from the separatory funnel was also returned to the Erlenmeyer flask (1 L) and the extraction procedure repeated twice more. All the methylene chloride layers were pooled together, dried with anhydrous magnesium sulfate and filtered through a Whatman No 1 filter paper into a round bottom flask (500 mL), and evaporated off by using a rotary evaporator to obtain an oily residue which was dissolved in n hexane solution. Aliquots of the shrimp extract in n-hexane (5 mL), and a solution of the internal standard (0.5 mL of 2.2 .tgImL as Sn) and methylmagnesium iodide in diethyl ether (3 mL) were added to the Erlenmeyer flask. The reaction mixture was left standing  with intermittent  shaking  for one  hour,  after  which excess  methylmagnesium iodide was destroyed by the gradual addition of dilute sulphuric acid (1 M) and de-ionized water (about 10 mL). The hexane solution in the Erlenmeyer flask was transferred to a separatory funnel (50 mL) where the aqueous bottom layer was removed, and the upper hexane layer passed through a silica gel column (2 cm x 0.5cm i.d) pre-equilibrated with n-pentane. The hexane solution was drained down the silica gel column, and then eluted with n-pentane (15 mL) into a sample vial. The solution in the sample vial was concentrated down to about 0.2 mL by blowing a gentle stream of nitrogen. About 0.3 mL of n-heptane were added to the sample vial, to bring the total volume of the solution to about 0.5 mL. The heptane solution (1 tL) was analyzed in duplicate by GC-MS SIM.  41 2.3.5 Extraction of the organotin compounds from marine animals. About 2-5 frozen marine bivalves were thawed, and then shucked. Portions of the soft tissue weighing between 18-137 g were placed in a blender together with 100 mL de-ionized water, and homogenized for about 3 minutes. The homogenized tissue slurry was transferred to an Erlenmeyer flask (1 L). Sodium chloride (20 g), concentrated hydrochloric acid (50 mL), and methylene chloride (100 mL) were added into the tissue slurry and mixed by shaking. The tissue slurry was then shaken on a mechanical shaker for one hour, and then extracted and processed as described in Section 2.3.4 above. A flow diagram for the extraction of organotin compounds from marine animals is given in Figure 2.1.  42 Tissue  I Homogenize  lOOmL deionized water 5OmL MCi 20g NaCI lOOmL CR2C1Z Shake for lhr Filter through glass wool Transfer into separatory funnel Remove CE2CI2 layer  Repeat CH2CI2 extraction twice  Pool CHzC1Z layer  CR2 C12 layer  Remove CH2CIZ Re-dissolve In u-Hexane Derivatize to tetraalkyltin  Silica gel clean up  Inject into GC-MS  Figure 2.1  Flow diagram for the extraction of organotin from marine animals.  43 2.4  RESULTS AND DISCUSSION.  2.4.1 Characterization of the standard tetraorganotin compounds.  The standard tetraorganotin  compounds were synthesized by Grignard  methylation of butyltin and cyclohexyltin halides as described in Section 2.2.3. above. The butylmethyltin and the tetrapropyltin compounds afford good elemental analyses. Tricyclohexylmethyltin was characterized by elemental analysis and mass spectrometry (Figure 2.2). Dicyclohexyldimethyltin was characterized by 1 H NMR (Figure 2.3) and mass speetrometry (Figure 2.4). The integrated peak area ratios of the methyl group protons, and the cyclohexyl group protons in the 1 H NMR spectra of dicyclohexyldimethyltin were used to confirm the number of protons on the cyclohexyl  group.  The  number  of protons  on  the  cyclohexyl group  of  dicyclohexyldimethyltin was 22 as expected. In the NMR spectra of the free , all the protons are equivalent, and give rise to a singlet at O = 1.4ppm 162 cyclohexane in CC! . In substituted cyclohexane such as bromocyclohexane, two groups of protons 4 are observed . In dicyclohexyldimethyltin three groups of cyclohexyl protons are 162 observed. The peaks are strongly coupled, and the multiplets observed cannot be explained on the basis of a first order coupling. The cyclohexyl group proton on the carbon atom directly bonded to the tin atom is deshielded and resonates at low field (o=1.8 ppm). Further  characterization  of the butylmethyltin  and cyclohexylmethyltin  Sn NMR (Table 2.1) as this information is not yet compounds was provided by 9  44 available in the literature. From the chemical shift data shown in Table 2.1, it can be inferred that the cyclohexyl group has a more deshielding effect on the tin atom, than the n-butyl group.  Table 2.1  Sn NMR chemical shifts for the slandard organotin compound?. 119 Compound  6 (ppm)  Conc. (M)  SnCH ) 9 H 4 (C 3  -6.128  1.10  Sn(CH 9 H 4 (C 2 ) 3  -2.539  0.58  , 1 H 6 (C S 3 nCH )  -44.378  0.77  H 6 (C S 2 ) 3 n(CH ,,)  -16.393  0.30  Sn 4 ) 7 H 3 (C  -18.072  1.98  Sn NMR was obtained on deuterated chloroform solutions of the 119 a= organotin compounds, and referenced relative to tetramethyltin.  45  + +  c-)  L)  C%1  ‘1-  —  ‘0  ‘0  L.)  c_) 100  219  +  c-) 135  50  +  L)  z  4.  z  I ?  ‘I)  L  ‘0  384  0 100  Figure 2.2  150  200  250  300  350  mi  400  Mass spectra (El) of iricyclohexylmethyltm.  TT  450 MJZ  500  46  6  Figure 2.3  5  4  3  2  1  H NMR spectra of dicyclohexyldimethyltm. 1  0 P PM  47  + N,  + N,  c-)  ‘0  L)  C#•)  100  + r.j N,  + +  N, +  c-)  Cl)  z z  c-)  N, N,  Cl) rj  C,, ci)  C%J ‘0  +  +  L)  ‘0  C,)  L)  ‘0  L) ‘0  C.)  0 100  Figure 2.4  150  200  250  300  Mass spectra (El) of dicyclohexyldimethyltm.  MJZ  350  48 2.4.2 Fragment ions and intensities of the standard tetraorganotin compounds. The standard tetraorganotin compounds were subjected to low resolution mass spectrometry. The molecular ions were of low intensity, and therefore were not used for the GC-MS analysis. The fragmentation involves the loss of butyl or cyclohexyl or methyl groups from the tin atom. The fragment ions and intensities relative to the base peak are given in Tables 2.2, 2.3 and 2.4.  Table 2.2  Major fragment ions of tributylmethyltin.  Standard  M/Z  Fragment ion  compound  Relative Intensity  (% )  SnCH 3 ) 9 H 4 (C  121  4 SnH  79  8 (MW=306)  135  CH S 3 n  100  177  C S 9 H 4 n  65  193  3 S 9 H 4 C nHCH  99  235  Sn 2 ) 9 H 4 (C  12  249  3 S 2 ) 9 H 4 (C nCH  93  291  (C S 3 ) 9 H 4 n  9  306  SnCH 3 ) 9 H 4 (C  0.7  a  =  Molecular weight and all fragment ion assignments are based on  49 Major fragment ions for dibutyldimethyltm and tricyclohexylmethyltin.  Table 2.3  Standard compound  M/Z  Fragment ion  Relative Intensity  (%) n(CH 3 S 9 H 4 (C 2 ) (MW  =  264)  ) nCH , 1 H 6 (C S 3 (MW  =  a  384)  =  121  SnW  35  135  n CH S 3  70  150  n (CH S 2 ) 3  99  177  n C S 9 H 4  14  193  SnHCH 9 H 4 C 3  52  207  n(CH 3 S 9 H 4 C 2 )  100  249  nCH 3 S 2 ) 9 H 4 (C  39  264  n(CH 3 S 9 H 4 (C 2 )  5  121  SnW  17  135  n CH S 3  52  219  1 SnHCH 1 H 6 C 3  100  301  1 nCH 3 1 H 6 (C S 2 )  98  369  n 1 ) 1 H 6 (C S 3  3  384  ,,) nCH H 6 (C S 3  4  0 Sn. 2 Molecular weight and all fragment ion assignments are based on ‘  50 Major fragment ions for dicyclohexyldimethyltin and tetrapropyltin.  Table 2.4  Standard compound  MIZ  Fragment ion  Relative Intensity  (%) ) n(CH 3 , 1 H 6 (C S 2 ) (MW  =  316)  n (C S 4 ) 7 H 3 292)  (MW  a  =  121  4 SnH  13  135  n’ CH S 3  35  150  n (CH S 2 ) 3  94  203  1 Sn 1 H 6 C  5  219  1 SnHCH 1 H 6 C 3  15  233  1 Sn(CH 1 H 6 C 2 ) 3  100  301  1 nCH 3 1 H 6 (C S 2 )  10  316  1 n ) 1 H 6 (C S 3  19  121  SnH  46  135  n CH S 3  9  163  .Sn C H 3  91  207  nW (C S 2 ) 7 H 3  91  249  n (C S ) 7 H 3  100  292  n (C S 4 ) 7 H 3  3  Sn Molecular weight and all fragment ion assignments are based on 120  51 2.4.3 GC-MS elution profile and masses of fragment ions used for selected ion  monitoring. The elution profile of the standard tetraorganotin compounds is shown in Figure 2.5. The retention times and the retention time window (the time frame within which the retention time can vary and still be valid) used for the GC-MS analyses of the organotin compounds are shown in Table 2.5.  Table 2.5  Retention time and retention time window used for (3C-MS SIM  analysis. Compound  Retention time  Retention time window  (mm)  (mm)  3 S 9 H 4 (C 2 ) n(CH  12.42  11.50-13.33  (C S 4 ) 7 H 3 n  13.97  13.33-14.25  SnCH 3 ) 9 H 4 (C  14.60  14.25-15.50  3 1 H 6 (C S 2 ) n(CH 1  16.13  15.50-17.00  1 H 6 (C S 3 ) nCH 1  18.93  17.00-20.00  The fragment ions chosen for GC-MS SIM were selected on the basis of their high intensities in low resolution mass spectrometry (Tables 2.2, 2.3, 2.4). The fragment ions and masses monitored for each organotin compound are shown in Table 2.6.  52  11:29  1  Figure 2.5  13:10  100  14:5 1  Retention Time (min:sec) 18:14 16:33 19:56  200 300 Scan Number  400  500  GC-MS elution profile of the standard tetraorganotin compounds. Peaks a, b, c, d, e, correspond to dibutyldimethyltin, teirapropyltin, tributylmethyltin, dicyclohexyldimethyltin, and tricyclohexylmethyltm respectively.  53 For each fragment ion, masses corresponding to tin 116, 118 and 120 isotopes were monitored (Table 2.6).  Table 2.6  Fragment ions and masses used to detect and quantify each organotin compound in GC-MS SIM. Compound  M/Z monitored  Fragment ion  SnCH 3 ) 9 H 4 (C  193, 191, 189  SnHCH 9 H 4 C 3  n(CH 3 S 9 H 4 (C 2 )  207, 205, 203  n(CH 3 S 9 H 4 C 2 )  ,) .nCH 1 H 6 (C S 3  219, 217, 215  1 SnHCH 1 H 6 C 3  , n(CH 3 H 6 (C S 1 2 )  233, 231, 229  1 Sn(CH 1 H 6 C 2 ) 3  n (C S 4 ) 7 H 3  249, 247, 245  n (C S ) 7 H 3  2.4.4 Suitability of tetrapropyltin as internal standard as studied by using gas chromatography (GC). The use of internal standards eliminates the effect of variations in the instrument’s operating parameters on the analyte. In GC or GC-MS, variations in injection volume, ion source temperature,  carrier gas flow rate, could cause  considerable errors from run to run, if not eliminated by the method of internal standardization. Ideally, compounds used as internal standards should be structurally  54 similar to the analyte and their spectroscopic response should also be similar to that of the analyte. Also, the internal standard should not be naturally present in the analyte. Tetrapropyltin satisfies these conditions closely. No natural source of tetrapropyltin in the marine environment is known, and its gas chromatographic retention time is in the range of the retention times of the analytes (Fig 2.5). This ensures that the internal standard and the analytes experience similar broadening effects on the capillary column. Also, calibration graphs obtained for tributylmethyltin solutions by using different concentrations of tetrapropyltin (2 50 tg/mL as Sn) as -  internal standard, gave high correlation coefficients (Table 2.7).  Table 2.7  Regression data for graphs obtained with various concentrations of  the internal standard tetrapropyltin. Conc. (C Sn 4 ) 7 H 3  Regression equation for  Regression  (/.Lg/mL as Sn)  SnCH ) 9 H 4 (C 3  coefficient  2  Y=0.4964X 0.0924  0.9980  4  Y=0.2514X-0.0521  0.9910  6  Y=0.1659X 0.0322  1.0000  50  Y=0.0190X 0.0023  1.0000  -  -  -  The calibration curves obtained for tributylmethyltin by using different concentrations of the internal standard are shown in Figures 2.6 and 2.7. The error  55 a e I  5.0 4.5  c.) 4.0  (a)  3.5 3.0 0  I  2.5 2.0 1.5 1.0  0.5 0.0  0  2  4  6  8  10  12  Conc. 3 H 4 ( S nCH ,) (ppm Sn)  a  (b)  I Figure 2.6  0  2  4  6  8  10  12  SnCH (ppm Sn) ) 9 H 4 (C Conc. 3 Effect of internal standard concentration on the linearity of calibration curves for tributylmethyltin. Graphs a and b were obtained by using 2 jtglmL (as Sn) and 4 jgImL (as Sn) tetrapropyhin respectively.  56 CiD  (a) c-)  C -.  0  2  4  6  8  10  12  Conc. (C SnCH (ppm Sn) 3 ) 9 H 4 020 CID  0.15  (b)  0.10  C  0.05  L.  0.00 0  2  4  6  8  10  12  Conc. 3 SnCH (ppm Sn) ) 9 H 4 (C  Figure 2.7  Effect of internal standard conceniration on the linearity of calibration  curves for tributylmethyltin. Graphs a and b were obtained by using 6 g/mL (as Sn) and 50 LgImL (as Sn) tetrapropyltin respectively.  57 bars in Figures 2.6 and 2.7 represent the standard error for three replicate determinations. All the calibration graphs for tributylmethyltin obtained by using the various concentrations of tetrapropyltin (2 tg/mL to 50 ig/mL as Sn) as internal standard gave good linearity (Figures 2.6 and 2.7). Thus, the internal standard could be applied in concentrations up to 50 tg/mL (as Sn) without introducing non linearity in the calibration curves. The regression equations obtained for the graphs are shown in Table 2.7. An ideal internal standard would have been an analogous stable isotope of the butyltin or cyclohexyltin compounds because it would be chemically very similar to the analyte. Deuterated tributylmethyltin was examined for use as internal standard in GC-MS SIM, but was found to be unsuitable because it caused a decrease in the instrument’s sensitivity to tributylmethyltin detection as explained in Section 2.3.1.3.  2.4.5 Detection limit, calibration curves, and precision for the GC-MS SIM analysis.  2.4.5.1  Detection limit and calibration curves obtained by GC-MS SIM. The limit of detection was obtained from a plot of the peak area ratio of the  standard tetraorganotin compounds to the internal standard versus the concentration of the standard tetraorganotin  solutions (Figures 2.8 and 2.9). The error bars  represent the standard error for three replicate determinations.  The detection limit defined as the analyte concentration which gives a signal equal to the signal of the blank, plus thrice the standard deviation of the blank, was  58 16 14 12 10  (a)  rJD ‘‘  8 6 4  0.0  0.2  0.4  0.6  0.8  1.0  1.2  Conc. ) 3 S 9 H 4 (C 2 n(CH (ppm Sn) 30 Ci2  25  (b)  20  15  0  10  0 0.0  Figure 2.8  GC-MS  0.2  0.4  0.6  0.8  1.0  1.2  1.4  Conc. (C SnCH (ppm Sn) 3 ) 9 H 4 calibration curves for (a) dibutyldimethyltm  tributylmethyltin.  and  (b)  59 10  8  ci  (a)  ci C  6  Cl)  N  ci  4  © Ct  2  Ct C? I..  Ct C?  0 0.0  0.2  0.4  0.6  0.8  1.0  1.2  1.4  Cone. ) 3 1 H 6 (C 5 2 n(CH 1 (ppm Sn) 4.0 C Cl)  3.5 ci  3.0  ci  (b)  2.5  Cl)  n  I  2.0 ci 1.5  —  C Ct  Z  1.0  CtC? I.. .  0.5  .4  0.0 0.0  0.2  0.4  0.6  0.8  1.0  1.2  1.4  Cone. ) 1 H 6 (C 5 3 nCfJ 1 (ppm Sn) Figure 2.9  GC-MS calibration curves for (a) dicyclohexyldimethyltin and (b) tricyclohexylmethyltin.  60 1 from calculated according to the method previously described by Miller and Mi1ler , at the 1M the calibration curves (Figures 2.8 and 2.9). According to these authors 8 + 3S where Y limit of detection the analyte signal is given by the equation Y=Y 8 is the 8 is the blank signal and S is the analyte signal at the limit of detection, Y standard deviation of the blank. The standard deviation of the blank SB can be calculated as the standard deviation of the y residuals  8 may The blank signal Y  be taken to be the intercept of the graph on the y axis. From the working calibration curves of the analytes (Figures 2.8 and 2.9), the intercept of the graph on the y-axis is obtained, and the standard deviation of the y-residuals  is calculated. Then, the  detection limit is determined (Table 2.8).  Table 2.8  Detection limits for organotin compounds by GC-MS SIM.  Compound  Detection limit (j.ig/mL as Sn)  SnCH ) 9 H 4 (C 3  0.053  Sn(CH 9 H 4 (C 2 ) 3  0.028  13 H 6 (C Sn(CH 1 2 )  0.049  1 1 H 6 (C S 3 ) nCH  0.064  61 Typical calibration curves obtained by the least squares method, for the quantitation of the organotin compounds from environmental samples by GC-MS SIM are shown in Figures 2.8 and 2.9. A linear relationship is obtained over the concentration  Table 2.9  Calibration equations used for the quantitation  of environmental  samples by GC-MS SIM. Compound  Calibration equation  Regression coefficient  SnCH 3 ) 9 H 4 (C  RA  =  4.0378RC  n(CH 3 S 9 H 4 (C 2 )  RA  =  2.9721RC  1 n(CH 3 1 H 6 (C S 2 )  RA  =  1.5812RC  , nCH H 6 (C S 3 ) 1  RA  =  0.6196RC  -  -  -  -  0.1554  0.9944  0.5492  0.9989  0.1093  0.9817  0.1742  0.9685  RA =Relative peak area ratio of organotin compound to internal standard, and is plotted as y-axis. RC =Relative concentrations of organotin compound to internal standard, and is the x-axis.  range studied. The regression equations obtained from a direct plot of the peak area ratios of the tetraorganotin compounds to the internal standard versus the various concentrations of the tetraorganotin compounds were modified to obtain the working  62 calibration equations by substituting RC (concentration ratio of tetraorganotin to internal standard) for X in the general form of a straight line equation Y=MX + C. The resulting calibration equations are shown in Table 29. An obvious characteristic of the calibration curves in Figures 2.8 and 2.9 is the failure of the regression line to pass through the graph’s origin despite various  optimization steps in the GC-MS operating conditions. This phenomenon was very reproducible in calibration curves obtained at various times and consequently is unlikely to affect its use for quantitation. A possible consequence of this effect is the difficulty in attaining a very low detection limit required for trace metal speciation.  Precision of the GC-MS SIM method.  2.4.5.2  The precision of the GC-MS SIM analysis was determined by analyzing replicate injections of mixtures of the standard tetraorganotin compounds and the internal standard dissolved in n-heptane. The precision was determined at two concentrations 0.2 g/mL and 1.2 j.ig/ mL (as Sn). The relative standard deviation for six replicate injections was calculated (Table 2.10). There was no significant difference in precision at the two organotin concentrations as tested by means of a . 163 two-tailed F-test at 5% probability level The sensitivity of the GC-MS SIM method as shown in the slope of the calibration  graphs  (Table  2.9)  follows  the  order  tributylmethyltin  dibutyldimethyltin > dicyclohexyldimethyltin > tricyclohexylmethyltin.  >  63 Table 2.10  Precision for six replicate injections of organotin compounds as determined by (JC-MS SIM. Precision (RSD %)  Compound  0.2ig/mL as Sn  l.2g/mL as Sn  SnCH ) 9 H 4 (C 3  5.4  8.1  Sn(CH 9 H 4 (C 2 ) 3  9.4  10.5  1 H 6 (C S 2 ) 3 n(CH 1  11.1  10.0  , 1 H 6 (C S 3 nCH )  14.8  7.4  2.4.6 Recovery studies on the extraction procedure. Recovery studies of the organotin compounds were carried out at the concentration level of 1.5 tg (as Sn) per 40 g (wet wt) of shrimp as described in section 2.3.4 above. The extraction procedure affords good recoveries for tributyltin, dibutyltin and dicyclohexyltin species  (Table  2.11).  The detection  of the  tricyclohexyltin species was hampered by the elution of other unidentified compounds from the sample matrix in its retention window (Fig 2.10). Thus in Figure 2.10, there is an unidentified peak E, of very high intensity which elutes at the same retention time as tricyclohexylmethyltin and completely masks the peak due to this tin compound. Therefore, the quantitation of tricyclohexyltin was not carried out. The  64 other peaks labelled A, B, C and D on the diagram (Figure 2.10) are due to  dibutyldimethyltin,  tetrapropykin,  tributylmethyltin  and dicyclohexyldimethyltin  respectively. The unlabelled peaks in Figure 2.10 are unknown and their mass spectra do not show tin isotope pattern. The mass spectra for peaks A, C and D (Figure 2.10) are shown in Figures 2.11 and 2.12 (i) and (ii) respectively.  Table 2.11  Recovery of organotin compounds spiked into Shrimp (Pandalus tridens) by extraction with methylene chloride.  Compound  Percentage recovery ± (RSD %)  SnCH ) 9 H 4 (C 3  96.9 ± 2.1  Sn(CH 9 H 4 (C 2 ) 3  99.9 ± 1.6  1 H 6 (C S 2 ) 3 n(CH 1  93.0 ± 9.1  a  =  Relative standard deviation of two extractions, each of two  replicate injections into the GC-MS. RSD = Relative standard deviation.  65  Retention Time (min:sec) 16:33 18:14  200 Figure 2.10  300 Scan Number  Selected ion current chromatogram of standard organotm compounds spiked into shrimp. Peak B corresponds to the internal standard.  207  100  0 100  150  250  200  300  350  400  MJZ  Figure 2.11  Mass spectra of peak A in Figure 2.10. The peak at m/z207 indicates the presence of dibutyltin.  66 100  193  -  (i)  50-  z z  I  I  —  100  I  150  I  I  l  200  I  I  250  I  I  300  I  I  350  MJZ  I  I  I  400  100  (ii)  50  z  0  111111111  —  100  150  200  III  1111111  250  lilt  300  111111111  11111111  400  350 Mu  Figure 2.12  Mass spectra of (i) peak C and (ii) peak D in Figure 2.10 above indicating the presence of tributyltin and dicyclohexyltin species respectively.  67 2.4.7 Organotin concentrations in so me marine organism s of British Columbi a, Cada The marine animal s selected for orga notin analyses consist ed mainly of bivalves molluscs. This was mainly be cause of their availa bility in the marine locations sampled. The analysis of mar ine organisms for orga notin compounds w as carried out with th e following objectiv es: (i)  To obtain an indica tion of the geograph ical spread of organo tin pollution in the coastal areas of British Columbia in terms of dibutyltin, tri butyltin, and the cyclohexyltin compo unds. The latter were of particular interest because dicyclohexyltin spec ies have been foun d in surface microla yers of some British Columbian marine waters, and . bio 1 1ta 7 (ii) To study organotin concentrations in pa rticular species of marine animals, over a period of th ree years. (iii)  To examine organotin concentrations in diffe rent animals from on e particular location to see if any particular anim al has the ability to accumulate organo tin compounds more th an the others. In practice, animal av ailability varied from location to location th ereby making it difficult to obtain data from the same sp ecies of animals in al l locations sampled. The map of the loca tions sampled is show n in Appendix A.  2.4.7.1  Organotin concentra tions in oysters.  The organotin concen trations in the Paci fic oyster Crassostrea gigas from  68 some locations in British Columbia, Canada are shown in Table 2.12.  Table 2.12  Organotin  concentrations  in the oyster Crassostrea  gigas from  some coastal areas of British Columbia. ng/gasSn (Wet weight) Year of  Location  Sn 2 ) 9 H 4 (C  Sn 3 ) 9 H 4 (C  ,) 1 H 6 (C S 2 n I  collection July, 1991  Denman  12.5 ± 1.8  ND  ND  Islandb Sept., 1991  Von Donop  17.3 ± 0.3  7.6 ± 0.3  ND  Inlet, Cortes Island May, 1991  Pendrell  ND  ND  ND  Sound  ND a  =  =  Not detected.  Standard error for two separate sample determinations.  All other  standard errors given are for two replicate injections of one sample. b= Oysters were purchased.  69 Oysters in particular have been shown to be very sensitive to tributyltin . Effects of tributyltin pollution on oysters include shell malformation and 24 pollution retarded growth . Oysters from Pendrell Sound British Columbia (Table 2.12) 22 showed no organotin pollution. Comparative data on the organotin body burden• of oysters from another Canadian location, Fanny Bay, British Columbia  has been  reported by Stewart and Thompson . According to them, the oyster Crassostrea 1M  gigas contained tributyltin and monobutyltin concentrations of 52 ng/g (dry wt as Sn) and 4.6 ng/g (dry wt as Sn) respectively. These organotin concentrations translate to approximately 10.4 ng/g (wet wt as Sn) tributyltin species and 0.92 ng/g (wet wt as Sn) monobutyltin species. No dibutyltin species were detected by the authors”. This shows that Fanny bay is comparable to Von Donop Inlet, British Columbia in tributyltin pollution (Table 2.12). Other organotin data for oysters from other parts of the world are available. Rice Li 165 have reported tributyltin concentrations of 9 ng/g (wet wt as tributyltin cation) or 3.7 ng/g (wet wt as Sn) for oysters from Sarah Creek, and 834 ng/g (wet wt as tributyltin cation) or 341.3 ng/g (wet wt as Sn) for oysters from Kings Creek, Virginia U.S.A.. Tributyltin concentrations of 49.74 189 -  ng/g (wet wt as tributyltin cation) or 20.4 77.3 ng/g (wet wt as Sn) have also been -  1 for the oyster Crassostrea gigas from Coos Bay reported by Wolniakowski et al Estuary, U.S.A.. A comparison of the butyltin body burden for oysters analyzed in this study 165 indicates that (Table 2.12) with the butyltin body burden reported by Rice et al oysters from Von Donop Inlet, British Columbia, contain higher tributyltin levels  70 than those from Sarah Creek, Virginia U.S.A., but less tributyltin levels than oysters from Kings Creek, Virginia, U.S.A.. Concentrations expressed in wet weight are not comparable to concentrations expressed in dry weight, unless appropriate conversion factors are applied. An approximate conversion factor applied in this study for oysters is x p.glg dry wt basis =  5x .tg/g wet wt basis. This conversion factor was arrived at, after freeze-drying  known weight of wet oyster samples. Waldock and Miller 167 have reported tributyltin levels of up to 4.5 ig/g dry weight or approximately (0.37 jg/g wet wt as Sn) for some oysters from England. Rapsomanikis and Harrison have also reported tributyltin levels of 0.027 jig/g (dry wt as tributyltin cation) or 2.2  -  -  1.66  135.9 ng/g (wet wt as Sn) and dibutyltin  levels of 0.012 0.402 tg/g (dry weight as dibutyltin cation) or 2.4 32.9 ng/g (wet -  -  wt as Sn) for some oysters from England. Other tributyltin levels in oysters have been reported by Stewart and de Mora for the Mangrove oyster Crassostrea mordax of Fiji. Tributyltin concentrations in the range 626 to 3180 ng/g (dry wt as tributyltin cation) or 51.2-260.3ng/g (wet wt as Sn) were obtained. In New Zealand, tributyltin concentrations in the range 0.033  -  1.38 g/g (dry wt as tributyltin) or 2.7  -  110.5  ng/g (wet wt as Sn) and 0.049 0.467 tg/g (dry wt as tributyltin cation) or 4.0- 38.2 -  ng/g (wet wt as Sn) have been reported for the oysters Crassostrea gigas and Saccostrea glomerata respectively, for the Tamaki Estuary of New Zealand . Han 169 and Weber 79 have determined dibutyltin and tributyltin concentrations of 840 and 2200 ng/g (dry wt as Sn) respectively for a French oyster sample. By using a dry  71 weight/wet weight conversion ratio of about 0.2 as given by these authors , 9 ’ 7 dibutyltin and tributyltin concentrations  of 168 and 440 ng/g (wet wt as Sn)  respectively were obtained for the French oyster sample. A visual inspection of the oysters’ shells prior to their analysis did not reveal obvious shell malformations. Oysters from the Canadian location Denman Island did not show the presence of tributyltin species, but did show the presence of dibutyltin species which may have originated from the metabolism of tributyltin compounds. The available data in Table 2.12 do not indicate pollution by dicyclohexyltin species.  2.4.8 Spread of organotin compounds in the Canadian environment. The extent of organotin pollution in British Columbia was monitored by sampling various available marine organisms from different locations in British Columbia. The results obtained are shown in Table 2.13 below. The concentrations and species of organotin compounds detected in any one location should be a reflection of the type of industrial activity going on in that environment. By the very nature of introduction of tributyltin compounds into the environment, areas of high boating or shipping activity should show high tributyltin concentrations. Data in Table 2.13 show the occurrence of tributyltin species in a substantial number of locations sampled. An interesting feature of Table 2.13 is the observation that the highest concentration of dibutyltin species 67.3 and 39.6 ng/g (wet wt as Sn) were found in the Blue mussels Mvtilus edulis from Anyox shore, and Kitimat respectively. The highest concentration of tributyltin species (37.3 ng/g wet wt as Sn) was also found  72 Table 2.13  Organotin concentrations, spread and speciation in some marine locations of British Columbia.  Organism  Location and  Conc.(nglg  date of  wet wt Sn)  collection  Blue mussel  Marklane  (Mytilus edulis)  Point, Kitimat  Sn ) 9 H 4 (C 2  Sn 3 ) 9 H 4 (C  Sn 2 ) 11 H 6 (C  39.6 ± 0.6  ND  ND  ND  19.4 ± 0.7  ND  ND  ND  ND  6.7 ± 0.1  14.4 ± 0.4  3.5 ± 0.1  ND  ND  ND  (1990) Soft shell Clam  Dundas Island  (Mya arenaria)  (1990)  Shrimp  Holberg  (Pandalus tridens)  Sound (1991)  Blue mussel  Wreck Beach,  (Mytilus edulis)  Vancouver (1989)  Bentnose Clam  Alice Arm  (Macoma nasuta)  (1989)  Table 2.13 continued on next page.  73 Table 2.13 continued. Location  Sn 2 ) 9 H 4 (C  n (C S 3 ) 9 H 4  n ) 2 , 1 H 6 (C S  & date of  (nglg wet wt  (ng/g wet wt  (ng/g wet wt  collection  as Sn)  as Sn)  as Sn)  Bentnose Clam  Hilton  ND  ND  ND  (Macoma nasuta)  Point,  ND  ND  ND  67.3 ± 8.6  37.3 ± 15.0  21.3 ± 0.1  1.9 ± 0.2  0.7 ± 0.2  ND  ND  7.7 ± 2.8  ND  Organism  Kitimat. (1990) Soft shell Clam  Hastings  (Mya arenaria)  Arm  (1990) Blue mussel  Anyox  (Mytilus edulis)  Shore (1990)  Soft shell Clam  Anyox  (Mya arenaria)  Shore (1989)  Basket Cockles  Anyox  (Clinocardium  Slag shore  nuttallii)  (1989)  74 Table 2.13 contd.  California mussel  Quatsino  (Mytilus  Sound  californianus)  (1990)  ND  9.2 ± 1.5  ND  ND = Not detected in the same Blue mussels from Anyox shore. Blue mussels from Wreck beach, Vancouver, also have moderately high concentrations of tributyltin species (14.4 ng/g wet wt as Sn) when compared to other organisms studied, except the Soft shell clam Mya arenaria from Dundas Island. There appeared to be a tendency for Blue mussels to accumulate relatively higher concentrations of organotin compounds than the other bivalve molluscs studied. The occurrence of dicyclohexyltin species is not widespread. Dicyclohexyltin species were found only in the Blue mussels from Wreck Beach, Vancouver, (Figure 2.13) and Anyox shore. Also interesting, is the absence of dicyclohexyltin species in clams and cockles from the same Anyox location. It seems possible that Blue mussels have greater ability to accumulate dicyclohexyltin compounds than the other bivalves studied. This may indicate the incapability of Blue mussels to effectively metabolize or excrete dicyclohexyltin compounds, thereby suggesting different metabolic pathways between blue mussels and other bivalves with regard to organotin metabolism. If this relation holds true, mussels may become good biological indicators for monitoring cyclohexyltin pollution. An examination of Fig  75  2.13(a), demonstrates the superiority of mass spectrometric detection over most non specific detectors. With non-specific detectors, the dicylohexyltin peak D, could have been discarded as baseline noise. , no 7 ’ 1 Apart from the present work, and an earlier report by Cullen et al concentrations of dicyclohexyltin species have been reported for mussels in British Columbia. However, tricyclohexyltin concentration of 36 ng/g (dry wt as Sn) has . Recently, 170 been found for sediments from Esquimalt Harbour, British Columbia cyclohexyltin species have been reported for environmental samples from St John’s . Other butyltin concentrations in 172 ’ and Spain 7 harbour, New Foundland, Canada’  73 (Table 2.14) for Blue the range obtained in this study have been found by Garrett’ mussels from Nanoose Bay and Wood Bay, British Columbia, Canada. Stallard Li 122  have reported dibutyltin concentrations in the range 0.087 0.169 .ig/g (wet wt -  as dibutyltin cation) and tributyltin concentrations in the range 0.068  -  1.067 j.ig/g  (wet wt as tributyltin cation) for Blue mussels from San Diego bay, U.S.A. (Table 2.14). Higashiyama  et  174 al  have  also  reported  dibutyltin  and  tributyltin  concentrations in the range 0.04-0.54.ig/g, and 0.02-0.24g/g (wet wt as organotin cation) respectively for mussels from Tokyo bay, Japan (Table 2.14). To afford a comparison of the British Columbian mussels with the reported organotin concentrations in Table 2.14, the organotin concentrations for mussels in this study were converted from ng/g (wet wt as Sn), to g/g (wet wt as organotin cation) in Table 2.15. A comparison of organotin data in Tables 2.14 and 2.15  76  100  Retention Time (min:sec) 18:14 19:55 16:32  14:51  13:09  11:29  I  I  I  (a) Cl)  Z50  1  100  500  400 300 Scan Number  200 233  100  (b)  1 F0  0 100 Figure 2.13  150  200  250  I  3Ô0  350  400 MIZ (a) Selected ion current chromatogram of extract from Blue mussel  Mytilus edulis from Wreck Beach, Vancouver. (b) Mass spectra of peak  D, revealing the presence of dicyclohexyltin species.  77 Table 2.14  Some organotin concentrations reported for the Blue mussels Mytilus edulis. .tg/g (wet wt as organotin cation)  Location  Sn ) 9 H 4 (C ’ 2  Sn 3 ) 9 H 4 (C  )Sn 9 H 4 (C 3  Reference  0.169-0.087  0.068- 1.067  0.076-0.257  Stallard  U.S.A San Diego Bay  122 a1 Japan Tokyo Bay  0.04- 0.54  0.02 0.24 -  0.02- 0.12  Higashiyama 17’4 et al  Canada Nanoose Bay  0.002  0.007  0.003  3 ’ 17 Garret  Wood Bay  0.02  0.037  0.003  173 Garret  indicates that the concentration of tributyltin species found in Anyox, British 122 and Higashiyama Li’ Columbia is in the range reported by both Stallard j 74  for San Diego Bay, U.S.A and Tokyo Bay, Japan respectively. The level of tributyltin species present in Anyox British Columbia is higher than the levels in both Nanoose Bay, and Wood Bay British Columbia respectively. The occurence of dibutyltin  78 species in Blue mussels from Markiane point, Kitimat and Wreck Beach, Vancouver without a corresponding occurence of the tributyltin species (Table 2.15)  is  surprising, and may indicate that for Blue mussels, the excretion of dibutyltin species  Table 2.15  Organotm concentrations in the Blue mussel Mytilus edulis converted to pgIg wet wt as organotin cation.  Organism  pg/g Wet wt. (as  and (Location)  Organotin cation) n 24 S 2 ) 9 H 4 (C  Blue mussel  Sn 3 ) 9 H 4 (C  Sn 2 ) 11 H 6 (C  0.08  ND  ND  0.01  0.04  0.01  0.13  0.09  0.05  (Markiane Point, Kitimat) Blue mussel (Wreck beach, Vancouver) Blue mussel (Anyox shore)  is slower than that of tributyltin species. For soft shell clams, the opposite trend was observed because dibutyltin species were generally not detected (Section 2.4.10, Table 2.18).  79 2.4.9 Organotm concentrations in various organisms from the same locations. Marine animals from the same locations were sampled for the presence of organotin compounds with a view to finding their distribution among organisms. The  Table 2.16  Organotm distribution  in marine animals from Camano Sound,  British Columbia. Organism  Butter clam  Year of  Conc. (ng/g as  collection  Sn wet wt) Sn ) 9 H 4 (C 2  Sn 3 ) 9 H 4 (C  Sn 2 ) 11 H 6 (C  1989  ND  ND  ND  1989  ND  ND  ND  1989  ND  7.0 ± 2.6a  ND  (Saxidomus giganteus) Basket cockle (Clinocardium nuttallii)  Soft shell clam (Mya arenaria)  a= Standard error for two determinations, of two replicate injections each. ND = Not detected.  80 organotin distribution in organisms from Camano Sound and Tasu Sound is shown in Tables  2.16 and 2.17 respectively. In Camano  Sound, no dibutyltin or  dicyclohexyltin compounds were detected in the three animals sampled. Only the Soft shell clam Mya arenaria showed the presence of tributyltin species (Table 2.16). The occurrence of tributyltin species in the Soft shell clam, without a corresponding occurrence in the butter clam is surprising, and may indicate different mechanisms of tributyltin detoxification even in different species of the same animal. Of the two animals from Tasu Sound (Table 2.17), the little neck clam showed a much higher concentration of tributyltin species than the Blue mussel. Unfortunately, little neck clams from other locations were not available to study this trend further.  Table 2.17  Organotin distribution in marine anima1 from Tasu Sound, British Columbia.  Conc. ng/g as Sn (wet wt.)  Blue mussel  Sn ) 9 H 4 (C ’ 2  Sn’ 3 ) 9 H 4 (C  12 H 6 (C Sn ) 1  ND  13.2 ± 3.1  ND  ND  177.0 ± 11.9  ND  (Mya arenaria) Native littleneck clam (Protothaca staminea)  81 2.4.10 Distribution of organotin compounds in marine anim2ls studied over a period of three years. The concentration and speciation of organotin compounds in Soft shell clams from Quatsino Sound, British Columbia were monitored for over a period of three years (Table 2.18). An examination of the data in Table 2.18 indicates a very  Table 218  Organotin body burden for Soft shell clams Mya arenaria from Quatsino Sound, British Columbia studied over a period of three years.  Year of collection  ng/g Sn (wet wt) ()a  Sn ) 9 H 4 (C 2  Sn 3 ) 9 H 4 (C  12 H 6 (C Sn ) 1  1989  ND  26.3 ± 0.6  ND  1990  ND  12.8 ± 0.4  ND  1991  ND  •s ±  ND  b 18  a=Standard error for two replicate injections. b=Standard error for two separate determinations significant decrease in the concentrations of tributyltin species with time. Such a very significant decrease in tributyltin concentration could only be possible if the input source of tributyltin compounds in this location is decreasing with time. According  82 re in 1989 the Canadian Government regulated tributyltin compounds 155 Magui to , under the Pest Control Products Act (Canada Department of Agriculture 1989). Under this regulation, the permitted daily release rate of tributyltin species is 4jtg per square centimetre of hull surface. This regulation also prohibits the use of antifouling paints containing tributyltin compounds on vessels less than 25 metres in length. As Maguire these regulations should minimize the environmental impact , surmised by 155 of antifouling uses of tributyltin compounds in Canada. The decreasing concentration of tributyltin species with time as shown in Table 2.18 may represent the impact of the Government’s regulation on the input of tributyltin compounds into the marine environment. An important trend that is observable from Table 2.18 is the absence of dibutyltin species in the clams. Usually dibutyltin species should co-exist with tributyltin species in organisms because dibutyltin species are metabolites  of  tributyltin compounds in animals. This trend may indicate that Soft shell clams generally do not metabolize tributyltin species or that dibutyltin species are very quickly excreted from the clams. This observation is in contrast to the trend found for blue mussels (Table 2.15), where all the Blue mussels analyzed contained dibutyltin species. This observation pQints to different detoxification mechanisms for soft shell clams and blue mussels. The occurrence of organotin species in some remote British Columbian locations such as Anyox, Hastings Arm, Alice Arm, and Tasu Sound is surprising because of the very low boating and agricultural activity in these locations: perhaps, aerial transport of these compounds needs to be considered.  83 CHAPTER 3 EFFECT OF TRIBUTYLTIN CHLORIDE, MONOBUTYLTIN TRICHLORIDE AND TRIM[THYLTIN HYDROX[DE ON THE PERMEABILiTY OF EGG PHOSPHATIDYLCHOLINE  3.1  LIPOSOMES.  INTRODUCTION This chapter describes the effect of some organotin compounds namely  tributyltin chloride, monobutyltin trichioride, and trimethyltin hydroxide on model biological membranes formed by the hydration of egg phosphatidyicholine (EPC) or a mixture of organotin compound and EPC in tris buffer to form liposomes, also known as vesicles. The experiment was originally designed to study the permeation of these organotin compounds through these liposomes. However at the high concentrations of tributyltin chloride and monobutyltin trichioride needed inside the liposomes for their 1 H NMR signals to be observed, the liposomes do not form. Therefore, the approach adopted was to use a molecular probe which is capable of easy permeation through the liposomes, to monitor the effects of low concentrations of organotin compounds on the permeability of these model biological membranes. The compound chosen as a probe was dimethylarsinic acid (DMA). The reasons for choosing DMA as a probe molecule are given below in Section 3.2. In an earlier study  by  Tosteson  tetraphenylarsonium  and  156 Weith  the  probe  ions  tetraphenylboron,  and  were used to study the effect of tributyltin chloride on the  membrane potential of a phosphatidylethanolamine planar lipid bilayer. A decrease  84 of about 70 mV in the intrinsic dipole potential of the planar lipid bilayer caused by tributyltin chloride was observed by Tosteson and Weith . 156 The experimental  technique employed in the present study was NMR  spectroscopy. NMR spectroscopy is well suited for the study of solute or molecular permeation through liposomal membranes, provided the NMR signals of the solute inside and outside the liposomes can be differentiated  from each other. The  differentiation of the outside and inside NMR signals is usually achieved by the use of spectroscopic shift reagents; these reagents are usually first row transition metal complexes and complexes of the lanthanide , 1m elemen ts and are usually added to the sample prior to the NMR experiment. By using NMR spectroscopy the permeation of molecules or solutes can be followed to equilibrium without the intermittent withdrawal of samples from the reaction system. In addition, the technique is capable of providing information on the state of the liposomes, particularly liposome lysis during the experiment, because the NMR signal inside and outside the liposome would collapse into a single peak if the liposome disintegrates or bursts.  3.2  DIMETHYLARSINIC ACID (DMA) AS A PROBE FOR STUDYING THE EFFECT OF ORGANOTIN COMPOUNDS ON THE MEMBRANES OF  LIPOSOMES. The permeation of dimethylarsinic acid (DMA) through EPC liposomes has been studied by Herring ci . DMA has the following properties which make it 176 suitable as a probe molecule for further studies:  85 (i)  DMA permeates across EPC liposomes by passive diffusion  (ii)  DMA has good aqueous solubility which enables high concentrations to be  trapped in the small aqueous volumes of the liposomes. This in turn makes it easy to observe the NMR signals of DMA in the liposomes. Butyltin compounds do not possess high enough aqueous solubility to enable the NMR signals of trapped butyltin compounds to be detected. (iii)  The rate of efflux of DMA from EPC liposomes is slow enough to permit its  study by NMR spectrometry. (iv)  The methyl hydrogen atoms of DMA give rise to a simple NMR spectra  (singlet) which can be shifted by using spectroscopic shift reagents.  3.3  LIPOSOMES AS MODELS FOR BIOLOGICAL MEMBRANES. Biological membranes are made up of two major components, phospholipids  and proteins”’’, and other components such as oligosacchrides . The major barrier 178 to membrane permeability is provided by the phospholipid bilayer. The proteins are inserted into the phospholipids which are oriented in the bilayer. An important property of the phospholipids is the possession of hydrophillic and hydrophobic ends (FiHxre 3.1). WhHx hydrated in aqueous solutions, most phospholipids form closed structures called vesicles or liposomes which possess internal aqueous volumes (intraliposomal compartment) which can be used to trap many compounds (Figure 3.2). The permeability properties of the liposomes are similar to those of biological . The advantage of using liposomes over biological membranes for 79 membranes”  86 permeability studies is that experimental results are easier to interpret because proteins and oligosacchrides, which might otherwise complicate permeation processes  ) 3 N(CH  (a)  CH2 CH2  ci 0 H H—C  2 CH C—H  0  =o  c=o  CH  CH,  CH,  H.  2 CH  2 CH  : :  Figure 3.1  H  H  CH,,  C1-1.  (b) 3 C -CH N 2 H H  Ethanolamine  3 C 2 -CH ) COO H(NH CH(OH) 2 -CH O H CH  (a) Structure of a phospholipid (phosphatidyicholine) commonly occuring head groups on the phospholipid.  Glycerol  and (b) other  87  are absent. Also, the liposomes can easily be prepared  in a controlled and  reproducible manner.  3.4  TYPES OF LIPOSOMES AND METHODS OF PREPARATION.  Various methods  for the preparation of liposomes are available. These  methods of preparation have been a subject of reviews by Hope LJ , and Szoka 180  Qc c5  Figure 3.2  D  Extraliposomal volume  Liposome, showing the intraliposomal compartmentlvolume  where  molecules of a permeant can be encapsulated.  and 18 . Papaha djopol 1 ous  Three types of liposomes namely multilamellar vesicles  (MLVs), small unilamellar vesicles (SUVs), and large unilamellar vesicles (LUVs) have been popularly used and the methods available for the preparation of these  88 vesicles are given below.  3.4.1 Multilamellar vesicles (MLVs). 182 in 1965. His method involved Bangham prepared the first vesicles (MLVs) a gentle dispersion of a lipid in buffer. The vesicles that formed were heterogenous in size, and aqueous volumes of the different lamellae were later reported by Gruner to be depleted in solutes relative to the buffer in which they are made, and as such, are under osmotic compression. MLVs having uniform solute distribution in the lamellae can be prepared by the methods reported by Gruner  j183  and Mayer  et a1. The methods involve the evaporation of ether from an ether-buffer-lipid 183 or the mixture, followed by resuspension of the sonicated emulsion in buffer repeated freezing and thawing of the lipid-buffer preparation. Another method of , involved the dehydration of 85 MLV formation reported by Kirby and Gregoriadis’ lipids from an aqueous solution by using either freeze-drying or direct vacuum evaporation, followed by controlled rehydration. The major draw back in the use of MLVs for permeation  studies is the presence of multilamellae,  and the size  inhomogeneity of the liposomes.  3.4.2 Small unilamellar vesicles (SUVs). Early methods employed for the preparation of SUVs were based on the , the size of the SUVs 1 sonication of multilamellar vesicles. According to Johnson is dependent on the lipid composition, with the vesicle diameter varying from 204  A  89 for egg phosphatidyicholine (EPC) vesicles to 362 A for EPC vesicles containing 50% cholesterol. Preparation of SUVs can also be accomplished by the French press . The very small trapped volumes of the SUVs (<O.2iLper 187 method of Barenholtz tmo1 phospholipid) and vesicle instability are the major draw back to their use in permeation studies.  3.4.3 Large unilamellar vesicles (LUVs). Large unilamellar vesicles (LUVs) are the liposomes of choice for most permeation experiments, and were the liposomes used in the present study because of their unilamellarity and large trapped volumes. The LUVs can be prepared by the the reverse phase evaporation method  ethanol injection method of Kremer ulos’ 89 Papahadjopo of Szoka and ,  the ether injection method of Deamer and  , and the rapid extrusion 191 , the detergent dialysis method of Madden 90 Bangham’ method of Olson  192  Hope  93 LV  and Meyer  The ethanol injection, ether injection, and the reverse phase evaporation methods involve the dispersion of lipids in an appropriate organic solvent, and the subsequent injection into a buffer. The organic phase is evaporated off at the time of hydration for the ether injection method, removed under reduced pressure for the reverse phase evaporation method, or is diluted into the buffer for the ethanol injection method. An additional step involving gel permeation chromatography is usually employed to remove organic solvents. The detergent dialysis method involves the detergent induced solubilization of the lipid into micelles and the subsequent  90 removal of the detergent by dialysis. These methods described above are tedious, and usually entrap residual organic solvents or detergents. The presence of residual solvents in the LUVs is not desirable because it may change the properties of the liposomes. The entrapment of residual organic solvents can be avoided by rapidly . These authors 192 extruding MLVs under low pressure, as reported by Olson et a1 also reported that reverse phase vesicles exhibit greater size homogeneity after low pressure extrusion through a polycarbonate filter. The use of moderate pressure extrusion to produce defined pore sized, unilamellar vesicles from multilamellar vesicles has been reported by Hope Lili 193 and Meyer  3.5  TRANSPORT PROCESSES IN MEMBRANES.  Solute or ionic transport across biological membranes can be described in terms of the following: (i)  Simple or passive diffusion  (ii)  Facilitated diffusion  (iii)  Active transport  A brief description of these transport processes is given in the following sections:  3.5.1 Simple or passive diffusion. Passive diffusion occurs when a concentration  gradient exists across a  membrane. The movement of molecules through the membrane is due to thermal  91 . The direction of transport is determined by the concentration 194 molecular motion gradient, and diffusion is in the direction of lower solute concentration, until concentration on each side of the membrane is equalized. Passive diffusion obeys Fick’s first law:  J--D dX  /s) 2 where J, is the flux (mol/cm /s), D, is the diffusion coefficient (cm 2  ,  dX is the  membrane thickness, and dC/dX is the concentration gradient. In general, the rate of diffusion is determined by the concentration difference across the membrane, the molecular size of the permeant, the viscosity and width of the membrane, and on temperature. In passive diffusion, it is assumed that lipophilic solutes penetrate the membrane by dissolving in the hydrophobic layer and then diffusing across the bilayer, while hydrophilic solutes pass through aqueous pores on the membrane. permeation  This assumption is based on the observation that the rate of  is non-saturable, and permeation  is not inhibited competitively by  . A detailed description of passive diffusion 195 analogous compounds of the permeant . 195 has been given by Heinz  3.5.2 Facilitated diffusion. In facilitated diffusion, the transport of the permeant is aided by the presence of another molecule capable of acting as a carrier or capable of forming channels in  92 the membrane. The direction of transport is along the concentration gradient and . The mechanism of solute transport by facilitated 196 Fick’s first law is not obeyed diffusion is described in terms of the following models:  Solute iranslocation through channels.  3.5.2.1  In this model, the permeant moves across the membrane via channels. Channels are transient pores formed in the membrane by ionophoric substances. The transient pores appear to oscillate between two conformational states. Channels show specificity, for different permeants, and the specificity shown is not related to the size of the permeant. Channels are subject to competitive inhibition.  3.5.2.2  Translocation through carriers. The carrier model postulates that a carrier molecule binds specifically to the  permeant molecule at one side of the membrane barrier, transports it through the barrier, releasing it at the other side. The carrier molecule is able to move freely within the bilayer without leaving it. In situations where the size of the carrier exceeds the thickness of the lipid bilayer (30-50  A),  it has been suggested that the  whole carrier molecule does not move but only a loose chain or part of the carrier . 95 swings from one side of the membrane to the other, releasing the bound permeant’ , 94 A detailed description of facilitated diffusion has been presented by Hofer’ . A schematic diagram of the various steps involved in 197 , and Stein 195 Heinz facilitated diffusion is shown in Figure 3.3.  93  Lipid bilayer  -“-B  AB  A is the permeant B is the carrier molecule AB is the carrier-permeant complex Figure 3.3  Schematic diagram of facilitated diffusion (efflux) mediated by a carrier.  3.5.3 Active transport. In active transport, a permeant is moved across the membrane by a carrier molecule usually a protein against the permeant’s electrochemical potential gradient. The energy required for this process is provided by ATP hydrolysis, or electron flow connected with some redox reactions in the cell . A detailed description of active 196 transport has been given by 194 Hofer and Stein , . 197  94 3.6  SOLUTE TRANSPORT ACROSS LIPOSOMAL MEMBRANES.  3.6.1 Transport of solutes across membranes. When there is a difference in the electrochemical potential of a solute on both sides of a membrane, there will be a net diffusion of molecules of that solute across the membrane. This situation is represented as follows: -  -  0  where p. the chemical potential of the solute is given by the following, *+RT a  where a, is the activity of the solute,  E  +  ZFeV  +  Vp  is the charge on the electron, p is the applied  pressure, V is the volume of the solute, p. is the chemical potential of the solute in its standard state, ‘F is the electric potential, and F is the Faraday’s constant. If the  applied pressure and the electric potential are equivalent on both sides of the membrane, any observed chemical potential difference across the membrane is due to unequal activity of the solutes on either side of the membrane. The net flux of a solute across a membrane has been described by Stein , 197 and is given below:  J- —D— 8X  ) 2 where J (mol/s/cm  -  AC -D— AX  is the flux, D (cm /s) is the diffusion coefficient in the 2  95 membrane, and AX is the membrane thickness. The ease of permeation through a membrane is described in terms of permeability coefficients. For non-ionic solutes, there is a strong correlation between  the permeability coefficieHx for the transport of the solute across a lipid bilayer and . This observation is known as Overton’s rule. 198 the hydrophobicity of the solute A schematic diagram liposomal  lipid  membrane  for the permeation of a non-ionic solute across a from  the  intraliposomal  compartment  to  the  extraliposomal compartment is shown in Figure 3.4. As the solute permeates, it also partitions between the lipid bilayer and the aqueous phase. An equation that relates the permeability of a solute to its partition coefficient has been derived by Jain, and is given below;  AX where K is the partition coefficient of the solute in the bilayer (Figure 3.4), P (cm/s) /s) 2 is the permeability coefficient, AX (cm) is the membrane thickness and D (cm is the diffusion coefficient of the molecule in the membrane. The kinetics of this permeation process is described by the following rate constants: (i)  . 1 The rate constant for diffusion to the lipid bilayer k  (ii)  The rate constant for diffusion in the lipid bilayer k”.  (iii)  . 2 The rate constant for diffusion away from the lipid bilayer k The rate of diffusion in the bilayer is the slowest step and is therefore the  rate determining step.  96  Extraliposomal compartment  (a) 2 k  rout  —  1 r  =  AX  I  (b)  /  Figure 3.4  Passive diffusion of a permeant HA across a liposomal membrane  showing:- (a) the various permeation rate constants and (b) partitioning of the  permeant as it diffuses across.  97 The permeation of non ionic solutes is influenced by such factors as molecular 201 and hydrogen bonding capability ’ 200 size , 203 have . Orbach and Finkelstein 202 suggested that the effect of molecular size and hydrogen bonding capability is less important than the hydrophobicity of the molecule.  3.6.2 Transport of ions. In general, the flux of ions across lipid membranes is much lower than the flux of non ionic molecules. The permeability of water and ionic solutes across lipid membranes has been reviewed by Deamer and Bramhall’ . 79 Attempts to explain the low permeability of ions to lipid membranes were complicated by the observation that anions permeate lipid bilayers easier than cations. According to Hauser Li , at pH 5.5 the first order rate constant for the 204 escape of chloride ions from small unilamellar vesicles was three orders of magnitude higher than the rate constant for sodium ions. Many theories have been put forward to explain this difference  in cation and anion permeabilities.  According to  , the major energetic barrier to membrane transport of ionic solutes is 205 Persegian the Born energy: defined as the energy of an ion in an environment with a given dielectric constant . 205 Flewelling and Hubbel 206 have proposed a mechanism to account for the permeability  difference across lipid bilayers observed for cations and anions.  According to them, the observed permeability differences could be accounted for if other contributions to the Born energy such as image energy, dipole energy, and  98 neutral energy are considered. Their energy model produced reasonable agreement between the observed and calculated thermodynamic parameters for the translocation of tetraphenyiphosphonium cation and tetraphenylboron anion in a lipid bilayer. The image energy arises from the interaction between the charge of an ion in the lipid bilayer and the interfaces. The dipole energy arises from a two dimensional array of point dipole sources located at each membrane surface. This dipole source is believed to originate from the ester linkages of the fatty acid chain of the lipid bilayer. These dipoles give the interior of the bilayer a positive potential of several hundred millivolts. This has the effect of increasing the permeability of anions, and decreasing the permeability of cations. The neutral energy takes into account the non-electrical interactions between a permeant and the membranes. Such nonelectrical interactions include hydrophobicity, and steric factors. However, the Born energy considerations are not able to explain the anomalously high permeability of protons and hydroxyl ions when compared to other small monovalent ions. On the agreement  molecular mechanism of solute and ion transport, no general  has been reached. No one model has satisfactorily explained the  permeation of ionic solutes. For lipophilic solutes, permeation is explained in terms of the solubility-diffusion model, whereby the solute is thought to dissolve in the non polar region of the bilayer and cross the bilayer by simple diffusion. For hydrophilic solutes, permeation is usually explained in terms of diffusion through aqueous pores in the bilayer, or by permeation directly through the lipid bilayer via transient defects which occur in the bilayer as a result of thermal fluctuations.  99 3.7  PROPERTIES  OF  LIPOSOMES  CAPABLE  OF  YIELDING  INVESTIGATWE INFORMATION.  The properties of liposomes that can be used to study the effect of other compounds on the lipid bilayer are its material properties such as permeability, partition coefficient, electrical properties, elastic properties and gel to liquid (L-L) transitions. The material properties of the lipid bilayer have been described by . These material properties are a function of the vesicle composition, and 207 Gruner as such, any interaction of a “foreign” compound such as an organotin compound with the vesicle would exert some effects on these properties. This has been found to be true experimentally. Tosteson and Weith’ 56 have determined that tributyltin chloride affects the internal potential of a phosphatidylethanolamine planar lipid membrane, lowering its dipolar potential by 70 mV. In the present study, bilayer permeability was the material property of choice to be used for studying the effect of tributyltin chloride, monobutyltin trichioride and trimethyltin hydroxide on the permeability of egg phosphatidyicholine liposomes by using dimethylarsinic acid (DMA) as a permeability probe.  3.8  BUTYLTIN COMPOUNDS: THE NEED FOR THE PRESENT STUDY. 208 on the effect of the triorganotin ’ 15 The pioneering work of Selwyn et al  compounds on membranes has shown that the organotin compounds (trimethyltin, triethyltin, tripropyltin, tributyltin and triphenyltin species) partition into the membranes of mitochondria, liposomes, erythrocytes, and chloroplasts, and mediate  100 chloride-hydroxide transport across the membrane.  17 found that Motais et a1  tripropyltin chloride is able to act as carrier and mediate chloride-chloride and chloride-hydroxide exchanges in red blood cells. Although the ability of the triorganotin cations to act as carriers for CF and OW in the membrane has been established, the effect of the organotin compounds on the other properties of the membrane has received very little attention. Heywood 209  have provided evidence that tributyltin cation associates with the phosphate  head groups at the surface of liposomes. Such interactions are expected to modify the membrane properties of the liposomes. Alteration of membrane properties by tributyltin compounds is suspected to be responsible for the in vitro inhibition of . 157 2 mobilization reported by Arakawa cAi intracellular Ca 16 have reported that the tributyltin cation preferentially Tosteson and Weith 3 across a planar lipid bilayer. In the environment, the transports CF over N0 toxicity of other pollutants may be enhanced, if tributyltin cation preferentially mediates their diffusion across biomembranes. Hence, the present study aims to investigate the effect of organotin compounds on the permeability of biomembranes,  and any possible transport  mediating ability of the organotin cation on a probe permeant; dimethylarsinic acid which is also an environmentally occuring compound.  101 3.9  ThEORETICAL DESCRWrION OF THE DIFFUSION EXPERIMENT APPLICABLE TO NMR SPECTROMETRY.  3.9.1 Passive diffusion During the efflux experiments, as the DMA diffuses out of the vesicles, the intensity of the DMA peak inside the liposomes decreases, while the intensity of the  DMA peak outside the liposomes increases. A mathematical description for the first order efflux of DMA from egg phosphatidyicholine liposomes by passive diffusion has . The 210 been derived by Herring et aV , and has also been given by Nelson 76 mathematical equation describing the exponential decay of the DMA peak integral to equilibrium value is given below.  -  +  (I  -  1’)exp-{(1 .i-f)kt} [3.0]  I,  I° are the integrals of the DMA peak inside the liposome at time t,  equilibrium, and zero time respectively. Zero time refers to time before spectral acquisition; f, is the volume ratio of the intraliposomal  compartment  to the  extraliposomal compartment (i.e, 0 /V 1 f=V ) ; k is the observed rate constant for DMA efflux from the liposome and t, is the efflux time in seconds.  3.9.2 Facilitated diffusion. Another transport mechanism which was considered because some of the experimental data obtained in the present study did not fit equation [3.0], is  102 facilitated diffusion. In facilitated diffusion, the diffusing molecule enters into some form of reversible chemical association or complexation with a carrier molecule which transports it across the membrane. In solution at pH 7.4, the probe molecule dimethylarsinic acid (DMA) exists : the undissociated dimethylarsinic acid (DMAH), and the 210 as two chemical species  anionic species DMK. Both species are capable of passive diffusion in the membrane, but only DMK is likely to be transported by triorganotin cation during facilitated diffusion. Therefore, in this study, DMA refers to a mixture of DMAH and DMK present in solution. A proposed schematic diagram of tributyltin cation acting as a carrier is shown in Figure 3.5. In this scheme, DMA diffuses into the lipid bilayer where it associates with the tributyltin cation to form a tributyltin-DMA complex which is mobile within the lipid bilayer. At the interface of the liposome and the extraliposomal compartment, the tributyltin-DMA complex dissociates to liberate the DMK into the extraliposomal compartment. A theoretical treatment for facilitated diffusion based onHxhe adsorption ’ to account for the 2 2 has been described by Widdas equilibria of Langmuir . 3 placental diffusion of glucose, and by Hall and Baker  The theoretical  ’ is further developed below for 2 treatment by Widdas  trialkyltin cation mediated transport of DMK across a liposomal membrane. (a)  DMK can associate with the tributyltin cation which is the carrier to form a tributyltin-DMA complex.  103 Lipid bilayer Intraliposomal compartment  DMA  +  Extralqx)somaJ compartment  HSn 4 (C  HSn 4 (C  + DMA  —  1 K  H,)SnDMA 4 (C  1 k  B  HSnDMA 4 (C  kT  HSnDMA 4 (C  HSnDMA 4 (C  SnDMA. 3 ) 9 H 4 2 is the formation rate constant for (C k  =  1 k  —  =  SnDMA. 3 ) 9 H 4 2 is the dissociation rate constant for (C k  =  SnDMA. 3 ) 9 H 4 k/k is the equilibrium dissociation constant for (C  =  SnDMA. 3 ) 9 H 4 1 1k.. k 1 is the equilibrium formation constant for (C  B/ is the ratio of equilibrium formation constant to equilibrium dissociation constant of the carrier-permeant complex. K- is the partition coefficient of the carrier-permeant complex in the interface between the intraliposomal compartment and the bilayer. 0 is the partition coefficient of the carrier-permeant K  complex in the  interface between the extraliposomal compartment and the bilayer. kT and kT are the transfer rate constants  SnDMA 3 ) 9 H 4 of (C  to the  extral iposomal and intral iposomal compartments respectively.  Figure 3.5  Proposed mechanism of tributyltm mediated efflux of dimethylarsinate (DMA) from a liposome and the equilibria of the carrier-permeant interactions.  104 (b)  The tributyltin-DMA complex travels through the membrane and upon reaching the other surface dissociates and liberates the DMA  into the  adjacent medium. The free organotin cation returns to its original position may be, with a different anion in solution to start a new permeation cycle. (c)  The carriers are in equilibrium with the substrate at the interfaces.  (d)  The carriers pass backwards and forwards across the liposomal membrane.  (e)  The rate of transfer of the carrier-substrate complex is much smaller than the rate of formation and dissociation of the complex, and is the rate limiting step.  (f)  The net rate of transfer is proportional to the difference in the fraction of saturated carriers on both liposomal membrane interfaces. According to Langmuir , the adsorption equilibrium at any interface can be 211  expressed as:  -c 13 4)  —  13C÷4)  [3.1]  -c+i  4)  where  e  is the fraction of tributyltin saturated with DMK. C, is the concentration  of DMK in solution at the interface,  is the dissociation constant of the carrier  permeant complex, and B is the formation constant of the carrier-permeant complex. According to Widdas , equation [3.11 assumes the form of the expression of 212 214 concentration of enzyme combined with substrate in Michaelis-Menten’s equation 212 considered for enzyme kinetics (Appendix C). Therefore, Widdas  ,  analogous to  3 considered the dissociation ’ 2 the Michaelis-Menten’s constant. Also, Hall and Baker  105  constant of the carrier-permeant complex as a Michaelis-Menton’s constant. The rate of disappearance of the tributyltin-DMA complex at the interface between the lipid bilayer and the intraliposomal compartment (mi), is given by the expression:  -  an -  +  kO  [3.2]  —  where n, refers to the number of moles of DMA bilayer and the intraliposomal compartment. erni, and saturated  in the interface between the  e refer  to fraction of carriers  with DMK at the inside and outside interfaces of the liposome  respectively, and kT and k..T are the transfer rate constants of the tributyltin-DMA complex towards the outside and inside interfaces of the liposome respectively. Substituting equation [3.1] into equation [3.2], the rate of disappearance of the carrier-permeant complex at the inner interface (mi) becomes:  j-c  c -j 3  [331  Tp  +1  prni  If the concentrations Cmi and C are small such that (i3I)C <<1, equation [3.3] becomes  :-  an. -  +  k.TC)  [3.4]  13/ is the ratio of formation constant to dissociation constant for the tributyltin  106 DMA complex. Re-writing equation  [3.41 in terms of moles of carrier-permeant complex:  .S.  {kT(.?L)  +  kT(!L.)}  [5]  where Vmj and V refer to the volumes of the inner and outer interfaces of the liposomes respectively, n and n refer to the moles of carrier-permeant complex in the inner and outer interfaces respectively. C.  nV fl:Vnn  -  [3.6]  Where K. is the partition coefficient of the carrier-permeant complex in the inside  interface between the liposome and the intraliposomal compartment. Therefore,  ‘mi -  Icnvm, V  [3.71  Also,  -  -  mo  oIt mo  out  Therefore, KV —  I,  out  [3.8]  107 Substituting equation [3.7] and [3.8] into equation [3.5], and simplifying the resulting equation, the rate of disappearance of the carrier-permeant complex at the inner interface (rate of decrease of the DMK NMR signal inside the liposome) becomes:  an at  If k  =  k.T  =  n nI Ji_JTKin —a  V “  -  +  içv p  k, K 1  k-TK  a —  [3.91  and Vmj c V, equation [3.9] becomes:  =  nm.  an. -  V kZJ1’  l  n +  0 v  [3.10]  N, the total number of moles of DMA in both the intraliposomal and extraliposomal volumes, and the outer and inner interfaces is given by the equation: N=nin+no,#+nmj+n,,  0 because of the very But, n and n are very small when compared to nir. and n small volumes of the inner and outer liposomal interfaces. Therefore, 1 -N-n 0 n [3.11]  Substituting equation [3.111 into equation [3.10] and simplifying,  108  —  ãt  (N  +  V. k1--  [3.12]  Vout  Lfl4,lvUI  Simplifying equation [3.12],  Ni  i  1  -_Vk13f &  (  +  ç  +  [3.13]  Let,  [3.14]  V  1’  Substituting equation [3.14] into equation [3.13],  aflfl —  at  -  Vk  / N nV+—  vj  [3.15]  V.UI k--f&  [3.16]  Re-arranging equation [3.15] and integrating,  —  vi out,  ---  ‘  In  Ini’n V’  -  —-I] V OUt  -  t 1 V a k-  4)  [3.17]  109  in  [3.18]  —V’V-.kt  —  Re-arranging equation [3.18], [3.19]  k--t exp_ {v’v.n4f  -  N  Let V’ also be: 1+f  VI-  [3.20]  Where f is the volume ratio of the intraliposomal to the extraliposomal volumes (f= V N ) 0 Substituting equation [3.20] into equation [3.19] and re-arranging the resulting equation,  (1+f)  —  vin  (  N  j7  out  -  11  112jIo  +  N “ Iexp  f  J  —  v)  -  1 +f  {(i)  v  p  f  [3.211  At equilibrium,  t  (n(  1+!)  r  -,  N Vout  ,  fin  eq —  in  1+f  )  [3.22]  ckt}  Therefore the expression for N in equation [3.21] becomes,  -,  0  110 [3.23] N-  eq  Vin )I  Substituting equation [3.23] into equation [3.21] and simplifying,  -  -  (n  -  {( 1  n) exp  —  e n ) 1  _{(  +  f)  +  f )kt}  -  kt}  [3.24]  Re-arranging equation [3.24],  —  +  (n  —  1  [3.25]  The peak area of the proton resonance in ‘H NMR is directly related to the number of particles, therefore equation [3.25]can be re-written in terms of the integral of the methyl resonance of the DMK  -  When there is no facilitated diffusion, there is no B or  therefore, equation  [3.26] reduces to equation [3.0] for passive diffusion previously derived by Herring . Equation [3.26] predicts the exponential decay of the DMK resonance 76 et al’ inside the liposome with time, and shows that facilitated diffusion is controlled by the ratio of the formation constant to the dissociation constant B/ for the carrierpermeant complex.  111 3.10  EXPERIMENTAL  3.10.1 Instrumentation  3.10.1.1  Nuclear magnetic resonance spectromeiry (NMR).  A Bruker AM400 NMR spectrometer was used to obtain all NMR spectra. NMR facilities were provided by Professor F.G. Herring. The spectrometer was operated in the water suppression mode. The 5mm NMR tubes used for all the experiments were obtained from Norell Inc., Landisville, New Jersey, U.S.A.. The operating parameters that were used for spectral acquisition and water suppression are given in Appendix B.  3.10.1.2  Lipid extruder and membrane filters.  The lipid extruder used to produce unilamellar vesicles (liposomes) was provided by Professor F.G. Herring, and was purchased from Lipex Biomembranes Inc., Vancouver, Canada. The 200 nm pore-sized polycarbonate filters used with the extruder were purchased from Costar Corporation, Cambridge, Massachusetts, U.S.A..  3.10.1.3  UV-Visible spectrophotometry.  A Shimadzu 600 spectrometer was used for all phosphorus assays. All measurements were taken at 815 nm.  112 3.10.2 Chemicals and reagents. Tributyltin chloride was purchased from Ventron (Alfa Inorganics) Beverly, Massachusetts, U.S.A.. Monobutyltin trichioride and deuteriated 3-(trimethylsilyl) propionic acid sodium salt 2,2,3,3-d4 (TSP) were procured from Aldrich Chemical Company, Milwaukee, U.S.A.. Dimethylarsinic acid (DMA) was obtained from Fisher  Scientific  Company,  Tris(hydroxymethyl)aminomethane  Fairlawn,  New  Jersey,  U.S.A..  hydrochloride (tris buffer) and c-D(+)-glucose  were purchased from Sigma Chemical Company, U.S.A.. Egg phosphatidylcholine (EPC) was procured from Avanti Polar Lipids, Birmingham, Alabama, U.S.A.. Solutions of the tris buffer were prepared by dissolving appropriate amounts in de ionized water and adjusting the pH to 7.4 with sodium hydroxide. All solvents used for lipid extraction were Spectrograde. A stock solution of DMA (25 mg/mL) was prepared in tris buffer (300 mM), and its pH was adjusted to 7.4 with sodium hydroxide solution. The organotin compounds were freshly dissolved in tris buffer (40 mM), and their pH was adjusted to 7.4 with sodium hydroxide solution if necessary.  3.10.3 Preparation  of  large  unilamellar  vesicles  (LUVs)  from  egg  phosphatidyicholine (EPC) and the encapsulation of dimethylarsmic acid. A stock solution of EPC was prepared by dissolving EPC (1 g) in chloroform (10 mL). This stock solution was stored in the freezer until needed. The stock solution (2 mL) was pipetted into a test-tube, and the solvent was evaporated off by using a gentle flow of nitrogen gas, and then the resulting paste was dried for 3  113 hours on a vacuum line. Multilamellar vesicles (MLVs) were then prepared by adding 300 mM tris buffer (1 mL) containing dimethylarsinic acid (25 mg/mL) at  a pH of 7.4. The suspension was vortex mixed for 5 minutes, and then subjected to five freeze-thaw cycles, according to the method of Meyer et al  .  The sample was  dipped in liquid nitrogen for about 2 minutes and thawed in a water bath (30 °C). The freeze-thawed vesicles were then forced by using pressure from a nitrogen tank (200-500 psi), to pass through two stacked 200 nm pore sized polycarbonate filters in an extruder, to afford large unilamellar vesicles (LUVs). The LUVs were divided into two portions of about 0.4 mL, to allow duplication of each DMA efflux experiment. For the DMA efflux experiments, the LUVs (0.4 mL) were applied onto a Sephadex G-50 gel permeation column (1.5 cm i.d x 4 cm), pre-equilibrated in tris buffer (40 mM, pH 7.4). Elution was achieved by the use of further 40 mM tris buffer (pH =7.4). Upon the application of the LUVs onto the gel permeation column, timing was initiated. Only about the first 1 mL of the eluted LUVs were collected. An aliquot of the eluted LUVs (400 L) was pipetted into the NMR tube which already contained the following: of-D(+)-glucose (28 mg), manganese sulfate (40 jL of 30 mM), TSP (25 L of 40 mM), and tris buffer (135 L 40 mM, pH 7.4). The use of glucose was to control the osmotic pressure on the liposomes. The amount of glucose added was calculated to approximately balance the osmotic pressure acting on the liposomes. The time course for the efflux of DMA from the EPC LUVs was followed by acquiring NMR spectra at appropriate time intervals, until equilibrium was reached.  114 The operating parameters  that were used for spectral acquisition and water  suppression are given in Appendix B. The following experiments were performed on the liposomes in which DMA had been encapsulated: (i)  Efflux of encapsulated  DMA from the liposome in the absence of any  organotin compound. (ii)  Efflux of encapsulated DMA from the liposome, with organotin compound (trimethyltin hydroxide or tributyltin chloride or monobutyltin trichioride) added in the extraliposomal compartment.  3.10.4 Preparation of butyltin-EPC LUVs and the encapsulation of DMA. The stock EPC solution (2 mL) in chloroform was pipetted into a test tube. Also, aliquots of tributyltin chloride or monobutyltin trichioride (0.5, 1.5 or  5  gImL) in chloroform (1 mL) were pipetted into the same test-tube, and vortex  mixed. The chloroform was evaporated off, and the butyltin-EPC mixture dried on a vacuum line for 3 hours. DMA (1 mL of 25 mglmL solution) in tris buffer (300 mM, pH 7.4) was added to the dried butyltin-EPC mixture and vortex-mixed for about 5 minutes, to achieve the encapsulation  of DMA in the butyltin-EPC  multilamellar vesicles (MLVs) that were formed. The butyltin-EPC MLVs were forced to pass through two stacked 200 nm pore sized polycarbonate filters in the extruder, as described in Section 3.10.3 to produce large unilamellar vesicles (LUVs). The butyltin compounds possess highly hydrophobic butyl groups which  115 favour their incorporation into the lipid bilayer. However there will be some residual organotin compounds in the intraliposomal compartment. Butyltin-EPC liposomes of the following composition were prepared: (a)  0.5jg tributyltin chloride : 0.2g EPC (TBT-EPC A)  (b)  1.5ig tributyltin chloride : 0.2g EPC (TBT-EPC B)  (c)  5.0ig tributyltin chloride : 0.2g EPC (TBT-EPC C)  (d)  0.5/Lg monobutyltin trichioride : 0.2g EPC (MBT-EPC A)  (e)  1.5.ig monobutyltin trichioride : 0.2g EPC (MBT-EPC B)  The butyltin-EPC LUVs were divided into two portions of about 0.4 mL each, to permit the duplication of each DMA efflux experiment. A portion of the butyltin EPC liposomes (0.4 mL) was added onto a Sephadex G-50 gel permeation column (1.5 cm i.d x 3.0 cm) and eluted as described in Section 3.10.3. The first fraction (about 0.7 mL) of the eluted butyltin-EPC was collected. An aliquot of the eluted butyltin-EPC liposomes (400 .iL) was quickly pipetted into the NMR tube which already contained glucose (28 mg), aqueous manganese sulfate solution (40 L of 30 mM solution), TSP (25 L of 40 mM solution) and iris buffer(135 .iL of 40 mM solution, pH 7.4). When the presence of butyltin chloride was desired in the extraliposomal compartment, 135 L of 16.7 J.LM solution in iris buffer of the same butyltin chloride used to form the liposome was added. The DMA efflux experiment monitored by using the NMR spectrometer was conducted, as described in section 3.10.3 above. The following experiments were performed on the butyltin-EPC liposomes:  116 (i)  Efflux of encapsulated  DMA from the butyltin-EPC liposomes with no  butyltin chloride added into the extraliposomal compartment. (ii)  Efflux of DMA from the butyltin-EPC liposomes with tributyltin chloride or monobutyltin trichloride added into the extraliposomal compartment.  3.10.5 The NMR water suppression and spectral acquisition conditions for DMA efflux from EPC and butyltin-EPC liposomes. The Bruker AM400 NMR spectrometer was operated in the water suppression mode, which made it possible to obtain the NMR spectra of samples prepared in aqueous buffers. The water signal suppression was achieved by applying a narrow presaturation pulse at the frequency of the water signal, followed by a broadband excitation pulse which was applied at the frequency of the DMA resonance, while the water signal was still saturated. The time course for the efflux of DMA from the LUVs was followed by spectral acquisition at various time intervals. At each time interval, 48 scans were collected and averaged. The free induction decays were acquired by using a pulse width of 6 milliseconds, and Fourier transformed with a line broadening of 10 Hz. The spectrometer has variable temperature  capability, which was used in some  experiments. The micro-program used to operate the NMR spectrometer in the water suppression mode with automated spectral acquisition is given in Appendix B.  117 3.10.6 Determination of phospholipid concentrations by phosphorus assay.  3.10.6.1  Extraction of phospholipid  from liposomes prior to phosphorus  determination. Prior to determining the phosphorus concentration of the liposomes, the phospholipid (EPC) was separated from DMA by extraction into chloroform because  arsenic interferes with the subsequent phosphorus determination. The extraction was carried out according to the following procedure. The LUVs (0.5 mL) were diluted to 1 mL with deionized water. Methanol (2.2 mL) and chloroform (1 mL) were added to the vesicles, and the mixture was vortex-mixed. Deionized water (1 mL) and chloroform (1 mL) were further added into the mixture, causing it to separate into two phases. The top phase contained methanol, water, and DMA. The bottom phase contained chloroform and the lipid.  3.10.6.2  Lipid concentration determination.  Lipid concentrations of the vesicles were then determined by analyzing their ow and Bottcher 215 Subbar phosphorus content as previously described by Fiske and , . Lipid phosphorus was converted to phosphomolybdic acid which was 216 et a1 subsequently reduced by the Fiske-Subbarow reagent to a blue compound which can be measured colorimetrically. The procedure is as follows. Aliquots (25 L) of the chloroform extract of the vesicles were dispensed into test tubes and the chloroform was gently evaporated off with a stream of nitrogen gas. Perchioric acid (7.25 mL)  118 was placed into each of the test tubes which was then covered with a marble ball, and placed in a metal test tube-holding block which was heated (180°C  -  200°C, 1.5  hours). The test tubes were cooled, and 7.0 mL ammonium molybdate reagent (0.22 0 wlv) and 0.6 mL Fiske-Subbarrow 4 S 2 % wlv ammonium molybdate in 2% H O and 0.5g bis 1-amino-2-napthol-4-sulphonic acid 3 S 2 , ig Na 3 reagent (30g NaHSO in 200 mL water) were added into each of the test tubes. The contents of each test tube were vortex-mixed, heated for 15 minutes in a boiling water bath, cooled, and their absorbances measured at 815 nm. The samples were standardized against PO which had undergone similar chemical treatment 2 NaH known concentrations of 4 as the samples. Each assay was carried out in duplicate, and the average phophorus concentration determined. The phospholipid concentration was then calculated from the relationship that 1 mole of EPC contains 1 mole of phophorus.  3.10.7 ProcesHxng of the NMR spectra. For each experiment involving the efflux of encapsulated DMA from the liposomes, 25-30 data points collected over 11-17 hours were processed. Each data point, represents an average of 48 scans. The accumulated free induction decays (FID) were Fourier transformed with a line broadening of 10 Hz to produce the NMR spectra (Figure 3.6). In Figure 3.6, peak A (sharp singlet) is assigned to the DMA inside the liposome. Peak B (broad singlet) is assigned to DMA that has diffused out of the liposomes: the peak has been broadened and shifted by the manganese sulfate, a spectroscopic shift/broadening reagent added to the NMR tube  119  345 seconds (I)  6.0  50  4:0  3.0  1.o  2:0  2145 seconds (ii)  50  4.0  3.0  óo  2:0  VD  12345 seconds C A (iii)  Chemical shift (ppm) Figure 3.6  H NMR spectra of DMA as it diffuses out of EPC liposomes. Peaks 1 A and B are due to DMA inside and outside the liposomes respectively. Peak D is due to iris buffer. All peaks are referenced to TSP (peak C).  120 contents at the begining of the experiment. Peak D (6=2.4-4.5) is assigned to tris buffer. All peaks were referenced to TSP (peak C). As the experiment progresses, the peak due to the DMA inside the liposome decreases while the peak due to the DMA outside the liposome increases (Figure 3.6, i, ii, iii). Either of the peaks can be used to monitor DMA efflux from the liposomes. However, it was convenient to monitor the decrease of the peak due to DMA remaining in the liposome because the peak area was easier to obtain by integration. The peak due to tris buffer was used as an internal standard to nullify the effects of fluctuations in the instrument’s operating parameters. The peak area ratios of DMA inside the liposome to the tris buffer were calculated and plotted as a function of time to describe the efflux behaviour of the DMA molecules. Each data point is a combined signal from the methyl resonance of the two species of DMA namely; DMAH and DMK present in solution.  3.10.8 Analysis and treatment of data.  3. 10.8.1  Determination of rate constants and mode of permeation.  The experimental data for every efflux of DMA from EPC or butyltin chloride-EPC liposomes were analyzed for passive or facilitated diffusion by using equations [3.0] or [3.26] respectively.  —  +  (I  —  I)exp— f(1  +  f )kt}  [3.0]  121  =  A plot of ln( j  -  ,.eq  +  (I  -  1)exP_{(1 +f)J.kt}  [3.26]  eq) versus time gives a straight line with slope -(1+0k (Figure 1  3.7) for passive diffusion, or -(1 +O(BI)k for facilitated diffusion, where f is the ratio of the internal volume to the external volume. At equilibrium, the ratio of the peak integrals of the DMA signal inside the liposome to the signal outside the liposome corresponds to f. Therefore, eq in 1 e q out  0  —2  —4  —6  —8 0  200  400  600  800  1000 1200 1400  Efflux time (s) Figure 3.7  Log plot for the efflux of DMA from EPC liposomes.  [3.27]  122 where  and  ‘out  are the peak integrals (area) due to DMA inside and outside  the liposomes at equilibrium respectively, BI is the ratio of the formation constant to the dissociation constant of the carrier-permeant complex (see Figure 3.5). A method for estimating the ratio 8/, and k is described in Section 3.11.1. The total volume of reagents in the NMR tube for each experiment was 0.6 mL. Therefore, Vi,, +V =O.6mL 0  [3.28]  A combination of equations [3.27] and [3.28] allows V 1 and V 0 to be calculated. Hence f in equation [3.27] is determined, and k is calculated. From a plot of equation [3.0], the intercept ln (I°jnI’jr.) is obtained.  is  estimated from the peak integral of DMA inside the liposome at equilibrium. Then, 0 is calculated. Equation [3.0] was fitted onto the experimental data points by an J iterative procedure until convergence was obtained. This was done by using a commercially available mathematical software Sigmaplot 5.0 (Jandel Scientific). The calculated values of  f, and k were kept constant while Im° was permitted to  variable by about ± 0.005 units about the calculated value. A good fit of equation [3.0] onto the experimental data points indicates efflux by passive diffusion, provided the magnitude of the efflux rate constants is in the range expected for passive diffusion. This provision is necessary because in some situations (discussed in Section 3.11.1), equation [3.0] can also fit data for facilitated diffusion. If the experimental data points did not fit equation [3.0J,they were analyzed for facilitated diffusion by using equation [3.26].  123 At a pH of 7.4, which was the case for all experiments described in this  chapter, Herring Li 176 have determined that the major species of DMA  P  0 2 H3C—As—OH+H  I  CH3  ,0 -  pK= 6.28  DMAH Figure 3.8  3 H3C——O-+ O + H  I  CH3 DMK  Chemical species of dimethylarsinic acid (DMA) present at pH 7.4 (pH at which all experiments reported in this thesis were conducted).  permeating by passive diffusion is the undissociated acid represented as DMAH (Figure 3.8). The passive diffusion of the anionic species DMK is very slow. As the NMR signal obtained is composed of methyl resonances from both DMAH and DMK, the calculated values for the efflux rate constant k, and the permeation coefficient P, were corrected for the permeating species according to the 210 below: equations [3.29] k  -  [3.30] P- aP’  where a is the fraction of DMAH in solution, and is given by the relationship 176 a=[H} /(Ka + [H]) for a monoprotic weak acid. For DMAH , a is 0.0593 at pH 176  124 7.4. K is the dissociation constant. k’, and P’ are the corrected values for the permeating species DMAH. Fraction of DMK in solution is 1-a, and has a value of 0.9407.  3.10.8.2  Determination of permeability coefficients.  The permeability coefficients were calculated according to equation 3.31  k x Vjjimol.lipid —  A  [3.31]  where k (Is) is the efflux rate constant, V is the trap volume of the liposome per mol of phospholipid, A is area per j.tmol phospholipid, and has been calculated ° 21 /jmo1 phospholipid. 2 to be 1.81 x i0 3 cm The lipid concentration (I.Lmol phospholipid) was determined by phosphorus assay as described in Section 3.10.6.2.  125 3.11  RESULTS AND DISCUSSION.  3.11.lTheuseofDMAasaprobe inpermeabiity studies ofEPCliposomes, inthe  absence and presence of organotin compounds  in the extraliposomal  aqueous compartment. To observe the effect of organotin compounds on the permeability properties of the liposomes, experiments  encapsulated  were carried out by monitoring the efflux of  dimethylarsinic acid (DMA) from liposomes in the presence and  absence of the organotin compounds added into the extraliposomal compartment. At pH 7.4, two species of DMA namely DMAH and DMK are present in solution. The ‘H NMR spectra obtained are due to the combined proton resonances of the two species, therefore the graphs presented in this chapter describe the efflux of DMA, while the tables of data are for DMAH or DMK efflux. Under conditions of passive diffusion, DMAH is the major species permeating out of the liposomes. The , and is not treated in the 210 permeation of DMK by passive diffusion is very slow present study, except in situations where it permeates by facilitated diffusion. The time course for the efflux of DMA in the absence of any organotin compound at 24°C is shown in Figure 3.9. This efflux behaviour conforms to a first order passive diffusion as demonstrated by fitting a curve through the data points by using equation [3.0]. Previously, Herring Lai’ 76 had reported that DMA efflux from EPC liposomes is by passive diffusion. The fit of the data points around the curved portion of their graph is also similar to that shown in Figure 3.9. The rate constants  126 a  I  I  0.04  0.03  0.02  • Experimental data —Fit using eqn (3.0]  0.01  0.00 0.0  Figure 3.9  1.0  5.0 EFFLLIX TIME (s)  2.0  3.0  4.0  6.0  7.0  4 x10  Efflux of DMA from EPC liposomes (organotin compounds are absent in the extraliposomal compartment).  Table 3.1  Efflux data for the diffusion of DMAH from EPC liposomes in the absence of organotin compounds  Parameters  Valuea  (s) 12 t’  62±2  k’(/s)  (1.1 ± 0.04) x 10-2  P’(cm Is)  (1.7 ± 0.2) x 10-8  a  =  Data from ref 213  (0.97 ± 0.15) x 10-2  t’, k’ and P’ have been corrected for the major permeating specie  DMAH.  ,  127 permeation half-life and permeability coefficient for the efflux of DMAH in the absence of the organotin compounds are shown in Table 3.1. Also, data for DMAH ° are shown in 21 efflux from EPC liposomes in HEPES buffer obtained by Nelson Table 3.1, and are in close agreement with those obtained in this study. This indicates that tris buffer used in this study, or HEPES buffer do not introduce any significant effect in the efflux rate constants. The time course for DMA efflux in the presence of tributyltin chloride and monobutyltin trichioride is shown in Figures 3.10 and 3.11 respectively. The permeability data for DMAH efflux in the presence of tributyltin chloride in the extraliposomal aqueous compartment are shown in Table 3.2. A comparison of the efflux data of DMAH from EPC liposomes in the absence of any organotin compound (Table 3.1), and its efflux data in the presence  Table 3.2  Effect of 33.2 M tributyltin chloride on the efflux of DMAII from EPC liposomes.  Parameter  Value  t’ (if diffusion is passive)  25 ± 1  k’(Is) (if diffusion is passive)  (2.8 ± 0.1) x 10  P’ (cm/s) (if diffusion is passive)  (4.9 ± 1.0) x 108  128  0.020  I  I  0.015  0.010  0.005  • Experimental data —Fit using eqn [3.0] Fit using eqn [3.26] -  -  0.000  I  0.0  I  2.0  1.0  I  I  4.0  3.0  I  I  5.0  7.0  6.0  x 10  EFFLUX TIME (s)  Time course for the efflux of DMA from EPC liposomes (tributyltin  Figure 3.10  chloride present in the extraliposomal compartment).  I  —  z  0.005-  <  • Experimental data Fit using eqn 13.0]  -  0.000  I  0.0  0.5  I  10  I  1.5  x  EFFLUX TIME (s)  Figure 3.11  Time course for the efflux of DMA from EPC liposomes (monobutyltin trichioride present in the exiraliposomal compartment).  129 of tributyltin chloride (Table 3.2) shows that in the presence of tributyltin chloride, the permeation half-life t’ of DMAH efflux becomes about 2.5 times smaller than in its absence, indicating increased rate of permeation. The permeability coefficient is also about 2.9 times greater than in the absence of tributyltin chloride. The efflux rate constant k’ also increased by more than twice. This increased permeability of DMAH observed in the presence of tributyltin chloride suggests that some properties of the EPC liposomes have been changed by the tributyltin chloride. According to , the tributyltin cation causes membrane disruption and rupture 209 Heywood et a! (lysis) of EPC liposomes. Such rupture or pore formation in the liposomal bilayer would result in increased permeability to permeants. The increased permeability of the EPC liposomes to dimethylarsinic acid in the presence of tributyltin chloride could also arise if the tributyltin cation acted as a carrier, and mediated the transport of DMK by facilitated diffusion. The ability of the tributyltin cation to facilitate the diffusion of CF and OW has been reported . 6 n and Tosteson’ 08 15 Selwy 2 by ’ The equations [3.0] for passive diffusion and [3.26] for facilitated diffusion, I, -1’  +  (1 —1)exp -{(1+f)kt} [3.0]  -  +  (l  —  1  )exP_{(1  +  f)..P_kt}  [3.26]  used in this study for fitting curves onto the observed experimental data, are not capable of distinguishing between situations where there is a 100% passive diffusion  130 or 100% facilitated diffusion, unless the magnitudes of the expected rate constants  are known. This is because when there is 100% passive diffusion or facilitated 1 diffusion, a plot of ln(It  4el.r)  versus t, gives a straight line with slope (1+0k for  passive diffusion or (1 +f)(Bhk)k for facilitated diffusion. Each of the constants BIb and k for facilitated diffusion cannot be calculated separately, instead they are incorporated into each other as one parameter. The combined value of (i3/)k of equation [3.26] is equivalent to k of equation [3.0]. Under this condition, any curve generated by using either equation [3.0] or [3.26] should fit the experimental data (Figure 3.10). A method for obtaining the value of the constant 13/i when efflux takes place by a mixture of passive diffusion and facilitated diffusion is described later in this section. The time course for the efflux of DMA in the presence of monobutyltin trichioride is shown in Figure 3.11 above. The efflux of DMAH is by passive diffusion, but the rate of efflux has become slower (Table 3.3). The values of the rate constant and permeability coefficient shown in Table 3.3 are smaller than their values when no organotin compound was present (Table 3.1). The permeation half life of DMAH is about 3 times larger, indicating retarded permeation. Also, the permeability coefficient is decreased by about a factor of 4.3. The effect of externally added monobutyltin trichioride on the EPC liposomes is to decrease their permeability. A probable mechanism for this behavior is that monobutyltin trichloride permeates into the lipid bilayer and causes a decrease in the  131 membrane fluidity. Any compound capable of decreasing membrane fluidity such as 218 would decrease permeability. Such decrease in the 217 and o-tocopherol cholesterol membrane  fluidity caused  by dibutyltin  dichioride  has  been  observed  on  phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-diphosphate  Table 3.3  iM monobutyltin Effect of 33.2 1  irichioride  on the efflux of  DMAH from EPC liposomes. Value  Parameters  tlk  188 ± 6  (s)  k’ (Is)  (3.7 ± 0.1) x i0  P’ (cm/s)  (4.0 ± 1.2) x i0  20 Unfortunately, apart from the present study, there are no other reports . 2 9 ’ 2 vesicles of the interaction of monobutyltin trichloride with liposomal membranes. However, from the data presented in Tables 3.2 and 3.3, it seems that the monobutyltin species, unlike the tributyltin species, neither have the capability to induce membrane disruption nor act as carrier. Hence, the retarded permeation of DMAH. Monobutyltin is a degradation product of tributyltin in the environment and the scheme is tributyltin  -.  dibutyltin  -‘  monobutyltin  —‘  inorganic tin. These products  are progressively less toxic to life perhaps because debutylation leads to products less capable of causing membrane disruption.  132 The efflux of DMA across EPC liposomes in the presence of trimethyltin hydroxide (32.3 tiM) in the extraliposomal compartment was also studied. Analysis 1 of this permeation behaviour by plotting ln(It  -  Ifr) against t, of equation [3.0]  or [3.26] gave two straight lines of different slopes (Figure 3.12, slopes A and C). This behaviour is not predicted by equation [3.0], if permeation is by passive diffusion.  i  +  —  (1  —  1)exp —((1 +f)kt}  [3.0]  -  Jeq +  (I  -  1)exp _{(1  +  f )!kt}  [3.26]  If slope A (Figure 3.12) is considered a region of facilitated diffusion, the parameter (fi/)k of equation [3.26] is calculated. When the value of (BI)k was used in equation [3.26] to fit the experimental data, only the data points from the early and very late parts of the efflux experiment fitted (Figure 3.13  solid curve). Data  obtained at intermediate efflux times did not fit. If slope C (Figure 3.12) is considered a region of passive diffusion, the efflux rate constant k, for DMAH is calculated. When k was used in equation [3.0] to fit the experimental data, there was a close fit for data obtained at intermediate and late parts of the efflux (Figure 3.13 broken line). Towards equilibrium, equations [3.0] and [3.26] gave a close fit. A possible explanation for this phenomenon is that slope A (Figure 3.12) is a region dominated by facilitated diffusion of DMK mediated by trimethyltin cation, while slope C is a region dominated by passive  133  A A= Region of facilitated diffusion.  B  B = Mixture of facilitated & passive diffusion.  C  C = Region of passive diffusion.  -a  —5.0  I  I.  0  -  1000  2000  I  3000  Efflux time (s)  Figure 3.12  Log plot of DMA efflux from EPC liposomes (33.2 M Irimethyltin hydroxide present in extraliposomal volume).  0,03  0.02  - -  Fit using eqn [3.0]  —Fit using eqn [3.26] —  0.01  o  -‘-  Fit using eqn [3.31]  0.00 0  5000  10000  15000  20000  25000  (s) Time course for DMA efflux from EPC liposomes EFFLIJX TIME  Figure 3.13  trimethyltin hydroxide present in extraliposomal volume).  (33.2 tM  134 . Slope 210 diffusion of DMAH and DMK. The passive diffusion of DMK is very slow B (Figure 3.12) is a region dominated by a mixture of facilitated and  passive  diffusion. Thus, facilitated diffusion sets in at the early part of the efflux, and gradually gives way to passive diffusion as the efflux progresses. Assuming that the DMA is the major species permeating by facilitated diffusion while DMAH is the major species permeating by passive diffusion, the rate constant obtained from slope C (Figure 3.12) should be divided by 0.9407; the fraction of DMA (1-at) present at pH 7.4 (Section 3.10.8.1), to obtain the rate constant k’ for the passive efflux of DMA. Then, the parameter 8/ for facilitated diffusion of DMA can be calculated from slope A (Figure 3.12) by using the rate constant k’ (for passive diffusion of DMK) calculated from slope C. The calculated parameters  k’ (DMK), k (DMAH), B/, and l are  substituted into equation [3.31] which is a combination of equations [3.0] and [3.26], modified by introducing the parameters M and N. Equation 3.31 was fitted onto the experimental data by keeping every other parameter except M and N constant (Figure 3.13, dotted line).  -M(1+(I-1)exp-((1+t)kt})  +  1 N(1+(I  .  [3.31] (M is the percentage contribution of passive diffusion to efflux, while N is the percentage contribution of facilitated diffusion to efflux). The efflux parameters for this experiment are shown in Table 3.4.  135 Similar increased flux of chloride ions across liposomal membranes, mediated by trimethyltin cation as carrier has been reported by Selwyn . 15  Table 3.4  Data  for the  efflux of DMA  from EPC  liposomes  in the  presence of 33.2 jcM trimethyltin hydroxide. Parameter  Value  k’ DMAH (Is)  (2.5 ± 0.5) x  k’ DMK (Is)  (1.6 ± 0.3) x  BI (experimental)a  2.5 ± 0.5  B/ (curve fit)”  2.2 ± 1.0  N (% facilitated diffusion)b  66 ± 21  M (% passive diffusion)L  35 ± 21  a= Calculated from experimental data b=obtained from the curve fitting result.  3.11.2  Effect of organotin concentration on the efflux of DMA  The effect of tributyltin chloride concentration on the efflux of DMAH across EPC liposomes is shown in Table 3.5. As the concentration of tributyltin chloride in the extraliposomal compartment is increased from 0 to 8.3 pM, there is an initial decrease in the efflux rate constant and the permeability coefficient, followed by an  136 increase as the tributyltin concentration is raised from 8.3 to 33.2 tM. The values for the efflux half-life t’ also change in accordance with the changes in the permeability coefficients and the efflux rate constants, by becoming smaller as the  permeability of the liposome increases. The increase in the permeability coefficients, efflux rate constants, and the decrease in the efflux half-lives indicates that either the liposomal membrane has become more permeable to DMAH or that the tributyltin cation is facilitating the efflux of DMK. The effect of tributyltin chloride  on the permeation  of DMAH  concentration dependent. A plot of efflux rate constant versus tributyltin chloride  Effect of tributyltm chloride concentration on DMAII effluxa  Table 3.5  Cone.  P’ (cm/s)  (I.LM)  x 10  0.0  (1.7 ± 0.2)  62 ± 2  (1.1 ± 0.04)  8.3  (1.3 ± 0.1)  85 ± 11  (0.8 ± 0.1)  16.7  (1.6 ± 0.1)  45 ± 0.4  (1.5 ± 0.02)  33.2  (4.9 ± 1.0)  25 ± 1  (2.8 ± 0.1)  t’ (s)  k’(Is) x 102  a=It could not be determined if increased efflux was by passive or facilitated diffusion.  is  137 4 x 10  3 x 10  2 x 102  1 x 102  0 10  5  0  —5  15  20  25  30  35  SnC1 3 ) 9 H 4 M (C Figure 3.14  Effect of tributyltin chloride concentration on the permeability of EPC liposomes (tributyltm chloride was added into the extraliposomal compartment). 2 x 10-2  1 x 102  5 x io-  0 —5  Figure 3.15  0  5  10  15  20  25  30  35  40  JLM (C 3 ) 9 H 4 SnC1 Effect of monobutyltin trichloride on the permeability of EPC liposomes (monobutyltin trichioride was added in the extraliposomal compartment).  138 concentration shows a linear relationship (Figure 3.14) described by the equation Y=8.01 x i0’ X + 1.46 x iti, and a regression coefficient of 0.9990. The data point corresponding to zero concentration of tributyltin chloride does not fall on the regression line. This is probably because tributyltin chloride modified the properties of the liposomes. Therefore,  the data obtained  at zero tributyltin chloride  concentration, and the other data points effectively belong to different types of liposomes. Alternatively, non-linearity could also result if different modes of transport of DMA exist between the liposomes which are in contact with tributyltin chloride, and those not in contact with it.  Effect of monobutyltin trichioride concentration on the efflux of  Table 3.6  DMAH.  k’(Is)  Conc  P’ (cm/s)  (jiM)  x i0  0  (17.3 ± 0.2)  64 ± 0.5  (111.0 ± 0.04)  16.7  (2.8 ± 0.4)  188 ± 6  (3.7 ± 0.1)  33.2  (2.8 ± 0.1)  188 ± 6  (3.7 ± 0.1)  t’ (s)  x i0  The effect of monobutyltin trichloride concentration on the permeation of DMA across EPC liposomes is shown graphically in Figure 3.15  ,  while the  permeability data are shown in Table 3.6. As the concentration of monobutyltin  139 trichioride is increased from 0 to 16.7 jtM, there is a large decrease in the permeability coefficients and the rate constants. The permeation half-life increased by about thirty fold, indicating a very retarded permeability of the liposomal bilayer. No further change in the permeation parameters was observed as the concentration of monobutyltin trichioride was further increased.  3.11.3 Efflux of DMA from tributyltin chloride-EPC liposomes (with tributyltin chloride absent in the extraliposomal compartment). The efflux of DMA from liposomes composed of a mixture of tributyltin chloride (TBT) and egg phosphatidylchloine (EPC) was studied to establish the permeability properties  of these model membranes.  In these experiments no  tributyltin chloride was added to the extraliposomal compartment. The liposomes were prepared as described in Section 3.10.4 and are designated as TBT-EPC liposomes. The composition of the different TBT-EPC liposomes that were studied is as follows: (a) TBT-EPC A (0.5 g tributyltin chloride : 0.2 g EPC) (b) TBT-EPC B (1.5 ig tributyltin chloride : 0.2 g EPC) (c) TBT-EPC C (5.0 ig tributyltin chloride : 0.2 g EPC) The permeability properties of these liposomes were studied by measuring the efflux of encapsulated DMA from these liposomes. As an example, the efflux of DMA from TBT-EPC C liposomes is described. Analysis of this efflux experiment by plotting lfl(It  -  eq r)  of equations [3.0] or [3.26] as described in Section 3.11.1  140  —5  A= Region of facilitated diffusion.  —6  Mixture of facilitated & passive diffusion.  —7  Region of passive diffusion.  —s  0  5000  10000  15000  20000  25000  Efflux time (s)  Figure 3.16  Log plot of DMA efflux from TBT-EPC C liposomes.  003  • Experimental data 0.02 -.  —  0.01  Fit using eqn [3.0] Fit using eqn [3.26] Fit using eqn [3.31]  0.00 0  10000  20000  30000  40000  50000  60000  EFFLUX TiME (s)  Figure 3.17  Time course for DMA efflux from TBT-EPC C liposomes.  141 shows two lines with different slopes (Figure 3.16), which indicates that the efflux of DMA from TBT-EPC C is by a mixture of passive diffusion and facilitated diffusion. Facilitated diffusion is the major mode of DMA transport at the early stages of the efflux (Figure 3.17 solid line), while passive diffusion of DMAH dominates at the later stages (Figure 3.17 broken line). At intermediate times, a mixture of facilitated diffusion and passive diffusion dominate the efflux. The observed efflux behavior is therefore better described by fitting equation [3.31](a combination of equations [3.0] and [3.26], Section 3.11.1) onto the experimental data (Figure 3.17, dotted line).  Table 3.7  Diffusion parameters for the efflux of DMA from tributyltin-EPC liposomes by a mixture of passive and facilitated diffusiona.  Liposome  k’ (DMK)  k’ (DMAH)  x i(i (Is)  x iO (Is)  8/  % facilitated  passive  diffusion  diffusion  TBT-EPC A  2.0  3.1  2.2  66  34  TBT-EPC B  1.4  2.2  1.8  58  42  TBT-EPC C  1.1  1.8  2.2  54  46  a=Tributyltin chloride was not added into the extraliposomal compartment. Mean value BI  =  2.1 ± 0.2  142 The efflux of DMA from TBT-EPC A and TBT-EPC B, can also be accounted for by a mixture of facilitated diffusion and passive diffusion. The diffusion constants, and the percentage contributions of facilitated and passive diffusion to permeation are shown in Table 3.7. As the concentration of tributyltin chloride in the liposome is increased, facilitated diffusion decreases, while passive diffusion increases (Figure 3.18). 70  ,  60  i  50 I  — Facilitated diffusion Passive diffusion  40  30 0  Figure 3.18  1  2 3 4 5 tg tributyltm chloride : O.2g EPC  6  Contribution of passive and facilitated diffusion to the efflux of DMA from TBT-EPC liposomes of different tributyltm chloride composition.  143 When tributyltin chloride (16.7 tiM) was added into the extraliposomal compartment of TBT-EPC B liposomes, the plot of 1n(It  -  I  ) versus t, for DMA  efflux of either equation [3.0] or equation [3.26] gave one straight line of uniform slope (Figure 3.19).  —  1  0-  0  —  Figure 3.19  I  500  1000  I  I  1500 2000 Efflux time (s)  2500  3000  Log plot for efflux of DMA from TBT-EPC B liposomes when 16.7MM tributyltin chloride is present in the extraliposomal compartment.  144 Thus, it seems that when tributyltin chloride is added into the extraliposomal compartment, dissociation of the tributyltin-DMA complex at the interface between the bilayer and the extraliposomal compartment  is no longer favourable. The  equilibrium for dissociation is shifted to the left thereby suppressing the release of the complexed DMK. If the tributyltin-DMA complex does not dissociate at the interface of the extraliposomal compartment, there will be no more free tributyltin cations to sustain the facilitated transfer of DMK, therefore the facilitated diffusion of DMK ceases. Under these conditions, the passive diffusion of DMAH dominates. 0.020  I 0.015  0.010  • Experimental data 0.005  —Fit using eqn [3.0]  0.000 0  5000  10000  15000  20000  25000  30000  EFFLUX TIME (s) Figure 3.20  Time course for efflux of DMA from ThT-EPC B liposomes when 16.7  M tributyltin chloride is present in the extraliposomal compartment.  145 The time course for the efflux of DMA from TBT-EPC B liposome in the presence  of tributyltin  chloride  (16.7 JLM) added  into the extraliposomal  compartment is shown in Figure 3.20. The efflux behavior is described by equation [3.0] for passive diffusion. The parameters for this efflux are shown in Table 3.8. The rate constant for  the efflux of DMAH from TBT-EPC B in the presence of externally added tributyltin chloride is 1.8 x 10-2 /s (Table 3.8), while in the absence of externally added tributyltin chloride, it is 2.2 x i0 Is (Table 3.7). The observed difference in the two situations is attributed to the unequal concentrations of tributyltin chloride involved in the experiments.  Table 3.8  Parameters for the efflux of DMAH from TBT-EPC B liposomes in the presence of externally added tributyltin chloride (16 SM).  Parameter  Value  k’  (1.8 ± 0.1) x 10-2 (Is)  p’  (2.8 ± 0.5) x 10-8 (cm/s)  3.11.4 Efflux of DMA from monobutyltin trichloride-EPC liposomes. Monobutyltin trichloride-EPC liposomes designated MBT-EPC liposomes prepared as described in Section 3.10.4 were used to study the permeation of DMA.  146 MBT-EPC liposomes having the following compositions were studied: (a) MBT-EPC A (0.5 j.g monobutyltin trichioride : 0.2 g EPC) (b) MBT-EPC B (1.5 g monobutyltin trichioride : 0.2 g EPC) The efflux of DMA from MBT-EPC B with no monobutyltin trichioride in the extraliposomal aqueous compartment was studied. The time course for this efflux is 1 shown in Figure 3.22. Analysis of the experimental data points by plotting 1n(It  ) versus  -  t, of either equation [3.0] or equation [3.26] gave only a straight line  of uniform slope (Figure 3.21). This indicates that only one mode of diffusion is involved. The efflux parameters for DMAH, assuming passive diffusion is shown in Table 3.9. The efflux of DMA from MBT-EPC B in the presence of externally added monobutyltin trichioride (16.7 .iM) was studied. Also, only one mode of DMA efflux was found. The efflux parameters for DMAH  ,  assuming passive diffusion is shown  in Table 3.10.  It seemed reasonable to consider the efflux of DMAH from MBT-EPC liposomes to be by passive diffusion either in the presence or absence of monobutyltin trichioride in the extraliposomal compartment, because a comparison of the efflux parameters in Tables 3.9 and 3.10, with the efflux parameters for an “EPC only” liposome (Table 3.1), shows that the permeability coefficient for the MBT-EPC liposomes either in the presence or absence of externally added monobutyltin trichioride is the same as the permeability coefficient for the efflux of  147 -4.0  I  -4.2  —4.4  1  I 1  —4.6  —4.8  -5.0  0  500  1000  1500  Efflux time (s)  Figure 3.21  Log plot of DMA efflux from MBT-EPC B liposomes.  0.02  I  0.01  • Experimental data —Fit using eqn [3.0]  0.00 0  Figure 3.22  10000 20000 30000 EFFLUX TIME (s)  40000  Time course for DMA efflux from MBT-EPC B liposomes.  148 Table 3.9  Permeability data for efflux of DMAII from MBT-EPC B (1.5 g butyltin  trichloride:  2 g EPC)  liposomes  (with monobutyltin  tricbloride absent in the extraliposomal compartment). Parameter  Value  k’(Is)  (8.3 ± 0.4) x i0  t’(s)  84±4  P’(cm/s)  (1.7 ± 0.1) x 10-8  Table 3.10  Permeability data for efflux of DMAH from MBT-EPC B (l.5jcg monobutyltin trichioride: 0.2g EPC) liposomes with monobutyltin Irichloride present in the extraliposomal compartment.  Parameter  Value  k’(/s)  (9.1 ± 0.5) x i0  t’(s)  76±4  P’(cm/s)  (1.7 ± 0.1) x 10.8  DMAH from “EPC only” liposomes. The efflux rate constants in Tables 3.1, 3.9 and 3.10, are close. Therefore, monobutyltin trichioride does not have the ability to act as carrier for DMK.  149 It seems that the incorporation of monobutyltin trichioride into the lipid bilayer of EPC liposomes has no effect on its membrane permeability, but the membrane permeability is greatly retarded if the monobutyltin trichioride is added externally into the extraliposomal compartment (Section 3.11. 1,Table 3.3).  3.11.5 Effect of the butyltin chloride concentrations of the liposome on permeability  properties of TBT-EPC and MBT-EPC liposomes. The experiments reported in this section were conducted with tributyltin chloride (16.7 jIM) added into the extraliposomal compartment  of TBT-EPC  liposomes because under these conditions, passive diffusion of DMA is induced. Monobutyltin trichioride (16.7 M) was also spiked into the extraliposomal compartment of MBT-EPC liposomes to maintain similar experimental conditions with the MBT-EPC liposomes. The efflux of encapsulated DMA from butyltin-EPC liposomes was monitored at 24 °C. Studies were conducted by using TBT-EPC A, TBT-EPC B, MBT-EPC A, and MBT-EPC B liposomes. The permeability data for the efflux of DMAH from TBT-EPC liposomes are shown in Table 3.11. As the tributyltin chloride concentration of the liposome is increased on going from TBT-EPC A to TBT-EPC B, the permeability of the liposomes to DMAH also increases by about a factor of 1.6 (Table 3.11). The permeability data for MBT-EPC liposomes (with monobutyltin chloride present in the extraliposomal volume) are shown in Table 3.12. The permeability  150 coefficients, permeation half-lives, and rate constants show very little variation with increase in the monobutyltin trichioride composition of the liposome.  Table 3.11  Effect of tributyltin chloride concentration of TBT-EPC liposomes on permeability (tributykin chloride solution was also added to the extraliposomal volume.  Liposome  P’ (cmls)  k’(Is)  t’js)  x 10-2  x 10-8 TBT-EPC A  (1.7 ± 0.2)  57 ± 4  (1.1 ± 0.1)  TBT-EPC B  (2.8 ± 0.5)  39 ± 1  (1.8 ± 0.1)  Table 3.12  Effect of monobutyltin liposomes  trichioride  on permeability  concentration  (monobutyltin  of MBT-EPC  trichioride  added to the extraliposomal volume). Liposome  P’ (cm/s)  t’%(s)  k’(Is) x i0  x iO MBT-EPC A  (4.9 ± 0.8)  195 ± 7  (3.6 ± 0.1)  MBT-EPC B  (4.4 ± 0.3)  218 ± 19  (3.2 ± 0.2)  was also  151 3.11.6 Effect of temperature on the permeability of organotin-EPC liposomes. The effect of temperature on the permeability of TBT-EPC A and TBT-EPC B liposomes is shown in Tables 3.13 and 3.14 respectively, while the effect of  Table 3.13  Effect of temperature on the permeability properties of TBT-EPC A liposomes. Temperature °C  P’ (cmls)  k’(Is)  x 10-8  x 10.2  24  (1.7 ± 0.2)  (1.2 ± 0.1)  28  (3.1 ± 0.2)  (2.3 ± 0.1)  32  (3.4 ± 1.0)  (4.7 ± 0.1)  Effect of temperature on the permeability properties of TBT-EPC B  Table 3.14  (1.5 jg iributyltin chloride  O.2g EPC) liposomes.  P’ (cm/s)  k’ (Is)  x 10.8  x 10-2  24  (2.8 ± 0.5)  (1.8 ± 0.1)  28  (4.0 ± 0.03)  (3.5 ± 0.06)  32  (4.9 ± 0.5)  (5.4 ± 1.3)  • Temperature °C  152 Table 3.15  Effect of temperature  on the permeability properties of MBT  EPC A (0.5 jig butyltin Irichionde  0.2 g EPC) liposomes.  Temperature  P’ (cmls)  k’(/s)  (°C)  x i0  x i0  24  (4.9± 0.8)  (3.6±0.1)  28  (11.9 ± 1.0)  (7.0 ± 0.1)  32  (15.1 ± 1.2)  (14.9 ± 0.6)  Effect of temperature on the permeability properties of MBT-EPC B  Table 3.16  (1.5 jig monobutyltin trichioride : 0.2g EPC) liposomes. Temperature  P’(cm/s)  k’ (Is)  (°C)  x i0  x 10-2  24  (4.4 ± 0.4)  (5.4 ± 1.3)  28  (8.7 ± 0.1)  (7.6 ± 1.1)  32  (15.8 ± 1.0)  (1.1 ± 0.1)  temperature on MBT-EPC A and MBT-EPC B liposomes is shown in Figures 3.15 and 3.16 respectively. As the temperature  is increased, the permeability of both TBT-EPC and  153 MBT-EPC liposomes to DMAH also increases. Generally, permeation rates increase with increase in temperature. This is due to either increased partitioning of the permeant into the lipophilic bilayer or the increased ease of diffusion through the liposomal bilayer as temperature increases.  3.11.7  Activation energies for the permeation  of butyltin chloride-EPC  liposomes. The activation energies for the efflux of DMAH from liposomes of various butyltin chloride/EPC equation:  compositions were determined  by using the Arrhenius  -  P  —  AemT  or  InP----÷1nA RT A plot of In P against lIT gives a straight line from which the activation energy can be calculated  (Figures 3.23 and 3.24 for TBT-EPC and MBT-EPC liposomes  respectively). The activation energies are shown in Table 3.17  for tributyltin  chloride-EPC liposomes and Table 3.18 for monobutyltin trichloride-EPC liposomes. For the TBT-EPC liposomes, the activation energy for the permeation of DMAFI decreases with increasing tributyltin chloride concentration in the liposome. When compared to the activation energy for the efflux of DMAH from an “EPC , tributyltin chloride reduced the activation 210 only” liposome (86 ± 20 kJ/mol) energy required for DMAH efflux.  154 -16.0  -16.5  -17.0  -17.5  -18.0 3.25  3.35  3.30  3.40  (l/T x Figure 3.23  Arrhenius plot for DMAH efflux from TBT-EPC B liposomes.  -16.0  -17.0  -18.0  -19.0  -20.0 3.28  3.32  3.36  (l/T x 10) °K Figure 3.24  Arrhenius plot for DMAH efflux from MBT-EPC B liposomes.  155 Table 3.17  Effect of tributyltin chloride content of liposome on the activation  energy for efflux of DMAI{ from TBT-EPC liposomes. Liposome  Activation energy (kJ/mol)  TBT-EPC A  64.4  TBT-EPC B  52.3  Table 3.18  Effect of monobutyltin trichloride content of liposome on the activation energy for efflux of DMAI{ from MBT-EPC liposomes.  Liposome  Activation energy (kJ/mol)  MBT-EPC A  106.8  MBT-EPC B  121.5  , has described the activation energy for a permeant to comprise the 221 Cohen following:  -  (i)  adsorption of the solute at the lipid membrane/water  (ii)  dehydration of the solute  (iii)  diffusion through the hydrocarbon chain (lipid bilayer)  interphase  156 Thus the activation energy for permeation of a solute should increase as its ability to form hydrogen bonds increases. This is simply related to the number of hydrogen bonds the permeant has to break before it diffuses across the hydrophobic hydrocarbon  chains of the lipid bilayer. If the permeating  probe molecule  dimethylarsinic acid (DMAH) is kept constant, the contribution of the dehydration step to the activation energy should be constant for both TBT-EPC and MBT-EPC liposomes. Therefore, the observed difference in activation energies for the two types of liposomes must be due to either the effect of the organotin compounds on the adsorption of DMAH at the lipid/water interphase, or the effect on diffusion through the liposomal lipid bilayer. The low activation energy observed for the permeation of DMAH across tributyltin chloride-EPC liposomes (Table 3.17) supports the argument that tributyltin chloride modified the liposomal membrane. For the MBT-EPC liposomes, the activation energy for the permeation of DMAH increases as the concentration of monobutyltin trichloride in the liposomes increases (Table 3.18). This observation indicates that it became more difficult for the DMAH molecules to diffuse across the lipid bilayer and is further evidence that the monobutyltin cation is neither capable of inducing pore formation on the liposomes nor able to act as a carrier for DMA. The pre-exponential factors of the Arrhneius equation are shown in Tables 3.19 and 3.20 for TBT-EPC and MBT-EPC liposomes respectively. According to , the pre-exponential factor is related to the molar 202 , and De Gier et a1 221 Cohen  157  Arrhenius pre-exponential factor for TBT-EPC liposomes  Table 3.19  Pre-exponential factor (Is)  Liposome  x 10-8  TBT-EPC A  (2.8 ± 0.5)  TBT-EPC B  (4.0 ± 0.4)  Table 3.20  Arrhenius pre-exponential factor for monobutyltin MBT-EPC. Pre-exponential factor (Is)  Liposome  x 10-8 MBT-EPC A  (1.1 ± 0.3)  MBT-EPC B  (1.0 ± 0.4)  entropy change of the permeation process by the equation:  In A  -  constant  +  AS R  —  where A is the pre-exponential function, AS is the molar entropy change and R is the molar gas constant. The values of the pre-exponential factors obtained for the TBT-EPC and MBT-EPC liposomes are different from each other, but are fairly constant for each  158 type of liposome. Therefore it seems that entropy for the permeation process is  different for both the TBT-EPC and MBT-EPC liposomes, but remains constant for each type of liposome irrespective of the butyltin chloride concentration of the liposome. The slight variation in the values of the pre-exponential factors shown in Table 3.19 for the TBT-EPC liposomes may be attributed to experimental errors. The observed differences in the values of activation energies and pre exponential factors for the TBT-EPC and MBT-EPC liposomes may indicate that these butyltin chlorides act on the model membrane by different mechanisms. 221 has shown that the magnitude of the activation energy is related to Cohen the physical state of the hydrocarbon chains in the lipid bilayer. Thus, as the amount of cholesterol content of the vesicle is increased, the activation energy for its permeation also increases . 221 221 demonstrate that the The results of the present study and those of Cohen composition of the liposomes contributes very significantly to the magnitude of activation energy. This is contrary to the report by De Gier Li 202 that activation energy is solely determined by the capability of the permeating molecules to be involved in hydrogen bonding.  3.11.8 Relevance of this NMR study to the environmental  toxicity of butyltin  compounds. A number of chemical reactions of the trialkyltin compounds with other  22 Trialkyltin . 2 ’ 219 organic molecules of biological relevance have been reported  159 compounds derange mitochondrial  function by discharging a hydroxyl-chioride  gradient across the membranes, and by inhibiting ATP synthesis . They also cause 18 swelling and disruption of the mitochondrial membranes , and the rupture of human 18  red blood cells . 223 Dialkyltin compounds react with enzymes possessing thiol groups . The 18 biochemical effect of this is an interference with a-keto acid oxidation , while the 18 mono-organotin compounds do not show any significant toxicity. The effect of butyltin compounds on membranes has not been extensively studied. Early studies by Selwyn et , 208 Tosteson and Weith ’ 5 al’ , Motais ailV 16 7 show that tributyltin chloride and trimethyltin chloride can mediate chloride-hydroxide exchange across mitochondrial tripropyltin chloride mediates membranes.  The present  membrane  and model cell membranes, while  chloride-chloride  exchange across mitochondrial  study clearly shows that  tributyltin  chloride  and  monobutyltin trichioride exert different and opposite effects on the model cell membranes. Tributyltin chloride makes the model membranes more permeable while monobutyltin trichioride makes them less leaky. Since monobutyltin trichioride is by far less toxic than tributyltin chloride, the observed decrease  in membrane  permeability is likely a phenomenon that leads to reduced toxicity. The present study concludes that tributyltin and trimethyltin cations are able to function as mobile carriers for dimethylarsinate while monobutyltin cation lacks this ability.  160 CHAPTER 4 HYDRIDE GENERATION METHODS OF ATOMIC ABSORPTION  SPECTROPHOTOMERY  4.1  FOR TOTAL TIN DETERMINATION.  INTRODUCTION. With the advent of organotin pollution in the marine environment, many  workers in the field of environmental analysis have devoted their energies to the detection and quantitation of the more toxic organotin species. The determination of the total tin content in marine samples has been largely neglected. Consequently another objective of the present study was to provide information on the total tin content of some marine animals in British Columbia, Canada. Determination  of total tin content in environmental  samples is usually  accomplished by the use of atomic absorption spectrophotometry (AAS). Methods of sample preparation, prior to total tin determination, usually involve the extraction of the tin compounds into organic solvents by the use of complexing agents, or the digestion of the samples with mineral acids, to convert the various forms of tin to inorganic tin. The total tin content of the digested sample or the organic extract can 224 AAS 2 25 26 then be determined either directly by the use of conventional flame ’ and graphite  furnace atomic absorption  spectrophotometry  (GFAAS), or by  conversion to volatile derivatives such as inorganic tin hydride which can be analyzed by hydride generation-atomic hydride generation-graphite  absorption spectrophotometry  228 or ’ 227 (FIG-AAS)  furnace atomic absorption spectrophotometry  (HG  161 . The HG-GFAAS method has also been used in the analysis of the 229 GFAAS) . 2 1 and selenium following elements; bismuth°, antimony Conversion of tin compounds in environmental samples to inorganic tin hydride is usually preferred over direct determination,  because the analyte is  removed from the matrix of the digested sample, thereby minimizing matrix interferences during the HG-AAS or HG-GFAAS analysis. In this study, two methods  of hydride generation  atomic absorption  spectrophotometry were optimized and used for total tin determination in marine animals:  one  based  on  continuous  hydride  generation  atomic  absorption  spectrophotometry (HG-AAS) and the other on batch hydride generation-graphite furnace atomic absorption spectrophotometry (HG-GFAAS). The continuous hydride generation method (HG-AAS) utilizes the hydride 233 (Fig 4.1), for use in the generator previously reported by Cullen and Dodd determination of arsenic. Atomization of tin compounds was achieved inside a quartz cell, which was heated by the air-acetylene  flame of the atomic absorption  spectrophotometer. The batch hydride generation method (HG-GFAAS) involved the in situ generation of tin hydride which was then trapped, or adsorbed  onto a graphite  . The graphite furnace tube 229 furnace tube according to the method of Sturgeon eta1 served as a preconcentration device and also enabled high atomization temperatures to be reached. The overall reaction for the production of tin(IV) hydride (stannane) is given  162 below; BH l+l6H n 4Na 4HC 2 S + + 4 O  4.2  -.  +4H 2 12H 0 +4H 3 0 H 4NaCZ Sn + 4 B 3 +  EXPERIMENTAL.  4.2.1  Instrumentation.  4.2.1.1  Continuous hydride generation atomic absorption spectrophotometry (HG-AAS). The continuous hydride generator employed in this study was a home built  233 (Fig 4.1) for arsenic glass apparatus described previously by Cullen and Dodd determination. The operation of this hydride generator is similar to the type reported 228 for total tin determination. The hydride 22T and Subramanian by Vijan and Chan generator consisted of a 20 turn reaction glass coil (A, in Figure 4.1) connected to a gas-liquid separator (B, in Figure 4.1) by Teflon® tubing. Reagents were pumped into the glass reaction coil by means of a peristaltic pump (Gilson, Middleton Wisconsin, U.S.A.). The generated tin hydride was carried by a flow of nitrogen via a Teflon® tubing, into an open-ended T-shaped quartz cell which was heated by the air-acetylene flame of the atomic absorption spectrophotometer. The light from the tin hollow cathode lamp, and the deuterium background corrector were aligned to pass through the T-shaped quartz cell positioned in the optical path of the atomic absorption spectrophotometer.  The atomic absorption spectrophotometer  is  Varian 1275 model, operated at a slit width of 1 urn. Argon was used as the internal  a  163  Peristaltic Pump Reaction Coil Sample Acid 4 NaBH Gas-Liquid Separator Pressure Regulator  Drain  Figure 4.1  Schematic diagram of the apparatus used for the HG-AAS method  reported by Cullen and Dodd.  5.2cm(I.D.) 4 SnH  C E E E  S 0  F  It•)  c.’J  G  QUARTZ 22cmxl .lmm(I.D.)  4 NoBH OPTICAL PATH of A.A. Spectrometer  D H  GRAPHITE FURNACE TUBE  4mm stopcock  WATER VACUUM  Figure 4.2  Schematic diagram of the hydride generator used for HG-GFAAS.  164 purge gas. The tin hollow cathode lamp was purchased from Hamamatsu Photonics of Japan. Analyses were carried out at the 224.6 nm spectral line.  4.2.1.2  Batch hydride generation-graphite furnace atomic absorption spectrophotometry (HG-GFAAS).  The batch hydride generator used for the HG-GFAAS is a glass apparatus shown in Figure 4.2, and was modelled to be slightly different from the design for arsenic and selenium determination, and later for  reported by Sturgeon  n The lower portion of the batch hydride generator was 229 determinatio total tin . constructed of a 25 mL Buchner funnel (Corning Glass Works, Corning, U.S.A) with medium porous glass fit (pore size 10-15 J.Lm). This hydride generator was designed to accomodate larger volumes of reagents than the one reported by Sturgeon et a1, and the larger surface area of the glass fit should enable easier mixing of the reagents, and purging of the generated  tin hydride out of the batch hydride  generator. The tin hydride produced in the hydride generator was swept by an upward flow of nitrogen via a Teflon® tubing to a narrow quartz tube of inner diameter 1.1 mm, which was inserted into the heated graphite tube of the graphite furnace  atomizer,  spectrophotometer.  aligned  in the  optical  path  of the  atomic  absorption  The graphite furnace tubes were pre-used Varian Techtron®  graphite tubes, which were pre-coated with either sodium tungstate or palladium modifiers. When no pre-used tubes were available, fresh graphite tubes whose pyrolytic coatings had been roughened by using an abrasive (sandpaper), were coated  165 with solutions of these modifiers, and used. Sturgeon  have reported that pre  used graphite tubes are more efficient in trapping the tin hydride than fresh graphite tubes. The orifice on the wall of the graphite furnace tube was widened to a diameter of 2.3 mm to allow the insertion of the quartz tube. The graphite furnace atomizer  was a Varian GTA-95 instrument connected to a Varian 1275 atomic absorption spectrophotometer. The spectral line and slit width used are as described in Section 4.2.1.1.  4.2.2 Materials and reagents. The following chemicals; sodium tungstate dihydrate (Analar grade), L cysteine, sodium borohydride (Assured grade), potassium hydroxide (Aristar grade) hydrochloric acid (Analytical grade) were purchased from BDH Chemicals Ltd, Poole, England. Palladium powder was procured from Ventron Chemical Company, Danvers, Massachusetts, U.S.A. .Tin metal was obtained from Mallinckrodt Chemical Works, St Louis, Missouri, U.S.A.. Tort 1 (lobster hepatopancreas)  standard  reference material was obtained from the National Research Council, Canada. Hydrofluoric acid (doubly distilled in quartz) was obtained from Sea Star Chemicals, Victoria, Canada.  166 4.2.3 Methodology for the hydride generation atomic absorption spectrophotometry.  4.2.3.1  Continuous hydride generation method (HG-AAS). The reagents were pumped by means of a peristalic pump into the reaction  coil (A in Figure 4.1), where mixing of the reagents and the production of the tin hydride occurred. A flow of nitrogen gas ensured the purging of the generated tin hydride into the gas-liquid separator (B in Figure 4.1) which was further purged by nitrogen gas. The tin hydride was swept into the heated T-shaped quartz cell where atomization occurred. The absorbance reading was recorded after it became stable. All analyses by the HG-AAS method were carried out in triplicate.  4.2.3.2  Batch hydride generation-graphite furnace method (HG-GFAAS).  The batch hydride generator and the hydride transfer lines were silanized by using a 10% (vlv) triethylsilane solution in toluene as follows:- with all the transfer lines connected, and all taps closed except stopcock H at the bottom of the hydride generator, the quartz tube G was immersed in a solution of 10% triethylsilane in toluene with the water vacuum turned on. The triethylsilane solution was drawn into the hydride generator, and the spent solution was then pumped out via stopcock FL The hydride generator was then dried with a gentle flow of nitrogen, admitted through tap D, over a period of about fifteen minutes. This procedure minimized the adsorption of the tin hydride on the walls of the hydride generator. During the analysis, measured amounts of hydrochloric acid and the sample  167 were each pipetted onto the porous glass fit via a B24 joint at the top of the hydride generator, while an upward flow of nitrogen was maintained through tap D  Table 4.1  Graphite furnace atomization program for tin determination by (HG-GFAAS). Step  Temperature °C  Time (s)  Gas flow (Llmin)  1  700  19  3.0  2  700  40  3.0  3  700  40  3.0  4  700  40  3.0  5  700  40  3.0  6  700  4.0  3.0  7  2700  4.0  0.0*  8  2700  2.0  0.0*  9  2700  1.0  3.0  * =  When absorbance measurement was taken.  Steps 1-6 represent trapping and drying conditions. Steps 7-8 represent atomization conditions. Step 9 is clean up.  168 (Figure 4.2). Measured amounts of sodium borohydride solution were delivered into the hydride generator by means of a peristaltic pump via tap F. The tin hydride produced in the hydride generator was swept by an upward flow of nitrogen admitted through tap D, to a narrow quartz tube of inner diameter 1.1 mm, which was inserted into the heated graphite tube of the graphite furnace atomizer, and trapped by using the furnace program shown in Table 4.1. A gentle flow of argon maintained an inert atmosphere inside the graphite furnace tube, except during the atomization step when the argon flow was stopped. After the tin hydride had been trapped in the graphite furnace tube, the nitrogen flow into the quartz tube was stopped, and the quartz tube was manually removed from the graphite furnace tube which was then quickly heated to 2700 °C, to atomize the analyte. After each determination,  the solution remaining in the hydride  generator was pumped out, by using the water vacuum.  4.2.4 Preparation of matrix modifiers and standard tin solutions.  4.2.4.1  Preparation of palladium modifier.  The palladium  modifier solutions (2-10 % w/v) used to treat the graphite  tubes were prepared by dissolving palladium metal in 1 mL of a warm mixture of concentrated hydrochloric acid and nitric acid (1:5 vlv), and diluting with 2 % ascorbic acid solution in a 5 mL volumetric flask.  169 4.2.4.2  Preparation of sodium tungstate modifier.  Solutions of the sodium tungstate modifier (2  -  10 % w/v) were prepared by  dissolving sodium tungstate dihydrate in de-ionized water.  4.2.4.3  Preparation of standard tin solutions. Stock standard solutions were typically prepared by dissolving tin metal shot  in 2 mL of a warm mixture of concentrated HC1 and HNO 3 (1:1), and then diluting the resulting solution to 50 mL in a volumetric flask. Working standard solutions were prepared by diluting appropriate amounts of the stock solution in 0.5 M aqueous HC1 solution. The working standard solutions used for quantitation were prepared in 0.5 M HC1 solutions containing 2% L-cysteine.  4.2.5 Optimum concentration of reagents used in the continuous hydride generation method (HG-AAS). The generation of tin hydride is pH dependent . The optimum pH and 113 reagent concentrations were established as follows:-various concentrations of sodium borohydride in the range 0.5  -  2.5 % (w/v) were prepared in aqueous potassium  hydroxide solution (0.2% wlv). A standard tin solution in 0.5 M HC1, and different concentrations of hydrochloric acid in the range 0.1  -  1.0 M were each prepared in  different volumetric flasks. When required, these reagents were pumped into the reaction coil of the continuous hydride generator by using the parameters shown in Table 4.2.  170 At a fixed concentration of hydrochloric acid, standard tin solution, and varying concentrations  of sodium borohydride, simultaneously pumped into the  reaction coil, the absorbance of the tin hydride produced was measured by the atomic absorption spectrophotometer. The absorbance measured was taken to be an indication of the yield of tin hydride. The reagent concentrations giving the highest absorbance of tin hydride were then used for the determination of total tin.  Table 4.2  Operating conditions for the continuous hydride generation atomic absorption specirophotometry (HG-AAS).  Flow rate  Sample  4.4 mL/min  Flow rate  HCL  4.4 mL/min  Flow rate  4 NaBH  4.4 mL/min  Purging gas flow rate  0.6 L/min  4.2.6 Use of L-cysteine to remove interferences. During the atomic absorption analyses of the marine animal samples, it was observed that the absorbance signal started to decrease as the analysis progressed, until it finally disappeared. This phenomenon was more noticeable when digested environmental samples were introduced either into the batch or continuous hydride generators. Such behavior had previously been encountered including Brindle and Le , Beach and Shrader 236 , Le t13  Ii237,  by other workers and Quevauviller  171 . To eliminate this interference they added either L-cystine 112 a! 236 or L-cysteine 7 ’ 113 to the reaction mixture prior to hydride generation. Consequently, in the present  work a study was carried out to find the optimum concentration of L-cysteine needed to prevent the disappearance of the tin absorbance.  4.2.6.1  Optimum  concentration  of  L-cysteine  required  to  remove  interferences. Standard tin solutions (0.2 g/mL) containing 0.5- 3.0.ig/mL L-cysteine were prepared in 0.5 M hydrochloric acid. The absorbance corresponding to the tin hydride produced from the reaction between the standard tin solutions and sodium borohydride were measured by using HG-AAS. For the batch hydride generation method, no optimization was carried out, but the use of the optimum concentration of L-cysteine obtained for the continuous hydride generator was sufficient to prevent the disappearance of the tin absorbance.  4.2.7 Optimum conditions for the batch hydride generation-graphite  furnace  atomic absorption specirophotometry (HG-GFAAS). The batch hydride generation method was optimized for concentration and volume of reagents, trapping temperature, and trapping time of the tin hydride in the graphite furnace tubes coated with 8% sodium tungstate modifier solution.  172 Optimization of reagent concentrations for HG-GFAAS.  4.2.7.1  A standard tin solution (1 mL of 12 ng/mL tin solution), prepared in 0.5 M hydrochloric acid, was added into the batch hydride generator, and then reacted with various volumes of 0.2 M hydrochloric acid solution, and 2 % (wlv) sodium borohydride in 0.2 % potassium hydroxide solution. The absorbance of the generated tin hydride was measured, and taken to be an indication of the yield of tin hydride. The results obtained for this optimization are discussed in Section 4.4.4.1.  4.2.7.2  Optimization of trapping temperatures  and trapping time for tin  hydride in the graphite furnace tube. With the optimum volumes of reagents established, the trapping temperature for the generated tin hydride in the graphite furnace was varied, and the atomic absorbance of tin hydride measured. The experiment was repeated at other trapping temperatures. After the optimum trapping temperature had been established, the effect of the trapping time on the absorbance was also studied, by varying the trapping time of the tin hydride at a constant trapping temperature and reagent concentrations. The results obtained in this study are discussed in Section 4.4.4.3 and 4.4.4.4.  4.2.8 Treated graphite furnace tubes:- coating the graphite furnace tubes with solutions of sodium tungstate and palladium modffiers. The method used in this study for the treatment of graphite furnace tubes with  173 matrix modifiers, is similar to the procedure described by Fritzsche Lai . Pre-used 8 graphite furnace tubes were soaked for 26 hours in aqueous sodium tungstate solution (2 -10% wlv) or in a solution of palladium (2  -  10% w/v) in 2 % aqueous  citric acid. The preparation of the palladium and sodium tungstate modifiers is described in Section 4.2.5. The soaked graphite furnace tubes were dried in an oven at 125  -  129 °C for 4.5 hours. Prior to use, they were cleaned once by raising the  temperature of the graphite furnace to 3000 °C, and then conditioned by running the graphite furnace program (shown in Table 4.1) four consecutive times.  Optimum modifier treatment of graphite furnace tubes.  4.2.8.1  A standard tin solution was used to produce tin hydride which was trapped in the graphite furnace tubes treated with varying concentrations of sodium tungstate (2  -  10 % w/v) or palladium modifier (2  -  10 % w/v) solutions. The preparation of  the sodium tungstate and palladium modifiers is described in Section 4.2.4. A plot of absorbance versus modifier concentration (Section 4.4.4.5) revealed the optimum modifier concentration required to coat the graphite furnace tubes.  4.2.8.2  Calibration curves for the HG-GFAAS method.  Tin standards (2  -  14 ng/mL) in 0.5 M HC1 containing L-cysteine (2% w/v)  were introduced into the batch hydride generator, and reacted with 0.2 M HCI (5 mL), and 4 mL of 2 % sodium borohydride solution containing 0.2 % KOH, to produce tin hydride which was trapped on sodium tungstate-treated graphite tubes.  174 The measured absorbances were piotted as a function of the concentrations of the standard tin solutions, to obtain a calibration curve.  4.3  Sample digestion and preparation. Freeze dried oysters or the standard reference lobster hepatopancrease, Tort  1 (about 2.00 g) and 2% aqueous potassium hydroxide (20 mL) were placed in a 500 mL round bottom flask fitted with an air cooled reflux condenser previously 239 (Appendix D), and refluxed for lh 45 mm. The contents of the described by Dodd round bottom flask were cooled, and concentrated sulphuric acid (4 mL of 12 M) and concentrated nitric acid 30 mL of 15 M) were added to the round bottom flask, and further refluxed until all solution had gone into the reflux condenser, and the residue in the round bottom flask has charred. Heating was stopped, and after a few minutes, when the solution in the reflux condenser had dripped back into the round bottom flask, refluxing was resumed until the solution became clear and colorless, or very light yellow. The round bottom flask was cooled, and de-ionized water(10-20 mL) was added through the reflux condenser. Reflux was then continued until the solution turned colorless. The solution was cooled and then transferred to a 250 mL glass beaker where the solution was evaporated down to about 10 mL, by using a hot plate. The solution was transferred to another 250 mL beaker made of Nalgene®, followed by the addition of 1 mL hydrofluoric acid. The solution was further evaporated on the hot plate to about 5 mL. Concentrated hydrochloric acid, and de ionized water (50 mL) were added to the Nalgene® beaker, and further heated until  175 the volume of the solution has reduced to about 30 mL. The solution was cooled, transferred to a 50 mL volumetric flask containing L-cysteine, and made up to the mark with 0.5 M hydrochloric acid solution to form the digested sample in 2% w/v L-cysteine solution. A blank solution containing all the reagents used for sample digestion was also digested, by following the same digestion procedure described for the sample.  4.4  RESULTS AND DISCUSSION.  4.4.1 Optimum concentrations  of sodium borohydride and hydrochloric acid  necessary for the production of slnnnane in the continuous hydride generator. The effect of the concentrations of hydrochloric acid and sodium borohydride on the generation of tin hydride, as monitored by measuring the absorbance of the generated hydride, is shown in Figure 4.3. The error bars on all the graphs in this Chapter are the standard errors for three replicate determinations. At all the sodium borohydride concentrations  4 was generated as the sodium studied, more SnH  borohydride concentration was increased. At 2% sodium borohydride concentration, a maximum is reached, and further increase in sodium borohydride concentration 4 production as shown by a decrease in absorbance at 2.5 % leads to decreased SnH sodium borohydride. As the hydrochloric acid concentration  is increased, the  absorbance of the generated tin hydride decreased. L-cysteine was not used in this optimization study.  176  0.4  0.3  I  -  I  -  8  I  0.2  0.1  -  -  -I-  0,0  -  0.0  0.2  0.4  0.6  0.8  1 .0  1 .2  Concentration of HC1 (M)  A0.5 % % •1.5 % 02.0 % v 2.5 %  Figure 4.3  sodium sodium sodium sodium sodium  borohydride borohydride borohydride borohydride borohydride  Effect of sodium borohydride and HC1 on the absorbance of tm hydride produced from 4 tg/mL tin solution.  177 4.4.2 Optimum concentration of L.cysteine required to eliminate interfereDces in  HG-AAS. During the HG-AAS analysis of the marine animal samples, the tin absorbance started to decrease as the analysis progressed, especially when the oyster or Tort 1 digested solutions were introduced either into the batch or the continuous hydride generators or when the quartz cell of the continuous hydride generator became dirty with an insoluble material. Interferences capable of causing the decrease of the tin absorbance signals can be encountered in two stages of the hydride generation-atomic absorption analysis: (a)  In the hydride generator, where other metal ions could compete with Sn for  borohydride. (b)  In the heated quartz cell, where the formation of refractory tin carbide, which  does not atomize at the temperature of the air-acetylene flame would reduce tin absorption. ’ 24 ° and Thompson et al 24 , Nakahara 7 , Le et a1 6 Brindle and Le  have  reported that transition metal ions such as Fe(II), Fe(III), Co(II), Ni(II), and Cu(II) cause serious reduction of tin absorbance signals. Such interferences that inhibit the eine or L 113 L-cyst 7 4 had previously been eliminated by using ’ formation of SnH s L-cysteine also decreased the pH 113 author , 7 . According to these ’ 236 cystine dependency of the tin hydride formation. Therefore, a study was carried out to find the optimum concentration of L cysteine required to improve tin absorbance.  178 The optimum concentration of L-cysteine was found by analyzing a 0.2 .ig/mL tin standard solution containing varying concentrations of L-cysteine, and plotting the absorbances against L-cysteine concentrations (Figure 4.4). Figure 4.4 indicates that the optimum concentration  of L-cysteine is about 2 % (wlv). The optimum  concentration of L-cysteine is higher than the concentration reported by Beach and 113 and Le tai Shrader 237 in their methodologies, to improve tin absorbance. Both authors used a 1 % L-cysteine solution. Consequently, all standard tin solutions and digested animal samples were dissolved in solutions containing 2% L-cysteine. 0.20  0.15  0.10  0.05  0.00 0.0  0.5  1 .0  1 .5  2.0  2.5  3.0  3.5  Concentration of L-cysteine (% wlv) Figure 4.4  Effect of L-cysteine on absorbance of tin hydride.  179 4.4.3 HG—AAS determination of total tin in oysters and standard reference material (Tort 1). Digested sample solutions in 2% L-cysteine solution were pumped into the reaction coil of the continuous hydride generator, where a reaction occured between sodium borohydride and tin, to produce the stannane which was detected by the  atomic absorption spectrophotometer.  Quantitation of the total tin content of the  samples was accomplished by using the standard addition method as follows; 5 mL of the digested sample in 2% L-cysteine solution were spiked into 10 mL of standard tin solutions (0 8 tg/mL Sn) containing 2% L-cysteine in 0.5 M HC1 solution. The -  digested blank solution (5 mL) was also spiked into another set of calibration standards (0  -  8 jg/mL Sn) containing 2% L-cysteine in 0.5 M HC1. Each mixture  of the digested sample solution and the tin standard solution (4 mL) was pumped into the reaction coil of the hydride generator, and the absorbance of the generated tin hydride was measured by the atomic absorption spectrophotometer.  Similarly, the  absorbance of any tin hydride produced from each mixture of the digested blank and the standard tin solutions was also measured. Two replicate determinations of each sample mixture were carried out. The total tin contents of the samples were then calculated, after the blank values had been subtracted. Quantitation by using the more difficult and time consuming standard addition method reference  was preferred in this study, because repeated analyses of the certified material  Tort  1, by the  normal  calibration  method  consistently  overestimated its total tin content by about three fold. This situation could not be  180 improved upon, neither by the use of background correction nor blank subtraction. Without the addition of 2% L-cysteine into the sample and standard solutions, quantitation would not be possible, because the tin absorbance was completely suppressed in some determinations. The mechanism of action of L-cysteine is not  known with certainty. The total tin content of the samples and the standard reference materials are shown in Table 4.3. The total tin content of the standard reference material Tort 1 obtained in . Therefore, the 229 this study is in the range previously reported by Sturgeon et al digestion method and the HG-AAS method employed in this study are suitable for the determination of total tin in marine animals. Since the normal calibration method of quantitation gave a much higher total tin value than the certified value for Tort 1, another atomic absorption method capable of reproducing the total tin content of Tort 1, by normal calibration procedure was sought, because of the rapidity and ease of this quantitation method. Two non-conventional  hydride generation-atomic  absorption methods; a  7 and a batch HG-GFAAS continuous HG-AAS method developed by Le et aI method reported by Sturgeon a.i , were considered. The batch HG-GFAAS was 229 7 preferred over the non-conventional continuous HG-AAS method of Le et al because, their method was not validated for the quantitative determination of tin, as  7 is not neither its reproducibility nor detection limit was reported. Also, the method capable of reaching the very high atomization temperatures characteristic of the  181 Table 4.3  Total tin content of samples analyzed by the HG-AAS method.  Sample  Tort  b 1  Origin  NRC, Canada  Total tin content  Certified  (g/g dry wt)a  value (ig/g dry wt)  0.16 ± 0.04  0.139 ± 0.011c 0.144 ±  Pacific oyster  Cambell River  0.34 ± 0.09  Fanny Bay  0.35 ± 0.03  Jervis Inlet  0.13 ± 0.01  d 0016  Crassostrea gigas Pacific oyster Crassostrea gigas Pacific oyster Crassostrea gigas  a=Total tin content and the standard deviation for 3 replicate determinations b =Lobster hepatopancrease, a standard reference material from the National Research Council, Canada (NRC). c=Value certified by NRC, Canada. d=Value reported by Sturgeon Lal . 229  batch HG-GFAAS method. Therefore, molecular absorption or non-atomization of refractory tin compounds in the air-acetylene flame, in the quartz furnace of the nonconventional continuous HG-AAS might pose a problem.  182 Consequently, a batch HG-GFAAS apparatus (Figure 4.2) modelled on the principle reported by Sturgeon  but slightly different in design was constructed  and optimized for total tin determination.  4.4.4 Batch  hydride  generation-graphite  furnace  atomic  absorption  specirophotometry (HG-GFAAS). The use of the continuous HG-AAS method, has a major disadvantage of consuming large amounts of samples and reagents, and is therefore wasteful and expensive. Conversely, the batch hydride generation method consumes very small amounts of samples and reagents. The small amounts of tin hydride generated are suitable for trapping on a graphite furnace tube, where it is preconcentrated prior to atomization. The preconcentration step, and the ability of the inside surface of the graphite tube to reduce some refractory compounds, are expected to increase the sensitivity and the detection limit of this method. For normal operation in the graphite furnace mode, the steps involved are: sample drying, ashing, atomization, and tube cleaning. During the drying stage, solvent or water is removed from the sample. At the ashing step, organic and inorganic matrices are removed. However, in the hydride generation-graphite furnace method (HG-GFAAS),  organic and inorganic matrices are minimized. At the  atomization step, free atoms of the analyte are generated in the graphite tube, and their absorbances are measured by the atomic absorption spectrometer. During the graphite furnace operation, the incandescent  graphite tube is protected from  183 excessive corrosion by an upward flow of an inert gas such as argon or nitrogen (internal purge). The use of the graphite furnace tube to trap or adsorb tin hydride has been demonstrated by Sturgeon Lili 5 and was the basis for a HG-GFAAS ’ 229  method  reported by these authors . The extension of this methodology to trap tin 5 ’ 229 hydride in graphite furnace tubes precoated with sodium tungstate, and palladium modifiers is described in this section. In the method reported by Sturgeon Li , 229 no modifiers were used, probably because there was no interference from their sample matrix. In this study, the use of perchloric acid for sample digestion as used by Sturgeon et a1 229 was avoided because of its explosive nature, instead KOH, , 4 3 HNO S0 and HF were used in various stages of the sample digestion (Section 2 H , 4.3). The difference in reagents used for sample digestion may have contributed to the extent of interferences observed during the HG-GFAAS analysis reported in the present study. The initial approach taken to remove these interferences involved the manual injection of solutions of sodium tungstate or palladium modifiers into the graphite furnace tube prior to every absorbance measurement. 1_ater, the use of graphite furnace tubes pre-treated with solutions of palladium or sodium tungstate, and the presence of L-cysteine in the batch hydride generator, made it possible to analyze the environmental samples. The use of graphite furnace tubes pre-coated with solutions of these modifiers eliminates the inconvenience of manually injecting modifiers into the graphite furnace tube during each analysis.  184 4.4.4.1  Optimum  concentrations  of reagents  needed  for tin hydride  production in the HG-GFAAS method. At a trapping temperature of 700°C, standard tin solution in 0.5M HC1 (1 mL  of 12 ng/mL Sn), and measured amounts of 0.2 M hydrochloric acid (2  -  20 mL)  were pipetted into the batch hydride generator via a B24 joint at the top of the hydride generator. Measured amount of aqueous sodium borohydride (4mL of 2 % w/v in 0.2% KOH solution) was pumped into the batch hydride generator to react with a standard tin solution. The absorbance measurements represent the amount of tin hydride produced (Table 4.4).  Table 4.4  Reagent ratios needed to maximize tin hydride generation  Volume 0.2 M HC1 (mL)  Volume 2 % NaBH 4 (mL)  Absorbance  2  4  0.038 ± 0.007  5  4  0.040 ± 0.003  10  4  0.040 ± 0.007  20  4  0.028 ± 0.001  (±)a  a=Standard error for three determinations.  All the volume ratios of the reagents examined gave about the same absorbance values when 2  -  10 mL of 0.2 M HC1, and 4 mL sodium borohydride were used for  185 tin hydride production (Table 4.4). A probable reason for this observation is that in all the cases, the sodium borohydride was present in excess, therefore the reaction went to completion at all the reagent ratios studied. Since the reagent ratios used did not appear to be critical for tin hydride production, provided the sodium borohydride was in excess, all batch hydride generation, experiments were carried out at the reagent ratio of 0.2 M HC1 (5 mL) : 2% sodium borohydride (4 mL).  4.4.4.2  Optimum flow rate of sodium borohydride into the batch hydride generator.  The effect of the flow rate of sodium borohydride solution into the batch hydride generator, on the absorbance of tin hydride is shown in Figure 4.5. As the 0.05  0.04  0.03  0.02  0.01 4.0  4.5  5.0  5.5  6.0  6.5  7.0  4 flow rate (mLlmin) NaBH  Figure 4.5  Effect of sodium borohydride flow rate on absorbance. (The width of  the bars is arbitrary. The error bars are std error for 3 determinations).  186 sodium borohydride flow rate was increased from 4.4 to 6.3 mL/min, a maximum absorbance was observed at about 5.40 mL/min. This indicates that the flow rate of the sodium borohydride into the hydride generator affects the production of the tin hydride. In their work, Sturgeon  229  used a flow rate of 4 mL/min to deliver 2  mL of sodium borohydride solution into their hydride generator. The difference in flow rate between the batch hydride generator used in this study and the one 229 may be due to the difference in the size of the two reported by Sturgeon et a! hydride generators.  4.4.4.3  Optimum temperature  for trapping tin hydride in the pre—treated  graphite furnace tubes. The effect of temperature, on the ability of the graphite furnace tubes to trap tin hydride was studied by measuring the absorbance of tin hydride produced from a reaction between standard tin solution (1 mL of 14 g/mL solution) and sodium borohydride (4 mL of 2% solution), as the temperature of the graphite furnace tube is varied. The result obtained is shown in Figure 4.6. The absorbance of the tin hydride, as the temperature of the graphite furnace tube is varied, is an indication of the trapping efficiency of the graphite furnace tube. In the temperature range studied, maximum trapping efficiency was obtained at about 700 °C. This trapping temperaturç  is lower than the value reported by  , by 100 °C. The lower trapping temperature established in the 229 Sturgeon et a! present study may be due to the pre-treatment of the graphite tubes with sodium  187 tungstate matrix modifier, and is expected to prolong the “life span” of the graphite tube. 0.08  0.07  I  e  0.06  0,05  0.04  I  I  I  I  600  700  -  -  -  -  100  -  200  300  400  500  800  Trapping temperature °C Figure 4.6  Effect of trapping temperature on the atomic absorbance of tin hydride.  4.4.4.4  Optimum trapping time.  A study was carried out to find the trapping time needed to produce maximum absorbance. Figure 4.7 shows the effect of trapping time on the absorbance of tin hydride. Trapping efficiency as monitored  by absorbance  measurements  was  maximum at 250-260 seconds. Thereafter, the trapping efficiency decreased. The  188 decrease in absorbance as trapping time increased beyond 260 seconds may be due to the desorption and escape of the tin hydride from the graphite tube. 0.05 I I I t I I I -  0.04  0.03  -  -  0,02-— 200 210  I  I  220  230  240  250  260  270  280  Trapping time (s) Figure 4.7 4.4.4.5  Effect of trapping time on absorbance. Pre-treatment of graphite furnace tubes with modifiers.  Vickrey et a1 242 have coated some carbide forming elements  such as  zirconium, chromium, and molybdenum onto graphite furnace tubes by soaking the graphite furnace tubes in solutions of these elements. This coating method 242 was effective in reducing atomization interferences  in the GFAAS determination of  organotin compounds. Another method of coating graphite furnace tubes with carbide forming elements has been described by Almeida and Seitz . This method 243  involved soaking the graphite furnace tubes in solutions of titanium, molybdenum,  189 or tungsten salts under reduced pressure, in a vacuum line. This method was described by the authors as being more efficient in causing the penetration of the . 242 graphite furnace tubes by the modifier, than the method of Vickrey et a1 , the modifiers tungsten and titanium coat on the 243 According to Almeida and Seitz graphite furnace tubes as oxides, but are converted to the carbides at the high graphite furnace temperatures. The coating of graphite furnace tubes with carbide forming elements has also . His method involved the injection of an aqueous solution 244 been described by Lagas of lanthanum chloride into a graphite furnace tube. Drying, ashing, and atomization programs of the graphite furnace converted the injected lanthanum salt to its carbide. The physical basis for the action of carbide forming elements in GFAAS has . According to this author, the carbide coating prevents 244 been described by Lagas physical contact between the graphite tube and the analyte thereby preventing carbide formation by the analyte. Although the use of palladium modifiers to coat GFAAS tubes has not been reported, it was thought feasible to explore such methodology. It is expected that the mechanism of modifier action would be the same as when palladium solution is premixed with the analyte as is normally done in GFAAS. Its use as a modifier in the d 46 47 48 . reporte 245 2 analyses of an increasing number of elements has been ’ , 249 Palladium does not act by forming carbides. According to Volynsky palladium acts by forming intermetallic compounds and solid solutions with the analyte in the graphite furnace. To function as a modifier, palladium salts are usually  190 reduced to Pd(0) by using either a solution of citric acid or ascorbic acid. In the present study, palladium and sodium tungstate modifiers were coated onto graphite furnace tubes, by soaking the graphite furnace tubes in solutions of these modifiers over a period of 26 hours as described in Section 4.2.8 The effect of the concentration of the modifiers on the absorbance of tin hydride, produced by reacting 1 mL of 14 ig/mL tin solution with 4 mL of 2% sodium borohydride in the batch hydride generator is shown in Figures 4.8 and 4.9. For the graphite furnace tubes coated with sodium tungstate, treatment with 8 % sodium tungstate gave maximum absorbance, while treatment with 4% palladium modifier gave maximum absorbance for the palladium-treated graphite tubes. The exact amount of the modifier coated on each graphite furnace tube was not determined, but it was assumed that the graphite furnace tubes would absorb similar volumes of the modifiers, hence the amount of modifier coated onto these tubes should be proportional to the concentration of the modifier solution in which the graphite furnace tubes were soaked. This assumption seems reasonable because the pre-used graphite furnace tubes were in about the same physical condition, and were from the same batch supplied by the same manufacturer. Comparatively, at the optimum modifier concentrations, sodium tungstate treated graphite furnace tubes gave better tin absorbance than palladium treated tubes (Table 4.5). Also, analyses with the palladium treated graphite tubes was characterized by a loud popping sound resulting from the ignition of the air-hydrogen mixture in the heated graphite tube. Therefore, the graphite tubes treated with 8%  191 0.07  0.06  I  I  I  I  2  4  I  I  I  I  6  8  10  —-  -  I 0.05-  0.04 0  12  Concentration of sodium tungsiate (% wlv) Effect of sodium tungstate concentration on absorbance.  Figure 4.8  0.06  0.05  I  I  2  -  I  I  I  I  I  I  4  6  8  10  I  -  0.04  0.03  0.02  0.01  -  0.00 0  Concentration of palladium (% w/v) Figure 4.9  Effect of palladium on absorbance.  12  192  sodium tungstate were preferred over those treated with the 4% palladium solution.  Table 4.5  Comparison of palladium and sodium tungstate-lreated  graphite  furnace tubes showing the atomic absorbance of tin hydride generated from 14 JLg/mL tin solution (1 mL). (±)a  Graphite furnace tube treatment  Absorbance  4% Palladium treatment  0.054 ± 0.002  8% Sodium tungstate treatment  0.062 ± 0.003  a=Standard error for three determinations.  4.4.4.6  Determination of total tin content of a standard reference material by  the HG-GFAAS method. About 2.00 g of Tort 1 (lobster hepatopancrease),  a standard reference  material was digested as described in Section 4.3. A blank solution of all the reagents used to digest Tort 1 was similarly digested. The digested Tort 1 solution was transferred to a 25 mL volumetric flask containing L-cysteine, and dissolved in de-ionized water, so that the concentration of L-cysteine was 2% w/v. A solution of the digested blank, in 2% L-cysteine was similarly prepared. Aliquots of the digested sample or the digested blank solutions (3 mL), sodium borohydride (4 mL) and HC1 (5 mL) were reacted in the batch hydride generator, and the absorbance of the  193 generated tin hydride was measured. Quantitation of total tin in Tort 1 was carried out by the normal calibration method, by using the calibration curves obtained as described in Section 4.2.8.2. The total tin content obtained by using HG-GFAAS is shown in Table 4.6.  Table 4.6  Total tin content of a standard reference material Tort 1 obtained by different authors.  Technique (Source)  Value  HG-GFAAS, (the present study)  0.102 ± 0.018a  HG-GFAAS, (Sturgeon  0.144 ± 0.016k’  ii232)  HG-GFAAS, Isotope dilution ICP-MS  b 0.139 ± 0.01 1  (NRC, Canada)  a= Standard deviation of two replicate determinations. b = Standard deviation, but number of replicate determinations was not given by the authors.  The total tin content of Tort 1 determined by the batch HG-GFAAS method described in this study lies close to the lower range of values certified for Tort 1 (Table 4.6). The performance of this batch hydride generator may be improved further, by using other optimization techniques, such as the simplex method.  194 However, the batch HG-GFAAS method described in the present study offers an , especially in situations 229 alternative to the method described by Sturgeon et a1 229 was where matrix interferences are a problem. This is because their method reported to operate without encountering any interferences, therefore its operation in situations such as in the present study where intereferences posed a problem, has not been tested. When the continuous HG-AAS method and the batch HG-GFAAS used in this study are compared, the latter offers a faster method of quantitation by normal calibration. The continuous HG-AAS method is time consuming, involving standard additions. Other comparative data on the two atomic absorption methods of analysis used in the present study are shown in Table 4.7.  Table 4.7  Comparison of figures of merit obtained  with the two atomic  absorption spectrophotomelric methods used in this study. HG-AAS  HG-GFAAS  Detection limit  7.5 ng/ mL  1.8 ng/mL  Precision (for 10 runs)  4.3%  3.3%  195 CHAPTER 5  SUMMARY AND CONCLUSIONS. Studies involving the analyses of organotin compounds in marine organisms of British Columbia, Canada, and the effect of organotin compounds  on the  permeability of model biological membranes have been presented in this thesis. Analysis of organotin compounds in marine organisms by GC-MS SIM affords a very specific technique for the identification and quantitation of these organotin compounds  by using the peculiar  isotope pattern  for tin compounds.  This  methodology is therefore able to distinguish organotin compounds from other compounds that may co-elute with them from the GC. The major organotin pollutants found in this study for the coastal areas of British Columbia were tributyltin and dibutyltin species. Dicyclohexyltin levels of 3.5 ng/g (wet wt as Sn) and 21.3 ng/g (wet wt as Sn) were found in only two locations, namely  Wreck  Beach,  Vancouver  and  Anyox. Therefore,  pollution  from  dicyclohexyltin species is not widespread in the coastal areas studied. Contamination of mussels and clams by tributyltin and dibutyltin species is widespread, although some locations such as Hastings Arm, Hilton Point Kitimat, Alice Arm and Holberg Sound show no organotin pollution. The butyltin body burden for Blue mussels in the contaminated areas sampled range from 14.4 ng/g to 37.3 ng/g (wet wt as Sn) for tributyltin species and 6.7 to 67.3 ng/g (wet wt as Sn) for dibutyltin species. The trend observed is that most Blue mussels showed the  196 presence of both dibutyltin and tributyltin. This is expected because dibutyltin species are degradation products of the tributyltin species, and should be present if tributyltin compounds are present. The Soft shell clams analyzed showed tributyltin levels ranging from 0.7 to 19.4 nglg (wet wt as Sn). The dibutyltin species were mostly not detected even in the Soft shell clams that were contaminated by tributyltin species. This observation is contrary to expectation, and may indicate that dibutyltin species are easily eliminated from the soft shell clams when compared to the Blue mussels. For the other species of mussels and clams analyzed, paucity of samples could not allow for any trend to be established in terms of preferential accumulation of tributyltin or dibutyltin species. The study of tributyltin body content of Soft shell clams from Quatsino Sound showed a significant reduction in levels from 26.3 ng/g (wet wt as Sn) to 5.5 nglg (wet wt as Sn) over a period of three years. This reduction in the concentration of tributyltin species may be due to the Canadian Government’s regulation of the use of tributyltin compounds, which came into effect in 1989. When compared to some other locations of the world contaminated by tributyltin species, the mussels analyzed in this study show comparable pollution to mussels from San Diego Bay, U.S.A. 122 and Tokyo Bay, Japan”’ . 4 The use of 1 H NMR spectroscopy to study the effect of organotin compounds on the permeability properties of model biological membranes was presented in Chapter 3 of this thesis. This represents the first application of proton 1 H NMR to study the permeability properties of butyltin-EPC liposomes. Direct study of the  197 permeation of the organotin compounds from the model biological membranes was made difficult by their high hydrophobicity and low aqueous solubility. The use of dimethylarsinic acid (DMA) as a permeability probe afforded information on how the organotin compounds affect the permeability of model biomembranes. When tributyltin chloride was added to the extraliposomal compartment of an EPC liposome, the permeability of the liposomes to dimethylarsinic acid greatly increased. The mechanism of the increased permeability could not be determined because the diffusion equations [3.0] and [3.26] (Chapter 3, section 3.9), developed to describe this efflux behavior were unable to distinguish between situations in which there is 100% passive diffusion and 100% facilitated diffusion. However, the increased liposome permeability  shows a linear relationship  with the concentration  of  tributyltin chloride present in the extraliposomal compartment according to the relationship Y=8.01 x 10 X + 1.46 x i(I, where Y is the efflux rate constant and 4 X is the concentration of tributyltin chloride in the extraliposomal compartment. The permeability  coefficient for DMAH efflux from EPC liposomes  decreased from 1.7 x 10-8 cm/s to 4 x i0 cm/s. when monobutyltin trichloride was added to the extraliposomal compartment  of EPC liposomes. The decreased  permeability of the EPC liposomes to DMAH in the presence of monobutyltin trichloride does not show a linear relationship. Beyond 16.7 jM monobutyltin trichloride added into the extraliposomal compartment, no further decrease in permeability coefficient was observed. When trimethyltin hydroxide was added to the extraliposomal compartment  198 of EPC liposomes, the efflux of DMAH was by passive diffusion while the efflux of DMK (another species of DMA present in solution at pH 7.4) was by facilitated diffusion mediated by trimethyltin cation. The rate constant for the passive diffusion of DMAH (2.5 x iO Is) when in contact with trimethyl hydroxide was lower than in the absence of trimethyltin hydroxide (1.1 2 xlO Is). For the tributyltin chloride-EPC liposomes (TBT-EPC), it was found that DMAH permeated  by passive diffusion, while DMK permeated by facilitated  diffusion if tributyltin chloride was absent in the extraliposomal compartment. This facilitated diffusion was mediated by the tributyltin cations. The rate constant for the passive diffusion of DMAH was dependent on the tributyltin chloride content of the liposomes, and varied from 3.2 x i(i Is to 1.7 x i0 Is for TBT-EPC A and TBT EPC B liposomes of composition 5 g TBT  0.2 g EPC  ,  and 1.5 jg TBT  0.2 g  EPC respectively. The rate of facilitated diffision of DMK encountered in this study, was found to be controlled by the ratio of the formation constant to the dissociation constant (B/), of the trialkyltin-DMA complex. The ratio BI was found to be 2.5, and 2.1 for the trimethyltin-DMA  and tributyltin-DMA complexes respectively. When  tributyltin chloride was present in the extraliposomal compartment of a TBT-EPC liposome, the ability of tributyltin cation to mediate facilitated diffusion was removed. The activation energy for the passive efflux of DMAH from the TBT-EPC liposomes was also concentration dependent and decreased as the tributyltin chloride content of the liposome increased. The activation energies obtained were 64.4  199 U/mo! and 52.3 kJ/mol for TBT-EPC A and TBT-EPC B liposomes respectively. The Arrhenius pre-exponential factor for the TBT-EPC liposomes did not show much variation with the tributyltin chloride content of the liposome, and was calculated to be 2.8 x 10-8 and 4.0 x 10-8 /s for TBT-EPC A and TBT-EPC B respectively. For the monobutyltin trichloride-EPC liposomes (MBT-EPC), the efflux of DMA was by passive diffusion irrespective of whether monobutyltin trichloride was present in the extraliposomal compartment or not. The permeability coefficient of DMAH for the MBT-EPC liposomes showed slight dependence on the monobutyltin trichioride content of the liposomes, and was 4.9 x i0 cm/s and 4.4 x i0 cm/s for MBT-EPC A and MBT-EPC B liposomes of composition 0.5 g MBT: 0.2 g EPC, and 1.5 g MBT: 0.2g EPC  respectively. The activation energy for the efflux of  DMAH is 106.8 and 121.5 kJ/mol for MBT-EPC A and MBT-EPC B respectively. This indicates that the MBT-EPC liposomes become less permeable with increasing concentration of monobutyltin trichioride in the liposomes. The Arrhenius pre exponential factors calculated for the these liposomes are 1.1 x 10-8 and 1.0 x 10-8 /s for MBT-EPC A and MBT-EPC B liposomes respectively. From the results of these permeation experiments, it is concluded that tributyltin chloride and monobutyltin trichioride exert different effects on the permeability of both EPC liposomes and butyltin chloride-EPC liposomes. Whereas tributyltin cation has the ability to act as a carrier for DMK, the monobutyltin cation has no such ability. The present study is the first report of the facilitated transport  200 of an environmentally important compound, such as dimethylarsinate by trialkyltin cation. The observation that the Arrhenius pre-exponential factor for the butyltin chloride-EPC liposomes are approximately constant for each type of liposomes, but different for  the TBT-EPC and the MBT-EPC liposomes tends to support the  conclusion that tributyltin and monobutyltin species exert different effects on the permeability properties  of membranes. This difference may contribute to the  different toxic effects observed for these butyltin compounds on marine organisms. H NMR spectroscopy is a useful technique for studying the effect Although 1 of organotin compounds on model membranes, its low sensitivity precludes the direct study of organotin permeation at very low concentrations. There is also the possibility that the spectroscopic shift reagent used to distinguish between the proton resonance inside and outside the liposomes may also modify the properties of the liposomes, thereby influencing the results. The use of a radioisotope technique which would allow for low level organotin permeation studies would be desirable, to check on the results obtained in this investigation. Chapter 4 of this thesis is concerned with the determination of total tin in oysters from some locations in British Columbia, Canada. Two types of hydride generation-atomic absorption methods were employed for total tin determination: a continuous HG-AAS and a batch HG-GFAAS method. The HG-AAS method was optimized for experimental variables such as reagent concentrations, while the HG GFAAS method was optimized for reagent concentrations, reagent flow rates,  201 trapping time and temperatures. 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Schiemmer, G., Weltz, B., Spectrochim. Acta 41B, 1157 (1968).  249.  Volynsky, A., Analyst 116, 145 (1991).  220 APPENDiX A MAP OF BRiTISH COLUMBIA, CANADA, SHOWING SOME LOCATIONS SAMPLED FOR ORGANOTIN POLLUTION.  50  221 APPENDiX B  THE NMR SPECTRAL ACQUISITION AND WATER SUPPRESSION PARAMETERS FOR THE EFFLUX OF DIMETHYLARSINIC ACID FROM  LIPOSOMES. ;Baz:AU PROGRAM FOR DIFFUSION EXPERIMENTS II 2ZE VD 1DOHG 00=1 DO WR#1 IF #1 IN=2 EXIT VD VDLIST.001  1-27 appropriate delays (seconds) 1  0.001  10  1116.8  19  1716.8  2  216.8  11  1116.8  20  3516.8  3  216.8  12  1116.8  21  3516.8  4  216.8  13  1116.8  22  3516.8  5  216.8  14  1716.8  23  3516.8  6  516.8  15  1716,8  24  3516.8  222 7  516.8  16  1716.8  25  3516.8  8  516.8  17  1716.8  26  3516.8  9  516.8  18  716.81  27  3516.8  VD; the variable delay was obtained by subtracting the time required to obtain 48  scans from the intended delay. The time required for the NMR spectrometer to obtain 48 scans was 83.2 seconds. Other NMR parameters were: PW=3 DP=OL DR INITIAL= 16  FINAL =8  Dl =0.5 D3=3OS PW=PO=3.OO NS=48 DE=77.50 DS=2 Dl is the duration of the presaturation pulse D3 is the pulse delay time DS is the dummy scan PW is the pulse width DR is the digitizer resolution DP is the decoupler power  223 APPENDIX C MICHAELJS-MENTONS  EQUATIONS FOR ENZYME KINETICS  For enzyme catalyzed reaction:  k 1 2 k E+S”ES-’Product+E 1 k  where E is the enzyme and S is the substrate.  -  [ES]  E][S] [k 1 1 k  +  [ES] is the concentration of complexed enzyme 1 + k )/k, 2 where (k  =  km  km is Michaelis constant. The rate of the forward reaction is:  -  V[S]  km  +  [SI  224 APPENDIX D WET ASifiNG APPARATUS WiTH AIR COOLED REFLUX CONDENSER USED FOR DIGESTION OF MARINE ANIMALS.  i.d 15 mm  A  B  Wet ashing apparatus (Taken from reference 242) showing the following parts (A) Teflon cylinderical plugs, (B) Teflon diffusion funnel, (C) Teflon stopper with capillary, (D) 500 mL round bottom flask  

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