Distribution of Phthalate Esters in a Marine Aquatic Food-Web

Home , Benzyl butyl phthalate, Diisoheptyl phthalate

DISTRIBUTION OF PHTHALATE ESTERS

IN A MARINE FOOD WEB

Cheryl Mackintosh

B.Sc., University of British Columbia, 1996

A PROJECT SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE DEGREE OF MASTER OF RESOURCE

MANAGEMENT

in the School of Resource and Environmental Management

Report No. 295

Simon Fraser University

APRIL 2002

means, without the permission of the author.

APPROVAL

NAME: Cheryl Mackintosh

DEGREE: Master of Resource Management

TITLE OF PROJECT: Distribution of Phthalate Esters in a Marine Food Web

REPORT NO.: 295

EXAMINING COMMITTEE:

Dr. Frank A.P.C. Gobas Senior Supervisor Associate Professor School of Resource and Environmental Management Simon Fraser University

Dr. Margo Moore Associate Professor Department of Biological Sciences Simon Fraser University

Date Approved:______

ii ABSTRACT

Phthalate esters (PEs) are widely used chemicals, with over 4 million tonnes being produced worldwide each year. PEs exhibit octanol-water partition coefficients (Kow) ranging from

101.8 for dimethyl phthalate to 1010.6 for di-iso-decyl phthalate. Because of their hydrophobicity, some congeners have the potential to bioconcentrate and biomagnify in marine and aquatic food chains. There are currently no reported field studies on phthalate ester bioaccumulation. To investigate PE bioaccumulation in a marine food web, a field study was conducted in False Creek Harbour, Vancouver, Canada. The study involved collecting samples of seawater, sediment and eighteen marine species. Samples were analyzed by GC-LRMS for eight individual phthalate congeners (i.e., dimethyl, diethyl, di- iso-butyl, di-n-butyl, butyl-benzyl, di(2-ethylhexyl), di-n-octyl, di-n-nonyl), and by LC-

ESI/MS for five isomeric mixtures (i.e., di-iso-hexyl (C6), di-iso-heptyl (C7), di-iso-octyl

(C8), di-iso-nonyl (C9), di-iso-decyl (C10)). Environmental concentrations were determined and corresponding fugacities were calculated. PE fugacities in the sediment were greater than those in the freely dissolved water fraction. The degree of sediment-water disequilibrium decreased with KOW from a factor of 17,700 for dimethyl phthalate to values between 2.7 and 44 for the other twelve PEs. For the low KOW PEs (i.e., dimethyl and diethyl) fugacities in the biota were between those in the sediment and water, and did not exhibit a trend with trophic position in the food web. For the intermediate KOW PEs (i.e., di- iso-butyl, di-n-butyl, benzyl-butyl, C6, and C7), fugacities in the biota were lower than those in the sediment, comparable to those in the freely dissolved water fraction, and did not show a statistically significant pattern with trophic position. For the high KOW PEs (i.e., di(2- ethylhexyl), di-n-octyl, di-n-nonyl, C8, C9, C10), fugacity significantly declined with

iii increasing trophic position, and fugacities in the media appeared to decline from sediment ≥ freely dissolved water ≅ prey species > predator species. These results suggest that PEs do not biomagnify in the food web. Equilibrium partitioning between the organisms and the water appears to occur for the low and intermediate KOW phthalates, while trophic dilution in the food web occurs for the high KOW phthalates. Mean bioaccumulation factors (BAFs,

L/kg lipid) based on the “total” water concentration were generally below the Canadian

Environmental Protection Act (1999) bioaccumulation criteria of 100,000 L/kg lipid. BAFs for butyl-benzyl, di(2-ethylhexyl), di-n-octyl, di-n-nonyl, C6, C7, C8, C9, and C10, based on the “freely dissolved” water concentration, generally exceeded the CEPA criteria. Biota-

Sediment Accumulation Factors (BSAFs, kg OC/kg lipid) for the benthic species in False

Creek were generally less than one.

iv ACKNOWLEDGEMENTS

First off, I would like to sincerely thank my senior supervisor, Dr. Frank Gobas for his vast knowledge in the field of environmental toxicology, his valuable guidance with the project, and his continually positive attitude. I learned a great deal from him and am grateful for the opportunities he provided for presenting my research at conferences. I also wish to thank Dr.

Michael Ikonomou from the Institute for Ocean Sciences for overseeing the chemical analysis on the project and his expertise in analytical environmental chemistry, from which I greatly benefited. Many thanks to my second supervisor, Dr. Margo Moore, for her insightful comments on the project and thesis write-up. Thanks also to Tom Parkerton and Ken

Robillard, who provided comments throughout the project. There were several people who were instrumental in assisting with the hands-on aspects of this research project. I am grateful for the excellent efforts of Audrey Chong, Jing Hongwu, Jody Carlow, Natasha Hoover,

Zhongping Lin, and Linda White who conducted the chemical analysis for the project at the

Institute for Ocean Sciences. I would also like to thank several people for their assistance with the field collections: Shane Cuff, John Wilcockson, Dave Swanston, Barry Kelly, Laura

McLean, Jon Arnot, Glenn Harris, Kim Chapman, and Elsie Sunderland. Thanks also to

Laurie Wilson of the Canadian Wildlife Service for providing the surf scoter bird samples. I would like to thank Natural Sciences and Engineering Research Council of Canada (NSERC) for scholarship funding. Funding for the project was received from the American Chemistry

Council, Environment and Health Canada through the Toxic Substances Research Initiative

(TSRI), and from NSERC.

I would also like to thank my partner Daryl, my family, the “TOX” group, and my volleyball teammates for helping make these last four years rich and memorable. Finally, I would like to dedicate this work to the memory of my father Ted, who supported and encouraged me in all my endeavors. v TABLE OF CONTENTS APPROVAL...... II ABSTRACT...... III ACKNOWLEDGEMENTS ...... V TABLE OF CONTENTS ...... VI LIST OF FIGURES ...... X LIST OF TABLES...... XVII DEFINITIONS...... XXIII 1. INTRODUCTION...... 1

2. METHODS ...... 8

2.1. FIELD SAMPLING METHODS ...... 8 2.1.1. Study Site and Design...... 8 2.1.2. Preparation of Field Sampling Equipment...... 11 2.1.3. Sediment Sample Collection...... 11 2.1.4. Water Sample Collection...... 12 2.1.5. Biota Sample Collection...... 13 2.2. ANALYTICAL METHODS FOR DETERMINING PHTHALATE ESTER CONCENTRATIONS IN ENVIRONMENTAL SAMPLES...... 17 2.2.1. Materials ...... 17 2.2.2. Preparation of Glassware and Reagents...... 18 2.2.3. Extraction and Cleanup of Sediment and Biota Samples ...... 18 2.2.4. Extraction and Cleanup of Seawater Samples ...... 20 2.2.5. Quantification of Suspended Particulate Matter in the Seawater Samples...... 22 2.2.6. GC/MS Analysis of Environmental Samples ...... 25 2.2.7. LC/ESI-MS Analysis of Environmental Samples...... 25 2.2.8. Optimization of ESI-MS Parameters...... 27 2.2.9. LC/ESI-MS/MS Analysis of Environmental Samples...... 27 2.2.10. MS Calibration, Recovery and Procedural Blanks ...... 28 2.2.11. Quantitation of Phthalate Esters in Environmental Samples...... 29 2.2.12. Quantification of Diisodecyl Phthalate (C10) in Biota Samples...... 30 2.3. QUALITY ASSURANCE AND CONTROL OF DATA (QA/QC) ...... 32 2.3.1. Sediment & Biota Concentration Data ...... 32 2.3.2. Seawater Concentration Data...... 36 2.3.3. Summary of the Sediment, Biota and Seawater Data Quality ...... 43 2.4. MEASUREMENTS OF ORGANIC CARBON & LIPID CONTENTS IN SEDIMENT AND BIOTA SAMPLES ...... 45 2.4.1. Organic Carbon Content Analysis ...... 45 2.4.2. Lipid Content Determination ...... 47 2.5. DATA ANALYSIS AND NORMALIZATIONS ...... 47 2.5.1. Analysis of Concentration Distributions ...... 47 2.5.2. Sediment Organic Carbon Normalization...... 48 2.5.3. Biota Lipid Normalizations ...... 48 2.5.4. Fugacity Calculations ...... 50 2.5.5. Trophic Position Calculation ...... 51 3. RESULTS & DISCUSSION...... 57

3.1. SEDIMENT CONCENTRATIONS OF PHTHALATE ESTERS ...... 57 3.1.1. Concentration Summary...... 57 3.1.2. Spatial Variability ...... 61 3.2. SEAWATER CONCENTRATIONS OF PHTHALATE ESTERS...... 63 3.2.1. “Total” Seawater Concentration Summary ...... 63 3.2.2. Spatial Variability ...... 64 3.2.3. Ratio of Seawater Concentrations to Aqueous Solubilities ...... 65 3.2.4. Distribution of Phthalate Ester Internal Standards between the Glass Fibre Filter and C18 Extraction Disks ...... 66

vi 3.2.5. Distribution of Seawater Borne Phthalate Esters between the Glass Fibre Filter and C18 Extraction Disks ...... 68 3.2.6. Summary of the “Total”, “C18”, and “Freely Dissolved” Water Concentrations...... 75 3.2.7. Chemical Fugacities in the Water...... 77 3.3. SEDIMENT - WATER DISTRIBUTION OF PHTHALATE ESTERS...... 78 3.4. BIOTA CONCENTRATIONS OF PHTHALATE ESTERS ...... 82 3.4.1. Biota Concentration Overview...... 82 3.4.2. Spatial Variability ...... 83 3.4.3. Distribution of Phthalate Esters in Sediment, Seawater, and Biota and Chemical Transfer through the Food Web...... 86 3.4.4. Summary of Food Chain Bioaccumulation Results...... 121 3.4.5. Discussion ...... 123 3.5. BIOTA - WATER DISTRIBUTION OF PHTHALATE ESTERS ...... 131 3.5.1. Overview ...... 131 3.5.2. Bioaccumulation Factors (BAFs)...... 132 3.5.3. Chemical Distribution in the Food Chain...... 156 3.5.4. Relationship between the Lipid BAFs, based on the “Total” water concentration, and the Octanol – Seawater Partition Coefficient...... 158 3.5.5. Relationship between the Lipid BAFs, based on the “Freely Dissolved” water concentration, and the Octanol – Seawater Partition Coefficient...... 163 3.6. BIOTA - SEDIMENT DISTRIBUTION OF PHTHALATE ESTERS...... 166 3.6.1. Overview ...... 166 3.6.2. Biota - Sediment Accumulation Factors (BSAFs) ...... 166 3.6.3. Relationship Between the BSAF in Benthic Species and the Octanol-Seawater Partition Coefficient …...... 174 REFERENCES...... 178

APPENDIX A: BACKGROUND INFORMATION ON PHTHALATE ESTERS...... 195 1. INTRODUCTION ...... 1 2. METHODS...... 8 2.1. Field Sampling Methods...... 8 LATIN NAME...... 14 2.2. Analytical Methods for Determining Phthalate Ester Concentrations in Environmental Samples 17 2.3. Quality Assurance and Control of Data (QA/QC) ...... 32 2.4. Measurements of Organic Carbon & Lipid Contents in Sediment and Biota Samples ...... 45 2.5. Data Analysis and Normalizations...... 47 3. RESULTS & DISCUSSION...... 57 3.1. Sediment Concentrations of Phthalate Esters ...... 57 3.2. Seawater Concentrations of Phthalate Esters...... 63 3.3. Sediment - Water Distribution of Phthalate Esters ...... 78 3.4. Biota Concentrations of Phthalate Esters ...... 82 3.5. Biota - Water Distribution Of Phthalate Esters ...... 131 3.6. Biota - Sediment Distribution Of Phthalate Esters ...... 166 REFERENCES...... 178 I) NORMALITY TESTS ON DISTRIBUTIONS OF PHTHALATE ESTER CONCENTRATION DATA ...... 287

II) STATISTICAL TESTS ON THE SPATIAL DISTRIBUTION OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR ...... 287

III) STATISTICAL TESTS ON THE DISTRIBUTION OF PHTHALATE ESTERS IN THE ENVIRONMENTAL MEDIA AND SPECIES OF FALSE CREEK HARBOUR...... 287

vii I) MEAN PHTHALATE ESTER CONCENTRATIONS AND FUGACITIES IN SEDIMENT, SEAWATER AND BIOTA FROM FALSE CREEK HARBOUR...... 310

II) COMPARISON OF REPORTED PHTHALATE ESTER CONCENTRATIONS IN VARIOUS LOCATIONS THROUGHOUT THE WORLD TO OBSERVED CONCENTRATIONS IN FALSE CREEK HARBOUR...... 310

III) MEAN BIOACCUMULATION FACTORS...... 310

IV) MEAN BIOTA-SEDIMENT ACCUMULATION FACTORS...... 310

APPENDIX B: TROPHODYNAMIC INTERACTIONS AND LIFE HISTORY INFORMATION ON SELECTED RESIDENT MARINE SPECIES IN SOUTHWESTERN BRITISH COLUMBIA...... 210 1. INTRODUCTION ...... 1 2. METHODS...... 8 2.1. Field Sampling Methods...... 8 LATIN NAME...... 14 2.2. Analytical Methods for Determining Phthalate Ester Concentrations in Environmental Samples 17 2.3. Quality Assurance and Control of Data (QA/QC) ...... 32 2.4. Measurements of Organic Carbon & Lipid Contents in Sediment and Biota Samples ...... 45 2.5. Data Analysis and Normalizations...... 47 3. RESULTS & DISCUSSION...... 57 3.1. Sediment Concentrations of Phthalate Esters ...... 57 3.2. Seawater Concentrations of Phthalate Esters...... 63 3.3. Sediment - Water Distribution of Phthalate Esters ...... 78 3.4. Biota Concentrations of Phthalate Esters ...... 82 3.5. Biota - Water Distribution Of Phthalate Esters ...... 131 3.6. Biota - Sediment Distribution Of Phthalate Esters ...... 166 REFERENCES...... 178 I) NORMALITY TESTS ON DISTRIBUTIONS OF PHTHALATE ESTER CONCENTRATION DATA ...... 287

II) STATISTICAL TESTS ON THE SPATIAL DISTRIBUTION OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR ...... 287

III) STATISTICAL TESTS ON THE DISTRIBUTION OF PHTHALATE ESTERS IN THE ENVIRONMENTAL MEDIA AND SPECIES OF FALSE CREEK HARBOUR...... 287

I) MEAN PHTHALATE ESTER CONCENTRATIONS AND FUGACITIES IN SEDIMENT, SEAWATER AND BIOTA FROM FALSE CREEK HARBOUR...... 310

II) COMPARISON OF REPORTED PHTHALATE ESTER CONCENTRATIONS IN VARIOUS LOCATIONS THROUGHOUT THE WORLD TO OBSERVED CONCENTRATIONS IN FALSE CREEK HARBOUR...... 310

III) MEAN BIOACCUMULATION FACTORS...... 310

IV) MEAN BIOTA-SEDIMENT ACCUMULATION FACTORS...... 310

APPENDIX C: DIETARY MATRIX FOR CALCULATION OF TROPHIC POSITIONS ...... 267

viii APPENDIX D: QUALITY ASSURANCE AND CONTROL OF DATA (QA/QC) - TABLES AND FIGURES FROM SECTION 2.4...... 270

APPENDIX E:STATISTICAL ANALYSES ON PHTHALATE ESTER CONCENTRATION DATA 287

I. NORMALITY TESTS ON DISTRIBUTIONS OF PHTHALATE ESTER CONCENTRATION DATA…………….….288 SEDIMENT CONCENTRATION DATA...... 289 WATER CONCENTRATION DATA...... 290 BIOTA CONCENTRATION DATA ...... 291 II. STATISTICAL TESTS ON THE SPATIAL DISTRIBUTION OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR. ……………………………………………………………………………………………………………...298 SEDIMENT CONCENTRATION DATA ...... 299 BIOTA CONCENTRATION DATA ...... 300 III. STATISTICAL TESTS ON THE DISTRIBUTION OF PHTHALATE ESTERS IN THE ENVIRONMENTAL MEDIA AND SPECIES OF FALSE CREEK HARBOUR………………………………………………………………………..305 APPENDIX F: DATA TABLES FROM SECTION 3 (RESULTS & DISCUSSION)...... 310 1. INTRODUCTION ...... 1 2. METHODS...... 8 2.1. Field Sampling Methods...... 8 2.2. Analytical Methods for Determining Phthalate Ester Concentrations in Environmental Samples 17 2.3. Quality Assurance and Control of Data (QA/QC) ...... 32 2.4. Measurements of Organic Carbon & Lipid Contents in Sediment and Biota Samples ...... 45 2.5. Data Analysis and Normalizations...... 47 3. RESULTS & DISCUSSION...... 57 3.1. Sediment Concentrations of Phthalate Esters ...... 57 3.2. Seawater Concentrations of Phthalate Esters...... 63 3.3. Sediment - Water Distribution of Phthalate Esters ...... 78 3.4. Biota Concentrations of Phthalate Esters ...... 82 3.5. Biota - Water Distribution Of Phthalate Esters ...... 131 3.6. Biota - Sediment Distribution Of Phthalate Esters ...... 166 REFERENCES...... 178 APPENDIX G: ORIGINAL RAW DATA OF PHTHALATE ESTER CONCENTRATIONS IN SEDIMENT, SEAWATER AND MARINE BIOTA SAMPLES FROM FALSE CREEK HARBOUR ...... 349

ix LIST OF FIGURES

Chapter 1 Introduction FIGURE 1.1. GENERALIZED PHTHALATE ESTER CHEMICAL STRUCTURE...... 2 FIGURE 1.2. FUGACITY “F” ANALYSIS OF ALTERNATIVE HYPOTHESES OF CHEMICAL MOVEMENT THROUGH A FOOD CHAIN...... 5

Chapter 2 Methods FIGURE 2.1. MAP OF FIELD STUDY SITE: FALSE CREEK HARBOUR, VANCOUVER, BRITISH COLUMBIA, SHOWING LOCATIONS OF FOUR SAMPLING STATIONS (λ): “NORTH CENTRAL”, “MARINA – SOUTH”, “CAMBIE BRIDGE” AND “EAST BASIN”...... 10 FIGURE 2.2. FIELD SAMPLING EQUIPMENT...... 12 A) PETIT PONAR SEDIMENT GRAB SAMPLER, AND B) SEAWATER COLLECTION APPARATUS...... 12 FIGURE 2.3. GENERALIZED TROPHIC LINKAGES BETWEEN EIGHTEEN MARINE ORGANISMS COLLECTED FROM FALSE CREEK HARBOUR AND THE SPECIES TROPHIC POSITIONS (SEE SECTION 2.5.5)...... 16 FIGURE 2.4. WATER EXTRACTION APPARATUS CONSISTING OF FMI VALVELESS LABORATORY PUMP AND THREE 47MM STAINLESS STEEL IN-LINE FILTER HOLDERS HOUSING A GLASS FIBRE FILTER (0.45μM DIAMETER PORE SIZE) IN HOLDER #1, AND AN OCTADECYL (C18) EMPORE EXTRACTION DISK IN HOLDERS #2 AND #3...... 21 FIGURE 2.5. SUMMARY OF THE EXTRACTION AND ANALYTICAL PROCEDURES FOR THE ANALYSIS OF PHTHALATE ESTERS IN SEDIMENT, BIOTA AND SEAWATER SAMPLES. (POLYCHLORINATED BIPHENYLS (PCBS) WERE EXTRACTED CONCURRENTLY)...... 24 FIGURE 2.6. MEAN PHTHALATE ESTER CONCENTRATIONS (NG/G) IN SODIUM SULFATE PROCEDURAL BLANKS FOR SEDIMENT AND BIOTA ANALYSIS. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 33 FIGURES 2.7 - 2.10 (SEE APPENDIX D LISTINGS) FIGURE 2.11. MEAN TOTAL RECOVERIES (%) OF INTERNAL STANDARDS IN SPIKED WELL WATER BLANKS AND FALSE CREEK SEAWATER SAMPLES USING GC/MS ANALYSIS. BARS INDICATE FRACTIONS ON THE GLASS FIBRE FILTER (GF) AND C18 EXTRACTION DISKS (C18). ERROR BARS INDICATE ONE STANDARD DEVIATION...... 38 FIGURE 2.12. MEAN CONCENTRATIONS (NG/L) OF PHTHALATE ESTERS IN WELL WATER BLANKS. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 40 FIGURES 2.13 (SEE APPENDIX D LISTINGS) FIGURE 2.14. ILLUSTRATION OF THE PARTICULATE ORGANIC CARBON (POC) – BOUND CHEMICAL (LARGE DIAMETER SUSPENDED MATTER “LDSM”), DISSOLVED ORGANIC CARBON (DOC) – BOUND CHEMICAL (SMALL DIAMETER SUSPENDED MATTER “SDSM”), AND THE FREELY DISSOLVED CHEMICAL FRACTION IN THE WATER PHASE AND THE THREE WATER CONCENTRATIONS REPORTED IN THE STUDY...... 43

Chapter 3 Results & Discussion FIGURE 3.1A & B. PHTHALATE ESTER CONCENTRATIONS IN FALSE CREEK HARBOUR SEDIMENTS, EXPRESSED ON A DRY WEIGHT BASIS (NG/G DRY SEDIMENT) (A), AND ON AN ORGANIC CARBON NORMALIZED BASIS (NG/G ORGANIC CARBON) (B)...... 59 FIGURE 3.1.C & D. PHTHALATE ESTER FUGACITIES (NPA) IN FALSE CREEK HARBOUR SEDIMENTS (C), AND COMPARISON OF PHTHALATE ESTER CONCENTRATION (NG/N OC) AND FUGACITY (NPA) PROFILES IN FALSE CREEK HARBOUR SEDIMENTS (D)...... 60 FIGURE 3.2. SPATIAL VARIABILITY...... 62 FIGURE 3.3. TOTAL CONCENTRATIONS (MEAN ± STANDARD DEVIATIONS, NG/L) OF PHTHALATE ESTERS IN SEAWATER SAMPLES FROM FALSE CREEK HARBOUR. (NUMBER OF SAMPLES FOR WHICH WATER CONCENTRATION EXCEEDED THE MDL, IN BRACKETS)...... 64 FIGURE 3.4. RATIO OF THE SEAWATER CONCENTRATIONS (CW, NG/L) TO THE AQUEOUS SOLUBILITIES (SW, NG/L) OF PHTHALATE ESTERS, FOR THE TOTAL SEAWATER CONCENTRATION AND THE FREELY DISSOLVED SEAWATER CONCENTRATION, AS A FUNCTION OF THE OCTANOL - SEAWATER PARTITION COEFFICIENT..66 FIGURE 3.5. MEAN OBSERVED FRACTIONS (± STANDARD DEVIATION) OF SPIKED PHTHALATE ESTER INTERNAL STANDARDS ON THE GLASS FIBRE FILTER AND C18 EXTRACTION DISKS IN FALSE CREEK HARBOUR

x SEAWATER SAMPLES, AND THE MODEL-FITTED FREELY DISSOLVED (FDW MODEL) AND PARTICULATE- BOUND (PB MODEL) FRACTIONS, DETERMINED FROM EQUATION 3.3...... 68 FIGURE 3.6. MEAN OBSERVED FRACTIONS (± STANDARD DEVIATIONS) OF SEAWATER-BORNE PHTHALATE ESTERS ON THE C18 EXTRACTION DISKS IN SEAWATER SAMPLES FROM FALSE CREEK HARBOUR, THE 2-PHASE MODEL-FITTED FREELY DISSOLVED FRACTION (EQN. 3.3) AND THE 3-PHASE MODEL-FITTED C18 FRACTION (SDSM-BOUND + FDW) (EQN. 3.4) AND FREELY DISSOLVED FRACTION (EQN. 3.5)...... 73 FIGURE 3.7. FRACTION OF PHTHALATE ESTERS BOUND TO LARGE DIAMETER SUSPENDED MATTER (LDSM) (), BOUND TO SMALL DIAMETER SUSPENDED MATTER (), AND FREELY DISSOLVED () IN FALSE CREEK HARBOUR SEAWATER, DETERMINED FROM THE 3-PHASE SORPTION MODEL (EQN. 3.5). THE Y- AXIS ON THE RIGHT PANEL IS EXPRESSED ON A LOGARITHMIC SCALE...... 74 FIGURE 3.8. MEAN PHTHALATE ESTER CONCENTRATIONS (± STANDARD DEVIATIONS, NG/L) IN FALSE CREEK HARBOUR SEAWATER. “TOTAL” CONCENTRATIONS INCLUDE CHEMICAL BOUND TO LARGE AND SMALL DIAMETER SUSPENDED MATTER (LDSM, SDSM) AND FREELY DISSOLVED CHEMICAL. “C18” CONCENTRATIONS INCLUDE SDSM-BOUND AND FREELY DISSOLVED CHEMICAL. THE THIRD BAR REPRESENTS MODEL ESTIMATES OF THE “FREELY DISSOLVED” CHEMICAL CONCENTRATION...... 76

FIGURE 3.9. MEAN “TOTAL”, “C18”, AND “FREELY DISSOLVED” FUGACITIES (± STANDARD DEVIATIONS, PA) IN FALSE CREEK HARBOUR SEAWATER. “TOTAL” FUGACITIES INCLUDE CHEMICAL BOUND TO LARGE AND SMALL DIAMETER SUSPENDED MATTER (LDSM, SDSM) AND FREELY DISSOLVED CHEMICAL. “C18” FUGACITIES INCLUDE SDSM-BOUND AND FREELY DISSOLVED CHEMICAL. THE THIRD BAR REPRESENTS ESTIMATES OF THE FUGACITY BASED ON “FREELY DISSOLVED” CONCENTRATIONS...... 78 FIGURE 3.10. OBSERVED SEDIMENT-WATER PARTITION COEFFICIENTS (LOG KOC, L/KG OC), BASED ON THE TOTAL WATER CONCENTRATION “TOT”, AND THE FREELY DISSOLVED WATER CONCENTRATION “FD”, AND THE PREDICTED SEDIMENT-WATER EQUILIBRIUM COEFFICIENT (L/KG OC), BASED ON SETH ET AL. 1999...... 81 FIGURE 3.11. MEAN LIPID CONCENTRATIONS (± STANDARD DEVIATIONS, NG/G LIPID WT.) OF PHTHALATE ESTERS IN MARINE BIOTA SAMPLES FROM THREE SAMPLING STATIONS (“NC” = NORTH CENTRAL, “MA” = MARINA, AND “EB” = EAST BASIN) IN FALSE CREEK HARBOUR. SPECIES PRESENTED ARE: A) PLANKTON, B) GREEN ALGAE, C) GEODUCK CLAMS, D) PACIFIC OYSTERS, AND E) STRIPED SEAPERCH. STARRED BARS (*) INDICATE STATISTICALLY SIGNIFICANT DIFFERENCES IN CONCENTRATION BETWEEN 1 STATION AND THE OTHER 2 (SINGLE STAR PER CHEMICAL), OR BETWEEN 2 SPECIFIC STATIONS (TWO STARS PER CHEMICAL)...... 86 FIGURE 3.12. CONCENTRATIONS OF DIMETHYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP), LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 88 FIGURE 3.13. FUGACITIES (NPA) OF DIMETHYL PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 89 FIGURE 3.14. CONCENTRATIONS OF DIETHYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 90 FIGURE 3.15. FUGACITIES (NPA) OF DIETHYL PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 91 FIGURE 3.16. LOG FUGACITY (NPA) VERSUS TROPHIC POSITION FOR DIMETHYL PHTHALATE (LEFT) AND DIETHYL PHTHALATE (RIGHT)...... 92 FIGURE 3.17. CONCENTRATIONS OF DI-ISO-BUTYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 95 FIGURE 3.18. FUGACITIES (NPA) OF DI-ISO-BUTYL PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 96 FIGURE 3.19. CONCENTRATIONS OF DI-N-BUTYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 97 FIGURE 3.20. FUGACITIES (NPA) OF DI-N-BUTYL PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK. HARBOUR...... 98 FIGURE 3.21. CONCENTRATIONS OF BUTYLBENZYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 99 FIGURE 3.22. FUGACITIES (NPA) OF BUTYLBENZYL PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 100

xi FIGURE 3.23. CONCENTRATIONS OF DI-ISO-HEXYL PHTHALATE (C6) IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 101 FIGURE 3.24. FUGACITIES (NPA) OF DI-ISO-HEXYL PHTHALATE (C6) IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK. HARBOUR...... 102 FIGURE 3.25. CONCENTRATIONS OF DI-ISO-HEPTYL PHTHALATE (C7) IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 103 FIGURE 3.26. FUGACITIES (NPA) OF DI-ISO-HEPTYL PHTHALATE (C7) IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK. HARBOUR...... 104 FIGURE 3.27. FUGACITY (NPA) VERSUS TROPHIC POSITION FOR DI-ISO-BUTYL PHTHALATE (TOP LEFT), DI-N- BUTYL PHTHALATE (TOP RIGHT), BENZYLBUTYL PHTHALATE (MIDDLE), DI-ISO-HEXYL PHTHALATE (C6) (BOTTOM LEFT), AND DI-ISO-HEPTYL PHTHALATE (C7) ( BOTTOM RIGHT)...... 105 FIGURE 3.28. CONCENTRATIONS OF DI(2-ETHYLHEXYL) PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 108 FIGURE 3.29. FUGACITIES (NPA) OF DI(2-ETHYLHEXYL) PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 109 FIGURE 3.30. CONCENTRATIONS OF DI-N-OCTYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 110 FIGURE 3.31. FUGACITIES (NPA) OF DI-N-OCTYL PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 111 FIGURE 3.32. CONCENTRATIONS OF DI-ISO-OCTYL PHTHALATE (C8) IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 112 FIGURE 3.33. FUGACITIES (NPA) OF DI-ISO-OCTYL PHTHALATE (C8) IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 113 FIGURE 3.34. CONCENTRATIONS OF DI-N-NONYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 114 FIGURE 3.35. FUGACITIES (NPA) OF DI-N-NONYL PHTHALATE IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 115 FIGURE 3.36. CONCENTRATIONS OF DI-ISO-NONYL PHTHALATE (C9) IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 116 FIGURE 3.37. FUGACITIES (NPA) OF DI-ISO-NONYL PHTHALATE (C9) IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 117 FIGURE 3.38. CONCENTRATIONS OF DI-ISO-DECYL PHTHALATE (C10) IN MARINE BIOTA FROM FALSE CREEK HARBOUR EXPRESSED IN WET WEIGHT (NG/G WET WT.) (TOP) AND LIPID WEIGHT (NG/G LIPID WT.) (BOTTOM)...... 118 FIGURE 3.39. FUGACITIES (NPA) OF DI-ISO-DECYL PHTHALATE (C10) IN MARINE BIOTA (λ), SEDIMENT (ν), AND TOTAL (▬), C18 (°), AND FREELY DISSOLVED () WATER FROM FALSE CREEK HARBOUR...... 119 FIGURE 3.40. LOG FUGACITY (NPA) VERSUS TROPHIC POSITION FOR DI(2-ETHYLHEXYL) PHTHALATE (TOP LEFT), DI-N-OCTYL PHTHALATE (TOP RIGHT), DI-ISO-OCTYL PHTHALATE (C8) (MIDDLE LEFT) AND DI-N- NONYL PHTHALATE (MIDDLE RIGHT). AND DI-ISO-NONYL PHTHALATE (C9) (BOTTOM LEFT), AND DI-ISO- DECYL PHTHALATE (C10) (BOTTOM RIGHT)...... 120 FIGURE 3.41. FUGACITY VERSUS TROPHIC POSITION FOR INDIVIDUAL PHTHALATE ESTERS (DMP, DEP, DIBP, AND DBP (TOP), BBP, DEHP, DNOP, AND DNNP (BOTTOM)) IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 122 FIGURE 3.42. FUGACITY VERSUS TROPHIC POSITION FOR PHTHALATE ESTER ISOMERIC MIXTURES (C6, C7, C8, C9, AND C10) IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 123 FIGURE 3.43. CHEMICAL UPTAKE AND ELIMINATION ROUTES IN FISH ...... 124 FIGURE 3.44. BIOACCUMULATION FACTORS EXPRESSED ON A WET WEIGHT (L/KG WET WT.) (TOP), AND LIPID WEIGHT (L/KG LIPID WT.) (BOTTOM) BASIS FOR DIMETHYL PHTHALATE IN FALSE CREEK MARINE BIOTA.

THE BAFS ARE CALCULATED FROM “TOTAL” (▬), “C18” (σ), AND “FREELY DISSOLVED” (○) WATER

xii CONCENTRATIONS. THE CEPA BIOACCUMULATION CRITERION ( —°― ), AND OCTANOL-SEAWATER PARTITION COEFFICIENT (⎯) ARE PRESENTED. ERROR BARS REPRESENT ONE STANDARD DEVIATION.....134 FIGURE 3.45. BIOACCUMULATION FACTORS EXPRESSED ON A WET WEIGHT (L/KG WET WT.) (TOP), AND LIPID WEIGHT (L/KG LIPID WT.) (BOTTOM) BASIS FOR DIETHYL PHTHALATE IN FALSE CREEK MARINE BIOTA. THE BAFS ARE CALCULATED FROM “TOTAL” (▬), “C18” (σ), AND “FREELY DISSOLVED” (○) WATER CONCENTRATIONS. THE CEPA BIOACCUMULATION CRITERION ( —°― ), AND OCTANOL-SEAWATER PARTITION COEFFICIENT (⎯) ARE PRESENTED. ERROR BARS REPRESENT ONE STANDARD DEVIATION.....135 FIGURE 3.46. BIOACCUMULATION FACTORS EXPRESSED ON A WET WEIGHT (L/KG WET WT.) (TOP), AND LIPID WEIGHT (L/KG LIPID WT.) (BOTTOM) BASIS FOR DI-ISO-BUTYL PHTHALATE IN FALSE CREEK MARINE

THE BAFS ARE CALCULATED FROM “TOTAL” (▬), “C18” (σ), AND “FREELY DISSOLVED” (○) WATER CONCENTRATIONS. THE CEPA BIOACCUMULATION CRITERION ( —°― ), AND OCTANOL-SEAWATER PARTITION COEFFICIENT (⎯) ARE PRESENTED. ERROR BARS REPRESENT ONE STANDARD DEVIATION.....139 FIGURE 3.48. BIOACCUMULATION FACTORS EXPRESSED ON A WET WEIGHT (L/KG WET WT.) (TOP), AND LIPID WEIGHT (L/KG LIPID WT.) (BOTTOM) BASIS FOR BUTYLBENZYL PHTHALATE IN FALSE CREEK MARINE

THE BAFS ARE CALCULATED FROM “TOTAL” (▬), “C18” (σ), AND “FREELY DISSOLVED” (○) WATER CONCENTRATIONS. THE CEPA BIOACCUMULATION CRITERION ( —°― ), AND OCTANOL-SEAWATER PARTITION COEFFICIENT (⎯) ARE PRESENTED. ERROR BARS REPRESENT ONE STANDARD DEVIATION.....143 FIGURE 3.50. BIOACCUMULATION FACTORS EXPRESSED ON A WET WEIGHT (L/KG WET WT.) (TOP), AND LIPID WEIGHT (L/KG LIPID WT.) (BOTTOM) BASIS FOR DI-ISO-HEPTYL PHTHALATE IN FALSE CREEK MARINE

THE BAFS ARE CALCULATED FROM “TOTAL” (▬), “C18” (σ), AND “FREELY DISSOLVED” (○) WATER CONCENTRATIONS. THE CEPA BIOACCUMULATION CRITERION ( —°― ), AND OCTANOL-SEAWATER PARTITION COEFFICIENT (⎯) ARE PRESENTED. ERROR BARS REPRESENT ONE STANDARD DEVIATION.....149 FIGURE 3.53. BIOACCUMULATION FACTORS EXPRESSED ON A WET WEIGHT (L/KG WET WT.) (TOP), AND LIPID WEIGHT (L/KG LIPID WT.) (BOTTOM) BASIS FOR DI-N-NONYL PHTHALATE IN FALSE CREEK MARINE BIOTA.

xiii FIGURE 3.54. BIOACCUMULATION FACTORS EXPRESSED ON A WET WEIGHT (L/KG WET WT.) (TOP), AND LIPID WEIGHT (L/KG LIPID WT.) (BOTTOM) BASIS FOR DI-ISO-OCTYL (C8) PHTHALATE IN FALSE CREEK MARINE

BIOTA. THE BAFS ARE CALCULATED FROM “TOTAL” (▬), “C18” (σ), AND “FREELY DISSOLVED” (○) WATER CONCENTRATIONS. THE CEPA BIOACCUMULATION CRITERION ( —°― ), AND OCTANOL- SEAWATER PARTITION COEFFICIENT (⎯) ARE PRESENTED. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 155 FIGURE 3.57. LIPID BASED BIOACCUMULATION FACTORS (L/KG LIPID WT.) PLOTTED AS LOGARITHMS VERSUS TROPHIC POSITION FOR INDIVIDUAL PHTHALATE ESTERS (DMP, DEP, DIBP, AND DBP (TOP), BBP, DEHP, DNOP, AND DNNP (BOTTOM)) IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 157 FIGURE 3.58. LIPID BASED BIOACCUMULATION FACTORS (L/KG LIPID WT.) PLOTTED AS LOGARITHMS VERSUS TROPHIC POSITION FOR PHTHALATE ESTER ISOMERIC MIXTURES (C6, C7, C8, C9, AND C10) IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 158 FIGURE 3.59A. LIPID NORMALIZED BIOACCUMULATION FACTORS, BASED ON “TOTAL” WATER CONCENTRATIONS, OF PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR VERSUS THE

OCTANOL - SEAWATER PARTITION COEFFICIENT. THE CEPA CRITERIA (⎯) AND BAFLIPID = KOW LINE (▬) ARE PRESENTED...... 161 FIGURE 3.59B. LIPID NORMALIZED BIOACCUMULATION FACTORS, BASED ON “TOTAL” WATER CONCENTRATIONS, OF PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR VERSUS THE OCTANOL - SEAWATER PARTITION COEFFICIENT. THE CEPA CRITERIA (⎯) IS PRESENTED...... 162 FIGURE 3.60. LIPID NORMALIZED BIOACCUMULATION FACTORS, BASED ON “FREELY DISSOLVED” WATER CONCENTRATIONS, OF PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR VERSUS THE

OCTANOL - SEAWATER PARTITION COEFFICIENT. THE CEPA CRITERIA (⎯) AND BAFLIPID = KOW LINE (▬) ARE PRESENTED...... 165 FIGURE 3.61. BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DIMETHYL PHTHALATE (TOP), AND DIETHYL PHTHALATE (BOTTOM) IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 168 FIGURE 3.62. BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DI-ISO-BUTYL PHTHALATE (TOP), AND DI-N-BUTYL PHTHALATE (BOTTOM) IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 169 FIGURE 3.63. BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF BUTYLBENZYL PHTHALATE (TOP), AND DI(2-ETHYLHEXYL) PHTHALATE (BOTTOM) IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 170 FIGURE 3.64. BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DI-N-OCTYL PHTHALATE (TOP), AND DI-N-NONYL PHTHALATE (BOTTOM) IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 171 FIGURE 3.65. BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DI-ISO-HEXYL (C6) PHTHALATE (TOP), AND DI-ISO-HEPTYL (C7) PHTHALATE (BOTTOM) IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 172 FIGURE 3.66. BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DI-ISO-OCTYL (C8) PHTHALATE (TOP), AND DI-ISO-NONYL (C9) PHTHALATE (BOTTOM) IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 173

xiv FIGURE 3.67. BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DI-ISO-OCTYL (C8) PHTHALATE (TOP), AND DI-ISO-NONYL (C9) PHTHALATE (BOTTOM) IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 1734 FIGURE 3.68 BIOTA - SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) ON A LOGARITHMIC SCALE VERSUS LOG OCTANOL - SEAWATER PARTITION COEFFICIENTS FOR PHTHALATE ESTERS IN BENTHIC MARINE BIOTA FROM FALSE CREEK HARBOUR...... 177

Appendix A FIGURE A.1. CHEMICAL STRUCTURES OF SIX PHTHALATE ESTER CONGENERS ...... 199 FIGURE A.2. MEAN WET WEIGHT BIOCONCENTRATION FACTORS (L/KG WET WT.) OF PHTHALATE ESTERS AS A FUNCTION OF LOG KOW FROM LABORATORY STUDIES REVIEWED BY STAPLES ET AL. 1997A. “PARENT” BCFS REFER TO PARENT PHTHALATE ESTERS. “TOTAL” BCFS REFER TO THE PARENT COMPOUND AND RADIOLABELED METABOLITES...... 205

Appendix B FIGURE B.1. SUMMARY OF TROPHIC INTERACTIONS BETWEEN SELECTED MARINE SPECIES IN SOUTHWESTERN BRITISH COLUMBIA...... 263

Appendix D FIGURE D.2.7A. PROCEDURAL BLANK CONCENTRATIONS, MDLS AND SAMPLE CONCENTRATIONS IN 8 BIOTA BATCHES FOR: DIMETHYL PHTHALATE (TOP LEFT); DIETHYL PHTHALATE (TOP RIGHT); DI-ISO-BUTYL PHTHALATE (BOTTOM LEFT); AND DI-N-BUTYL PHTHALATE (BOTTOM RIGHT)...... 272 FIGURE D.2.7B. PROCEDURAL BLANK CONCENTRATIONS, MDLS AND SAMPLE CONCENTRATIONS IN 8 BIOTA BATCHES FOR: BENZYLBUTYL PHTHALATE (TOP LEFT); DI-2-ETHYLHEXYL PHTHALATE (TOP RIGHT); DI-N- OCTYL PHTHALATE (BOTTOM LEFT); AND DI-N-NONYL PHTHALATE (BOTTOM RIGHT)...... 273 FIGURE D.2.7C. PROCEDURAL BLANK CONCENTRATIONS, MDLS AND SAMPLE CONCENTRATIONS IN 8 BIOTA BATCHES FOR: DIISOHEXYL PHTHALATE (C6) (TOP LEFT); DIISOHEPTYL PHTHALATE (C7) (TOP RIGHT); DIISOOCTYL PHTHALATE (C8) (BOTTOM LEFT); AND DIISONONYL PHTHALATE (C9) (BOTTOM RIGHT)....274 FIGURE D.2.8. BLANK-CORRECTED SEDIMENT CONCENTRATIONS IN RELATION TO THE METHOD DETECTION LIMITS (MDLS) (▬) (I.E., 3 STANDARD DEVIATIONS) FOR EACH PHTHALATE ESTER (NG/G DRY WEIGHT). SEDIMENT SAMPLES ARE DIVIDED INTO TWO CATEGORIES: CONCENTRATIONS > MDL (z), AND CONCENTRATIONS < MDL (ς)...... 275 FIGURE D.2.9A. BLANK-CORRECTED BIOTA CONCENTRATIONS IN RELATION THE 10TH, 50TH AND 90TH PERCENTILES OF THE BATCH MDLS FOR: DIMETHYL PHTHALATE (TOP); AND DIETHYL PHTHALATE (BOTTOM). DATA ARE CLASSIFIED INTO THREE CATEGORIES: DATA > MDL (z), DATA < MDL (ς), AND DATA < MDL, BUT WITHIN THE RANGE OF OTHER DATA ({)...... 276 FIGURE D.2.9B. BLANK-CORRECTED BIOTA CONCENTRATIONS IN RELATION THE 10TH, 50TH AND 90TH PERCENTILES OF THE BATCH MDLS FOR: DIISOBUTYL PHTHALATE (TOP); AND DI-N-BUTYL PHTHALATE (BOTTOM). DATA ARE CLASSIFIED INTO THREE CATEGORIES: DATA > MDL (z), DATA < MDL (ς), AND DATA < MDL, BUT WITHIN THE RANGE OF OTHER DATA ({)...... 277 FIGURE D.2.9C. BLANK-CORRECTED BIOTA CONCENTRATIONS IN RELATION THE 10TH, 50TH AND 90TH PERCENTILES OF THE BATCH MDLS FOR: BUTYLBENZYL PHTHALATE (TOP); AND DI-2-ETHYLHEXYL PHTHALATE (BOTTOM). DATA ARE CLASSIFIED INTO THREE CATEGORIES: DATA > MDL (z), DATA < MDL (ς), AND DATA < MDL, BUT WITHIN THE RANGE OF OTHER DATA ({)...... 278 FIGURE D.2.9D. BLANK-CORRECTED BIOTA CONCENTRATIONS IN RELATION THE 50TH AND 90TH PERCENTILES OF THE BATCH MDLS FOR: DI-N-OCTYL PHTHALATE (TOP); AND DI-N-NONYL PHTHALATE (BOTTOM). DATA ARE CLASSIFIED INTO THREE CATEGORIES: DATA > MDL (z), DATA < MDL (ς), AND DATA < MDL, BUT WITHIN THE RANGE OF OTHER DATA ({)...... 279 FIGURE D.2.9E. BLANK-CORRECTED BIOTA CONCENTRATIONS IN RELATION THE 50TH AND 90TH PERCENTILES OF THE BATCH MDLS FOR: DIISOHEXYL PHTHALATE (C6) (TOP); AND DIISOHEPTYL PHTHALATE (C7) (BOTTOM). DATA ARE CLASSIFIED INTO THREE CATEGORIES: DATA > MDL (z), DATA < MDL (ς), AND DATA < MDL, BUT WITHIN THE RANGE OF OTHER DATA ({)...... 280 FIGURE D.2.9F. BLANK-CORRECTED BIOTA CONCENTRATIONS IN RELATION THE 10TH, 50TH AND 90TH PERCENTILES OF THE BATCH MDLS FOR: DIISOHEXYL PHTHALATE (C6) (TOP); AND DIISOHEPTYL PHTHALATE (C7)

xv (BOTTOM). DATA ARE CLASSIFIED INTO THREE CATEGORIES: DATA > MDL (z), DATA < MDL (ς), AND DATA < MDL, BUT WITHIN THE RANGE OF OTHER DATA ({)...... 281 FIGURE D.2.9G. BLANK-CORRECTED BIOTA CONCENTRATIONS IN RELATION TO THE MDL FOR DIISODECYL PHTHALATE (C10). CONFIRMED DATA > MDL (z), DATA ESTIMATED BY THE APPLICATION OF A RATIO (z), AND DATA < MDL (ς) ARE PRESENTED...... 282 FIGURE D.2.10. MEAN RECOVERIES OF INTERNAL STANDARDS IN SPIKED SEDIMENT SAMPLES AND SODIUM SULFATE BLANKS ANALYZED BY GC/MS (A), AND BY LC-ESI/MS (B), AND IN BIOTA SAMPLES AND SODIUM SULFATE BLANKS ANALYZED BY GC/MS (C), AND BY LC-ESI/MS (D). ERROR BARS REPRESENT ONE STANDARD DEVIATION...... 283 FIGURE D.2.13A. WELL WATER BLANKS, METHOD DETECTION LIMITS (MDLS), AND SEAWATER SAMPLE CONCENTRATIONS (NG/L) IN 4 BATCHES FOR DIMETHYL PHTHALATE (TOP LEFT), DIETHYL PHTHALATE (TOP RIGHT), DI-ISO-BUTYL PHTHALATE (BOTTOM LEFT), AND DI-N-BUTYL PHTHALATE (BOTTOM RIGHT)...... 284 FIGURE D.2.13B. WELL WATER BLANKS, METHOD DETECTION LIMITS (MDLS), AND SEAWATER SAMPLE CONCENTRATIONS (NG/L) IN 4 BATCHES FOR BUTYL-BENZYL PHTHALATE (TOP LEFT), DI(2-ETHYLHEXYL) PHTHALATE (TOP RIGHT), DI-N-OCTYL PHTHALATE (BOTTOM LEFT), AND DI-N-NONYL PHTHALATE (BOTTOM RIGHT)...... 285 FIGURE D.2.13C. WELL WATER BLANKS, METHOD DETECTION LIMITS (MDLS), AND SEAWATER SAMPLE CONCENTRATIONS (NG/L) IN 4 BATCHES FOR DIISOHEXYL PHTHALATE (C6) (TOP LEFT), DIISOHEPTYL PHTHALATE (C7) (TOP RIGHT), DIISOOCTYL PHTHALATE (C8) (BOTTOM LEFT), AND DIISONONYL PHTHALATE (C9) (BOTTOM MIDDLE), AND DIISODECYL PHTHALATE (C10) (BOTTOM RIGHT)...... 286

xvi LIST OF TABLES

Chapter 1 Introduction 3 TABLE 1.1. MOLECULAR WEIGHTS (G/MOL), LE BAS MOLAR VOLUMES (CM /MOL), AQUEOUS SOLUBILITIES 1 (MG/L), AND LOG OCTANOL – SEAWATER PARTITION COEFFICIENTS OF 13 SELECTED PHTHALATE ESTERS, AS REPORTED IN COUSINS & MACKAY (2000)...... 3

Chapter 2 Methods TABLE 2.1. TROPHIC CATEGORY, COMMON NAME, LATIN NAME, SAMPLE SIZE, MEAN LENGTH (CM), MEAN WET WEIGHT (G) AND SAMPLING METHODS FOR EIGHTEEN MARINE ORGANISMS COLLECTED FROM FALSE CREEK HARBOUR, VANCOUVER, BRITISH COLUMBIA...... 14 TABLE 2.2. COMPOSITION OF PHTHALATE ESTER (PE) AND POLYCHLORINATED BIPHENYL (PCB) STANDARDS, AND AMOUNTS (NG) ADDED TO SEDIMENT AND BIOTA SAMPLES...... 20 TABLE 2.3. MEAN PERCENTAGE OF C10 IN THE TOTAL PEAK (C10 + INTERFERENCE), THE COEFFICIENT OF VARIATION (%), SAMPLE SIZE (N), AND NUMBER OF SAMPLES WITH NON-DETECT C10 CONCENTRATIONS FOR BIOTA SAMPLES CONFIRMED USING LC/ESI-MS/MS...... 31 TABLE 2.4. (SEE APPENDIX D LISTINGS) TABLE 2.5. MEAN CONCENTRATIONS (NG/G) OF PHTHALATE ESTERS IN SODIUM SULFATE PROCEDURAL BLANKS FOR BIOTA AND SEDIMENT ANALYSIS, 3 STANDARD DEVIATIONS OF THE BLANKS, AND METHOD DETECTION LIMITS DEFINED AS THE MEAN BLANK CONCENTRATION + 3 STANDARD DEVIATIONS...... 35 TABLE 2.6. MEAN (+/- STANDARD DEVIATION) RECOVERIES OF INTERNAL STANDARDS FROM SPIKED FALSE CREEK SEDIMENT AND BIOTA SAMPLES AND SODIUM SULFATE BLANKS (%) ...... 36 TABLE 2.7. MEAN (+/- STANDARD DEVIATION) INTERNAL STANDARD RECOVERIES FOR FALSE CREEK SEAWATER SAMPLES AND WELL WATER BLANKS (%)...... 37 TABLE 2.8. (SEE APPENDIX D LISTINGS) TABLE 2.9. MINIMUM AND MAXIMUM METHOD DETECTION LIMITS (MDLS) IN NG/L AMONG 4 BATCHES OF WATER SAMPLES. MDLS REPRESENT THE MEAN PE CONCENTRATION IN THE BATCH BLANKS + 3 STANDARD DEVIATIONS...... 41 TABLE 2.10. NUMBER OF SAMPLES WITH DETECTABLE CONCENTRATIONS ABOVE THE METHOD DETECTION LIMITS (MDLS)...... 44 TABLE 2.11. MEAN LIPID CONTENTS (%, G LIPID/ G WET TISSUE) AND ORGANIC CARBON CONTENTS (% DRY WEIGHT AND % WET WEIGHT) (± STANDARD DEVIATION) IN BIOTA TISSUES THAT WERE ANALYZED FOR PHTHALATE ESTERS...... 49 TABLE 2.12. LATIN NAME, COMMON NAME, TROPHIC POSITION, PREY ITEMS AND THEIR DIETARY PROPORTIONS, AND PREDATORS OF KEY RESIDENT MARINE SPECIES IN THE GEORGIA BASIN ECOSYSTEM...... 53 TABLE 2.13. SUMMARY OF TROPHIC POSITIONS FOR SPECIES COLLECTED FROM FALSE CREEK ...... 56

Chapter 3 Results & Discussion TABLE 3.1. - 3.4. (SEE APPENDIX F LISTINGS) TABLE 3.5. MEAN FRACTIONS OF INTERNAL STANDARDS ON THE GLASS FIBRE FILTER (GF) AND C18 EXTRACTION DISKS (C18) IN WELL WATER BLANKS AND FALSE CREEK SEAWATER SAMPLES (%)...... 67 TABLE 3.6. MEAN OBSERVED FRACTIONS (%) (± STANDARD DEVIATIONS) OF SEAWATER BORNE PHTHALATE ESTERS ON THE GLASS FIBRE FILTER (GF) AND C18 EXTRACTION DISKS (C18) IN WELL WATER BLANKS AND FALSE CREEK SEAWATER SAMPLES...... 71 TABLE 3.7. MEAN FRACTIONS (%) OF PHTHALATE ESTERS BOUND TO LARGE AND SMALL DIAMETER SUSPENDED MATTER (LDSM, SDSM) AND FREELY DISSOLVED IN FALSE CREEK HARBOUR SEAWATER, DETERMINED FROM THE 3-PHASE SORPTION MODEL (EQN 3.5)...... 72 TABLE 3.8. (SEE APPENDIX F LISTINGS) TABLE 3.9. OBSERVED AND PREDICTED SEDIMENT-WATER PARTITION COEFFICIENTS (OBS KOC AND PRED KOC, L/KG OC) BASED ON THE FREELY DISSOLVED WATER CONCENTRATION, AND THE RATIO BETWEEN THE OBSERVED AND PREDICTED PARTITION COEFFICIENTS...... 81 TABLE 3.10 - 3.16. (SEE APPENDIX F LISTINGS) TABLE 3.17. STATISTICAL RESULTS OF REGRESSION: FUGACITY VERSUS TROPHIC POSITION (TP)...... 121 TABLE 3.18 - 3.30. (SEE APPENDIX F LISTINGS)

xvii TABLE 3.31. STATISTICAL RESULTS OF REGRESSION: LOG BAF (L/KG LIPID WT.) VERSUS TROPHIC POSITION 156

Appendix A 1 TABLE A.1. PHTHALATE ESTER (PE) END USE PRODUCTS ...... 196 TABLE A.2. PRODUCTION (PRDN), IMPORTS (IMP), AND CONSUMPTION (CONS) OF PHTHALATE ESTERS BY REGION (KTONNES PER YEAR) (PARKERTON AND KONKEL 2000)...... 197 TABLE A.3. PHYSIOCHEMICAL PROPERTIES OF 13 SELECTED PHTHALATE ESTERS AS REPORTED IN COUSINS & 3 MACKAY (2000): MOLECULAR WEIGHT (G/MOL), LE BAS MOLAR VOLUME (CM /MOL), AQUEOUS SOLUBILITY (MG/L), VAPOUR PRESSURE (PA), LOG OCTANOL – SEAWATER PARTITION COEFFICIENT, 3 3 HENRY’S LAW CONSTANT (PA-M /MOL), AND FUGACITY CAPACITY IN WATER (MOL/ PA-M )...... 200 1 2 TABLE A.4. “TOTAL ” AND “PARENT ” BICONCENTRATION FACTORS (L/KG WET WT.) AND WATER EXPOSURE CONCENTRATIONS (UG/L), FROM REPORTED PHTHALATE ESTER BIOCONCENTRATION STUDIES, EXPRESSED AS THE MEAN AND/OR (RANGE) FOR EACH TAXA...... 202

Appendix B TABLE B.1 SPECIES NAME, PREY ITEMS, DIETARY PROPORTIONS, AND PREDATORS FOR SELECTED RESIDENT MARINE SPECIES IN SOUTHWESTERN BRITISH COLUMBIA ...... 259 TABLE B.2. SUMMARY OF SPAWNING AND REPRODUCTIVE SCHEDULES OF SELECTED MARINE SPECIES IN SOUTHWESTERN BRITISH COLUMBIA ...... 265

Appendix C 1 TABLE C.2.1. DIETARY COMPOSITION AND TROPHIC POSITIONS OF 21 PREDATOR SPECIES / ORGANISMS IN THE GEORGIA BASIN ECOSYSTEM. PREY SPECIES, AND THEIR CORRESPONDING TROPHIC POSITIONS AND DIETARY PROPORTIONS ARE IDENTIFIED...... 268 TABLE C.2.2. IDENTIFICATION OF THE PREDATOR SPECIES PRESENTED IN TABLE C.2.1 (DIETARY MATRIX) AND THEIR CALCULATED TROPHIC POSITIONS. SPECIES / ORGANISMS IN BOLD TYPE ARE REPORTED ON IN THE CURRENT STUDY...... 269

Appendix D TABLE D.2.4. MEAN PHTHALATE ESTER CONCENTRATIONS (NG/G) IN SODIUM SULFATE PROCEDURAL BLANKS FOR BIOTA AND SEDIMENT ANALYSIS AND (LOWER – UPPER STANDARD DEVIATIONS)...... 271 TABLE D.2.8. GEOMETRIC MEAN CONCENTRATIONS (NG/L) OF PHTHALATE ESTERS IN 12 WELL WATER BLANKS AND (LOWER – UPPER STANDARD DEVIATIONS)...... 271

Appendix E TABLE E.2.1. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN SODIUM SULFATE BLANKS USED IN SEDIMENT ANALYSIS...... 289 TABLE E.2.2. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR SEDIMENT SAMPLES...... 289 TABLE E.2.3. RESULTS OF SHAPIRO-WILK NORMALITY TEST ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN WELL WATER BLANKS...... 290 TABLE E.2.4. RESULTS OF SHAPIRO-WILK NORMALITY TEST ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK SEAWATER SAMPLES.290 TABLE E.2.5. RESULTS OF KOLMOGOROV-SMIRNOV NORMALITY TEST ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN SODIUM SULFATE BLANKS USED IN THE BIOTA ANALYSIS...... 291 TABLE E.2.6. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR PLANKTON SAMPLES...... 291 TABLE E.2.7. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR GREEN ALGAE SAMPLES...... 292

xviii TABLE E.2.8. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR GEODUCK CLAM SAMPLES...... 292 TABLE E.2.9. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR BLUE MUSSEL SAMPLES...... 293 TABLE E.2.10. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR PACIFIC OYSTER SAMPLES...... 293 TABLE E.2.11. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR DUNGENESS CRAB SAMPLES...... 294 TABLE E.2.12. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR MINNOW SAMPLES...... 294 TABLE E.2.13. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR STRIPED SEAPERCH SAMPLES...... 295 TABLE E.2.14. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR PACIFIC STAGHORN SCULPIN SAMPLES...... 295 TABLE E.2.15. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR WHITESPOTTED GREENLING SAMPLES...... 296 TABLE E.2.16. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR SPINY DOGFISH LIVER SAMPLES...... 296 TABLE E.2.17. RESULTS OF KOLMOGOROV-SMIRNOV AND SHAPIRO-WILK NORMALITY TESTS ON ORIGINAL PHTHALATE ESTER CONCENTRATIONS AND LOG TRANSFORMED CONCENTRATIONS IN FALSE CREEK HARBOUR SPINY DOGFISH MUSCLE SAMPLES...... 297 TABLE E.3.1A. RESULTS OF ANOVA TESTS FOR DIFFERENCES IN THE ORGANIC CARBON NORMALIZED CONCENTRATIONS (NG/G OC) OF PHTHALATE ESTERS IN THE SEDIMENTS OF FOUR FALSE CREEK HARBOUR SAMPLING STATIONS...... 299 TABLE E.3.1B. TUKEY TEST RESULT MATRIX FOR DIFFERENCES IN CONCENTRATIONS (NG/G OC) OF PHTHALATE ESTERS IN SEDIMENTS FROM FOUR SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 299 TABLE E.3.2A. RESULTS OF ANOVA TESTS FOR DIFFERENCES IN THE LIPID NORMALIZED CONCENTRATIONS (NG/G LIPID) OF PHTHALATE ESTERS IN PLANKTON SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 300 TABLE E.3.2B. TUKEY TEST RESULT MATRIX FOR DIFFERENCES IN CONCENTRATION (NG/G LIPID) OF PHTHALATE ESTERS IN PLANKTON SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 300 TABLE E.3.3A. RESULTS OF ANOVA TESTS FOR DIFFERENCES IN THE LIPID NORMALIZED CONCENTRATIONS (NG/G LIPID) OF PHTHALATE ESTERS IN GREEN ALGAE SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 301 TABLE E.3.3B. TUKEY TEST RESULT MATRIX FOR DIFFERENCES IN CONCENTRATION (NG/G LIPID) OF INDIVIDUAL PHTHALATE ESTERS IN GREEN ALGAE SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 301 TABLE E.3.4A. RESULTS OF ANOVA TESTS AND TWO-TAILED T-TESTS FOR DIFFERENCES IN THE LIPID NORMALIZED CONCENTRATIONS (NG/G LIPID) OF PHTHALATE ESTERS IN GEODUCK CLAMS SAMPLES FROM TWO OR THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 302 TABLE E.3.4B. TUKEY TEST / T-TEST RESULT MATRIX FOR DIFFERENCES IN CONCENTRATIONS (NG/G LIPID) OF PHTHALATE ESTERS IN GEODUCK CLAM SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 302 TABLE E.3.5A. RESULTS OF ANOVA TESTS FOR DIFFERENCES IN THE LIPID NORMALIZED CONCENTRATIONS (NG/G LIPID) OF PHTHALATE ESTERS IN PACIFIC OYSTER SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 303

xix TABLE E.3.5B. TUKEY TEST RESULT MATRIX FOR DIFFERENCES IN CONCENTRATION (NG/G LIPID) OF PHTHALATE ESTERS IN PACIFIC OYSTER SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 303 TABLE E.3.6. RESULTS OF ANOVA TESTS FOR DIFFERENCES IN THE LIPID NORMALIZED CONCENTRATIONS (NG/G LIPID) OF PHTHALATE ESTERS IN BLUE MUSSEL SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 304 TABLE E.3.7. RESULTS OF ANOVA TESTS FOR DIFFERENCES IN THE LIPID NORMALIZED CONCENTRATIONS (NG/G LIPID) OF PHTHALATE ESTERS IN STRIPED SEAPERCH SAMPLES FROM THREE SAMPLING STATIONS IN FALSE CREEK HARBOUR...... 304 TABLE E.3.8. RESULTS OF ANOVA AND TUKEY TESTS ON DIFFERENCES IN THE CHEMICAL FUGACITIES BETWEEN THE FREELY DISSOLVED WATER FRACTION AND THE SEDIMENT OR MARINE BIOTA SPECIES. LEVEL OF SIGNIFICANCE IS 0.05 (* VALUES INDICATE Q<0.10)...... 306 TABLE E.3.9. RESULTS OF ANOVA AND TUKEY TESTS ON DIFFERENCES IN THE CHEMICAL FUGACITIES BETWEEN THE SEDIMENT AND MARINE BIOTA SPECIES. LEVEL OF SIGNIFICANCE IS 0.05 (* VALUES INDICATE Q<0.10)...... 307 TABLE E.3.10. RESULTS OF ANOVA AND TUKEY TESTS ON DIFFERENCES IN THE CHEMICAL FUGACITIES BETWEEN THE DOGFISH (MUSCLE) AND OTHER MARINE BIOTA SPECIES. LEVEL OF SIGNIFICANCE IS 0.05 (* VALUES INDICATE Q<0.10)...... 308 TABLE E.3.11. RESULTS OF ANOVA AND TUKEY TESTS ON DIFFERENCES IN THE CHEMICAL FUGACITIES BETWEEN THE DOGFISH (LIVER) AND OTHER MARINE BIOTA SPECIES. LEVEL OF SIGNIFICANCE IS 0.05 (* VALUES INDICATE Q<0.10)...... 309

Appendix F TABLE F.3.1. MEAN CONCENTRATIONS (NG/G) AND (LOWER - UPPER STANDARD DEVIATIONS), EXPRESSED IN DRY WEIGHTS (NG/G DRY WT.) AND ORGANIC CARBON WEIGHTS (NG/G OC), AND CORRESPONDING FUGACITIES (PA), OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR SEDIMENTS...... 311 TABLE F.3.2.A. REPORTED UPPER AND LOWER CONCENTRATION RANGES OR MEAN CONCENTRATIONS (NG/G DRY WEIGHT) OF PHTHALATE ESTERS IN SEDIMENTS FOR VARIOUS LOCATIONS IN THE WORLD...... 313 TABLE F.3.2.B. REPORTED UPPER AND LOWER CONCENTRATION RANGES OR MEANS (NG/G ORGANIC CARBON) OF PHTHALATE ESTERS IN SEDIMENTS FOR VARIOUS LOCATIONS IN THE WORLD...... 314 TABLE F.3.3. MEAN TOTAL CONCENTRATIONS (NG/L) AND (LOWER - UPPER STANDARD DEVIATIONS) OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR SEAWATER, AND NUMBER OF SAMPLES FOR WHICH WATER CONCENTRATION EXCEEDED THE MDL (N)...... 315

TABLE F.3.8A. MEAN “TOTAL”, “C18”, AND “FREELY DISSOLVED” CONCENTRATIONS (± STANDARD DEVIATION, NG/L) OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR SEAWATER...... 315

TABLE F.3.8B. MEAN “TOTAL”, “C18”, AND “FREELY DISSOLVED” FUGACITIES (± STANDARD DEVIATION, PA) OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR SEAWATER...... 316 TABLE F.3.4. REPORTED UPPER AND LOWER CONCENTRATION RANGES OR MEANS (NG/L) OF PHTHALATE ESTERS IN MARINE WATER AND FRESHWATER FOR VARIOUS LOCATIONS IN THE WORLD...... 317 TABLE F.3.10. MEAN WET WEIGHT CONCENTRATIONS (NG/G WET WT.) AND LOWER - UPPER STANDARD DEVIATIONS OF INDIVIDUAL PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR .....318 TABLE F.3.11. MEAN WET WEIGHT CONCENTRATIONS (NG/G WET WT.) AND LOWER - UPPER STANDARD DEVIATIONS OF PHTHALATE ESTER ISOMERIC MIXTURES IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 320 TABLE F.3.12. MEAN LIPID WEIGHT CONCENTRATIONS (NG/G LIPID) AND LOWER - UPPER STANDARD DEVIATIONS OF INDIVIDUAL PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR. ....322 TABLE F.3.13. MEAN LIPID WEIGHT CONCENTRATIONS (NG/G LIPID) AND LOWER - UPPER STANDARD DEVIATIONS OF PHTHALATE ESTER ISOMERIC MIXTURES IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 324 TABLE F.3.14. MEAN FUGACITIES (PA) AND LOWER - UPPER STANDARD DEVIATIONS OF INDIVIDUAL PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 326 TABLE F.3.15. MEAN FUGACITIES (PA) AND LOWER - UPPER STANDARD DEVIATIONS OF PHTHALATE ESTER ISOMERIC MIXTURES IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 328 TABLE F.3.16. REPORTED LOWER AND UPPER CONCENTRATION RANGES OR SINGLE OBSERVATIONS (NG/G WET WEIGHT) OF PHTHALATE ESTERS IN BIOLOGICAL SAMPLES FROM VARIOUS LOCATIONS IN THE WORLD.331

xx TABLE F.3.18. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DIMETHYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 333 TABLE F.3.19. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DIETHYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 334 TABLE F.3.20. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-ISO-BUTYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 335 TABLE F.3.21. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-N-BUTYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 336 TABLE F.3.22. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF BUTYLBENZYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 337 TABLE F.3.23. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-(2-ETHYLHEXYL) PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 338 TABLE F.3.24. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-N-OCTYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 339 TABLE F.3.25. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-N-NONYL PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 340 TABLE F.3.26. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-IOS-HEXYL (C6) PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 341 TABLE F.3.27. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-IOS-HEPTYL (C7) PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 342 TABLE F.3.28. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-ISO-OCTYL (C8) PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 343 TABLE F.3.29. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-ISO-NONYL (C9) PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 344 TABLE F.3.30. MEAN WET WEIGHT (L/KG WET WT.) AND LIPID WEIGHT (L/KG LIPID) BIOACCUMULATION FACTORS AND (LOWER – UPPER STANDARD DEVIATIONS) OF DI-ISO-DECYL (C10) PHTHALATE IN MARINE BIOTA FROM FALSE CREEK HARBOUR USING “TOTAL”, “C18” AND “FREELY DISSOLVED” WATER CONCENTRATIONS, EXPRESSED IN LOGARITHMS...... 345 TABLE F.3.32. BIOTA – SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DIMETHYL, DIETHYL, DI- ISO-BUTYL, AND DI-N-BUTYL PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR....346

xxi TABLE F.3.33. BIOTA – SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF BUTYLBENZYL, DI(2- ETHYLHEXYL), DI-N-OCTYL, AND DI-N-NONYL PHTHALATE ESTERS IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 347 TABLE F.3.34. BIOTA – SEDIMENT ACCUMULATION FACTORS (KG OC / KG LIPID) OF DI-ISO-HEXYL (C6), DI-ISO- HEPTYL (C7) DI-ISO-OCTYL (C8), DI-ISO-NONYL (C9), AND DI-ISO-DECYL (C10) PHTHALATE ESTER ISOMERIC MIXTURES IN MARINE BIOTA FROM FALSE CREEK HARBOUR...... 348

Appendix G TABLE G.1. STATION LOCATION, SAMPLE ID, SAMPLING DATE, ORGANIC CARBON CONTENT (TOC), INTERNAL STANDARD RECOVERY FOR GC-MS AND LC-ESI/MS ANALYSIS, AND BLANK AND RECOVERY CORRECTED PHTHALATE ESTER CONCENTRATIONS (NG/G DRY WT.) IN SEDIMENT SAMPLES FROM FALSE CREEK HARBOUR...... 350 TABLE G.2. STATION LOCATION, SAMPLE ID, SAMPLING DATE, CONCENTRATION OF LARGE AND SMALL DIAMETER SUSPENDED MATTER (LDSM & SDSM) (MG/L) IN THE SEAWATER, INTERNAL STANDARD RECOVERY FOR GC-MS AND LC-ESI/MS ANALYSIS, AND BLANK AND RECOVERY CORRECTED PHTHALATE ESTER CONCENTRATIONS (NG/L) IN SEAWATER SAMPLES FROM FALSE CREEK HARBOUR...... 351 1 TABLE G.3. SPECIES, LATIN NAME, SAMPLE ID , SAMPLING DATE, LIPID CONTENT (% WET WT.), TOTAL ORGANIC CARBON CONTENT (TOC) (% DRY WT.), INTERNAL STANDARD RECOVERY FOR GC-MS AND LC-ESI/MS ANALYSIS, AND BLANK AND RECOVERY CORRECTED PHTHALATE ESTER CONCENTRATIONS (NG/G WET WT.) IN BIOTA SAMPLES FROM FALSE CREEK HARBOUR...... 352

xxii DEFINITIONS

1) Octanol-Water Partition Coefficient “KOW” refers to the ratio of the concentration of a chemical in octanol (a surrogate for lipids) to its concentration in water, at equilibrium. It is a measure of the hydrophobicity of the chemical, and indicates the potential of a chemical to partition into the lipid tissue of organisms, and bioconcentrate (Mackay 1991, Connell 1990).

2) Fugacity (f) is equivalent to chemical activity. It is the pressure a chemical exerts in a particular medium and is expressed in units of Pascal. It is a function of the concentration of a chemical in a medium (C in units of mol/m3), and the fugacity capacity of that medium for that particular chemical (Z in units of Pa ⋅ m3/mol), i.e., f = C / Z (Mackay 1991).

3) Fugacity Capacity (Z) is a measure of the solubility of a particular chemical in a particular medium. It is defined as the amount of chemical that can be absorbed in a medium to increase the partial pressure in that medium by 1 Pascal, and is expressed in units of Pa⋅m3/mol (Mackay 1991).

4) Equilibrium Partitioning refers to a situation of chemical equilibrium, where the fugacities of a chemical in two or more media in the (eco)system are equal (Mackay 1982, 1991, Gobas et al. 1993).

5) Bioconcentration refers to the process of accumulation of a chemical substance in an organism, resulting from exposure of the organism to the substance in the water, typically under laboratory conditions. The driving force of bioconcentration is equilibrium partitioning of a substance between the organism and ambient water (Mackay 1982, Clark et al. 1990, Gobas et al. 1993).

6) Bioconcentration Factor (BCF) is the ratio of the chemical concentration in the organism to that in the water, under water-only exposure conditions, and may be

xxiii expressed in units of L/kg wet weight of L/kg lipid weight (Mackay 1982, Connell 1990).

7) Biomagnification refers to a process of accumulation of a chemical substance in an organism due to dietary exposure and absorption of the chemical. The driving force of biomagnification is a fugacity gradient in the gastrointestinal tract of an organism,

where fGIT > forganism (Gobas et al. 1993, Gobas et al. 1999).

8) Bioaccumulation refers to the process of accumulation of a chemical substance in an organism, resulting from chemical uptake through all routes of exposure (e.g. dietary absorption, transport across the respiratory surface, dermal absorption, and inhalation), and typically takes place under field conditions (Gobas and Morrison 1998, Clark et al. 1990).

9) Bioaccumulation Factor (BAF) is the ratio of the chemical concentration in the organism to that in the water, as the result of all routes of chemical exposure (e.g., water and diet), and is may be expressed in units of L/kg wet weight (Connell 1990).

10) Biota – Sediment Accumulation Factor (BSAF) is the ratio of the chemical concentration in the organism to that in the sediment. When normalized to organic carbon content in the sediment and lipid content in the organism, it is expressed in units of kg OC/ kg lipid (Morrison et al. 1996).

11) Biomagnification in the food chain refers to the process of chemical accumulation in the food chain, where the chemical fugacities in the organisms increase at each trophic level, due to biomagnification (dietary uptake) (Clark et al. 1990, Gobas 1993, Gobas et al. 1993, Gobas et al. 1999).

12) Trophic Dilution refers to a process where the chemical fugacities in organisms decrease at higher trophic levels in the food chain, generally due to metabolic transformation of the chemical within organisms.

xxiv 1. INTRODUCTION

Phthalates esters (PEs, Figure 1.1) are widely used as plasticizers in polyvinyl

chloride (PVC), polyvinyl acetates, cellulosics and polyurethanes. Additionally, they

have several other non-plasticizer applications including use in lubricating oils,

automobile parts, paints, glues, insect repellents, photographic films, perfumes, and food packaging (e.g. paperboard and cardboard) (Pierce et al. 1980). Current North American

production of phthalate esters is approximately 650,000 tonnes/year, while the global

production level is approximately 4,300,000 tonnes/year (Furtmann 1996, Parkerton and

Konkel 2000). Industrial formulations of phthalate esters include a large number of

congeners, which vary in alkyl chain length and branching and range in molecular weight

from 194 to over 600 g/mol. Phthalate esters are hydrophobic chemicals with octanol-

seawater partition coefficients (KOW’s) ranging between 101.8 for dimethyl phthalate to

1010.6 for diisodecyl phthalate (Table 1.1, Staples et al. 1997a, Cousins and Mackay

2000). Due to their hydrophobicity, phthalate esters are often assumed to have a high potential to bioconcentrate and bioaccumulate in biological organisms. A large number of laboratory studies have investigated the bioconcentration of phthalate esters in various fish species, algae, macrophytes, polychaetes, molluscs, crustaceans and aquatic insects

(Staples et al. 1997a). These studies indicate that phthalate esters may bioconcentrate in several taxa. However, quantification of reliable bioconcentration factors (BCFs) in most reported studies has been problematic due to several experimental artifacts, including the use of radiolabeled compounds, and conducting experiments at exposure concentrations in excess of the aqueous solubility of the test substance. Hence, the reported BCF values

1 may not accurately characterize the bioaccumulation potential of phthalate esters. In fish and certain invertebrate species, the BCFs that have been reported for certain phthalate ester congeners are less than expected from their Kow. The lower than expected BCFs of these substances have been linked to an organism’s ability to metabolize phthalate ester congeners. Bioavailability in the water phase has also been identified as another important factor affecting the measured BCFs in laboratory experiments. However, its role has never been determined or quantified. In terms of the environmental fate of phthalate esters, it has been suggested that these substances do not bioaccumulate in the food chain (Staples et al. 1997a, Macek et al. 1979, Belise et al. 1975). However, field studies to confirm this do not exist. Additionally, the majority of the data collected on the bioaccumulation of phthalate esters refers to a small number of congeners. Data on

DEHP are abundant, whereas similar data for other congeners are sparse or non-existent.

C R O

O C R′

Figure 1.1. Generalized Phthalate Ester Chemical Structure

2 Table 1.1. Molecular Weights (g/mol), Le Bas Molar Volumes (cm3/mol), Aqueous Solubilities (mg/L), and Log Octanol – Seawater Partition Coefficients1 of 13 Selected Phthalate Esters, as Reported in Cousins & Mackay (2000). Molecular Le Bas Molar AQ Solubility Salinity Phthalate Ester Weight Volume (mg/L) Corrected (g/mol) (cm3/mol) Log Kow1 Dimethyl DMP 194.2 206.4 5220 1.80 Diethyl DEP 222.2 254.0 591 2.77 Diisobutyl DiBP 278.4 342.8 9.9 4.58 Di-n-butyl DnBP 278.4 342.8 9.9 4.58 Butyl Benzyl BBP 312.4 364.8 3.8 5.03 Di(2-ethylhexyl) DEHP 390.6 520.4 2.5 ·10-3 8.20 Di-n-octyl DnOP 390.6 520.4 2.5 ·10-3 8.20 Di-n-nonyl DnNP 418.6 564.8 6.0 ·10-4 9.11 Diisohexyl C6 334.4 431.6 5.0 ·10-2 6.69 Diisoheptyl C7 362.4 476.0 1.1 ·10-2 7.44 Diisooctyl C8 390.6 520.4 2.5 ·10-3 8.20 Diisononyl C9 418.6 564.8 6.0 ·10-4 9.11 Diisodecyl C10 446.7 609.2 1.3 ·10-4 10.6 1 See Appendix A for calculation of the salinity corrected KOW.

The degree of bioaccumulation of phthalate esters is of considerable legal and regulatory importance. Both international legislation (UNECE Convention on Long on

Long Range Transboundary Air Pollution (1979) and its Protocol Persistent Organic

Pollutants, 1998), as well as domestic legislation in Canada (Canadian Environmental

Protection Act, 1999), the US (Toxic Substances Control Act, 1976; US EPA, 1998), and

Europe (UNECE, 1998) include provisions for eliminating substances from commerce that are “bioaccumulative”, “persistent” and “toxic”. Under the Canadian Environmental

Protection Act (CEPA), chemicals are considered “bioaccumulative” if they exhibit bioaccumulation factors (BAFs) or, alternatively, bioconcentration factors (BCFs) greater than 5,000 L/kg wet weight or 100,000 L/kg lipid weight in aquatic organisms. In the absence of a BAF or BCF, substances with octanol-water partition coefficients (KOW’s)

3 greater than 105 are classified as bioaccumulative. While certain phthalate esters meet

this hydrophobic criterion (Table 1.1), there is no evidence from field studies to support

categorizing the substances as bioaccumulative.

Since phthalate esters are hydrophobic chemicals, the research hypothesis is that

they will biomagnify in the food web (see preface pages for “definitions”).

Biomagnification occurs when organisms accumulate contaminants through dietary

sources, and are generally unable to metabolise the contaminants (Gobas et al. 1993,

Clark et al. 1990, and Gobas 1993). Biomagnification in the food web is defined to

occur if the fugacities of the test chemical increase at higher trophic levels in the food

chain, i.e., fpredator > fprey (Figure 1.2). Trophic dilution is an alternative hypothesis. It is defined to occur if fugacities decline with increasing trophic position, i.e., fpredator < fprey

(Figure 1.2). The lack of a fugacity increase or decrease in the food-chain (i.e., the null hypothesis), where the fugacities in the predator and prey are equal to those in the water, indicates that equilibrium partitioning of a substance between the lipid tissue of organisms and the water is occurring, i.e., fpredator ≅ fprey ≅ fwater (Figure 1.2, Mackay 1982,

1991, Gobas et al. 1993).

4 Biomagnification Trophic Dilution Equilibrium Partitioning

fpredator f fpredator predator

fprey2 fprey2 fprey2

fprey1 f fprey1 prey1

O O O fwater f O O fwater O O water O O

fsediment

Food Web Biomagnification Trophic Dilution Equilibrium Partitioning

fwater < fbiota fwater > fbiota fwater ≅ fbiota fprey1 < fprey2 < fpredator fprey1 > fprey2 > fpredator fprey1 ≅ fprey2 ≅ fpredator

Figure 1.2. Fugacity “f” Analysis of Alternative Hypotheses of Chemical Movement through a Food Chain.

The main purpose of this study is to determine the degree of food web bioaccumulation of phthalate esters. To test the alternative hypotheses of food web biomagnification versus trophic dilution versus equilibrium partitioning, a food web bioaccumulation field study was conducted in False Creek Harbour, Vancouver, British

Columbia, Canada. The bioaccumulation behaviour of eight individual phthalate ester

congeners (dimethyl (DMP), diethyl (DEP), di-iso-butyl (DiBP), di-n-butyl (DBP), butyl

benzyl (BBP), di 2-ethylhexyl (DEHP), di-n-octyl (DnOP), and di-n-nonyl (DNP)), and

five isomeric mixtures (di-iso-hexyl (C6), di-iso-heptyl (C7), di-iso-octyl (C8), di-iso- nonyl (C9), and di-iso-decyl (C10)) was investigated in this study (Table 1.1 and A.3 in

Appendix A). The author conducted a review of the literature on phthalate esters, related

to their production and use, chemical properties, bioaccumulation/ bioconcentration, and

5 ecological effects, which is presented in Appendix A. To ascertain the trophodynamic interactions and life history strategies of many of the key resident species in the southwestern British Columbia marine ecosystem, the author conducted an analysis of the relevant fisheries literature (Appendix B). Based on this information, a trophic position model was applied to quantify the positions of the species in the food web (Vander

Zanden and Rasmussen 1996, Section 2.5.5 and Appendix C). These trophic positions were used as the basis for assessing chemical movement in the food web. In addition to the scientific aspects of this research, the results are applied in a management context by comparing the observed bioaccumulation factors (BAFs) to the CEPA (1999) bioaccumulation criteria.

This study was conducted in collaboration between Simon Fraser University

(SFU) and the Institute for Ocean Sciences (IOS). The methods of this study involved (i) collecting environmental samples, conducted by the author (Section 2.1), (ii) filtering the seawater samples and measuring suspended particulate matter, conducted by the author

(Section 2.2.4 and 2.2.5), (iii) chemical extraction and analysis of the samples, conducted at IOS by Audrey Chong and Jody Carlow (laboratory equipment cleaning and sample preparations and extractions), and Hongwu Jing, Zhongping Lin, and Natasha Hoover

(GC-MS and LS-ESI/MS machine analysis) (Section 2.2), (iv) measurements of organic carbon and lipid contents in the sediment and biota samples, conducted at IOS by Linda

White (organic carbon contents), Audrey Chong and Jody Carlow (lipid contents)

(Section 2.4), (v) assessing data quality (QA/QC), conducted by the author (Section 2.3),

(vi) statistically analyzing the data, conducted by the author (Section 2.5), and (vii)

6 conducting a fugacity analysis of the concentration data, conducted by the author

(Section 2.5.4). The work at IOS was done under a grant to Dr. Frank Gobas, SFU.

7 2. METHODS

Overview: The methods section is divided into five parts describing the (i) field sampling, (ii) chemical analysis, (iii) methods for quality assurance and control (QA/QC) of data, (iv) supporting data measurement, and (v) methods related to the data analysis.

As stated in the Introduction, the chemical analysis and organic carbon and lipid content

measurements were conducted at the Institute for Ocean Sciences by Audrey Chong,

Jody Carlow, Hongwu Jing, Zhongping Lin, Natasha Hoover, and Linda White.

However, for completeness and because of their importance, I have included a

description of the methods related to the chemical analysis and supporting data

measurement in the Methods section. Additionally, as part of the overall study, the

methods that were developed for the chemical analysis of phthalate esters involved the concurrent extraction and analysis of polychlorinated biphenyls (PCBs). Therefore standards of both PEs and PCBs were added to the environmental samples, and are described in the methods section. However, all concentration results relating to PCBs in the environmental samples will be reported in Gobas et al. (in preparation).

2.1. Field Sampling Methods

2.1.1. Study Site and Design To assess the extent of phthalate ester bioaccumulation in a marine food web, a

field study was conducted in False Creek Harbour, a residential/ industrial embayment

located in downtown Vancouver, Canada (Figure 2.1.). False Creek is part of the Strait

of Georgia, where the mean summer temperature is 10.9°C, average salinity is 30 ppt,

and precipitation ranges from 90 to 200 cm/year. False Creek is shallow (i.e., mean

8 depth is ~ 20ft), and relatively well mixed. Within False Creek, three sampling stations

were selected to assess spatial variability: “North-Central” (49°16'13”N 123°07'40”W),

“Marina-South” (49°16'09”N 123°07'15”W), and “East-Basin” (49°16'28”N

123°06'18”W). Supplementary sediment and water samples were also collected from a

fourth station: “Cambie Bridge” (49°16' 18"N 123°07' 04"W). From each station, three independent samples of each media and species (i.e., sediment, water, and eighteen marine organisms) were collected to determine sampling and analytical variability. A limited number of samples of sediment (n=8), mussel (n=8), clam (n=3), and oyster (n=3) samples were collected from False Creek during a pilot study in July of 1998, and were used for analytical method development. The remainder of the sediment and biota samples, including the surf scoter samples from the Canadian Wildlife Service, were collected from May to October 1999, and were pooled with the 1998 samples. Water samples were collected in July of 2000, since they required additional time for sample filtration and extraction.

Figure 2.1. Map of field study site: False Creek Harbour, Vancouver, British Columbia, showing locations of four sampling stations (λ): “North Central”, “Marina – South”, “Cambie Bridge” and “East Basin”.

2.1.2. Preparation of Field Sampling Equipment Due to their widespread use, phthalate esters are commonly found in both sampling and analytical equipment, as well as in laboratory air and reagents. Consequently, reducing and determining the background contamination of samples is crucial for ensuring that environmental data on phthalate esters are acceptable, accurate and of high quality. Thus, several preparatory steps for cleaning field equipment were included in the protocol. All sampling equipment was made of glass or stainless steel. Glass vials (125 mL, 250 mL, 4L) were washed with lab grade detergent, rinsed twice with distilled hexane, iso-octane, and dichloromethane, and then heated in a muffler oven at 400°C for at least 10 hours. After baking, the vials were re-rinsed three times with distilled acetone, hexane, iso-octane, and dichloromethane, and then covered with clean aluminum foil and solvent rinsed metal lids.

Aluminum foil was rinsed with distilled acetone and distilled hexane and then heated at

350°C for 10 hours. Stainless steel sampling tools (e.g., spoons, knives, trays, and buckets) were cleaned following the procedures for the glass vials, and were wrapped with aluminum foil prior to sampling. The petit ponar sediment grab sampler was washed with lab-grade detergent and then rinsed three times with distilled acetone, hexane and dichloromethane.

2.1.3. Sediment Sample Collection Surficial sediment samples were collected using a petit ponar grab sampler and transferred onto clean aluminum foil (Figure 2.2). The top 0.5 to 1.0 cm, representing the

“active layer”, was removed with a metal spoon and transferred into a pre-cleaned glass vial, which was covered with aluminum foil and sealed with a metal lid. Vials were immediately placed on ice and were then kept at - 20°C in the dark prior to analysis.

11 2.1.4. Water Sample Collection Water samples were collected in 4L amber glass bottles from mid-ocean depth (~10-

12 ft) using a 12-foot extendible stainless steel pole (Figure 2.2). After collection, the bottles were sealed with a foil-lined lid, placed on ice, and then transferred to a 4°C refrigerator in the laboratory. Well water, used for procedural blanks, was collected from

Lynn Headwater Regional Park, North Vancouver. From each sample or blank, 1L of seawater or well water was quantitatively measured and spiked with 100ng of each DMP-d4,

13 13 13 DBP-d4, and DnOP-d4, 1.2ng of each C-PCB 52, C-PCB 128, C-PCB 209, and 5mL

HPLC grade methanol 1hr prior to extraction. The sample extraction occurred within 12 hours of collection, and is explained in the analytical methods section (2.2.4).

A) B)

12 ft. stainless steel extractable pole

Foil lined cork with monofilament line attached

Stainless steel wire attachment 4L amber glass bottle Figure 2.2. Field Sampling Equipment. Stainless steel A) Petit Ponar Sediment Grab Sampler, connecting and B) Seawater Collection Apparatus. clamp

12 2.1.5. Biota Sample Collection Eighteen marine organisms (Table 2.1, Figure 2.3) from various trophic levels in the food chain were collected from False Creek Harbour. These species represent both the benthic and pelagic food webs, and exhibit a variety of feeding strategies, sizes, and life- histories. For example, primary producers (e.g., plankton and algae) as well as both filter feeders (e.g. blue mussels (Mytilus edulis)) and deposit feeders (e.g., geoduck clams

(Panope abrupta)) were collected. The fish that were collected range from rapidly maturing, short-lived species with high fecundity rates such as the striped seaperch

(Embiotoca lateralis), to slow growing and long-lived species such as the spiny dogfish

(Squalus acanthias), whose gestation period lasts two years and natural life expectancy is greater than 50 years. Additionally, the selected species were “resident” or non-migratory, so that the False Creek sediment and water concentrations represented the phthalate ester levels to which the organisms were being exposed. The only exception was the dogfish, which inhabit larger range sizes and move inshore with the tide to forage. The selected species were also relatively abundant and widespread in False Creek, which facilitated collection. The methods of collection are described in Table 2.1. Plankton samples were collected in pre-cleaned 250mL glass vials. All other biota samples were wrapped in solvent- rinsed aluminum foil and frozen at - 20°C, prior to analysis.

13 Table 2.1. Trophic Category, Common Name, Latin Name, Sample Size, Mean Length (cm), Mean Wet Weight (g) and Sampling Methods for Eighteen Marine Organisms Collected from False Creek Harbour, Vancouver, British Columbia Trophic Common Name Latin Name Sample Mean Length Mean Weight Sampling / Collection Methods Group Size (Range) (cm) (Range) (g) Primary Green Algae Enteromorpha 9 NA NA Collected from shore at low tide Producers intestinalis Brown Algae Nereocystis luetkeana 1 NA NA Collected from water Fucus gardneri 1 Collected from shore at low tide Phytoplankton1 9 NA NA Plankton tow net - 236 μm mesh size Benthic Blue Mussels Mytilus edulis 9 NA Individual Collected from pilings during low tide Invertebrates Pacific Oysters Crassostrea gigas 9 shellfish were Collected off rocks during low tide Geoduck Clams Panope abrupta 9 pooled to Dug up from mud shore during low tide Manila Clams Tapes philippinarum 5 obtain Dug up from mud shore during low tide samples of ≥ 10g. Dungeness Crabs Cancer magister 9 12.4 252 Stainless steel crab traps and bait (9.3 – 16.0) (102 – 514) carapace width Purple Starfish Pisaster ochraccus 3 NA NR Collected from rocks and pilings during low tide Forage Fish Minnows 16 Beach seining net – ¼ inch mesh size Shiner Perch Cymatogaster aggregata 6 Individuals Individuals Pacific Staghorn Leptocottus armatus 3 ranged in size were pooled Sculpin from approx. to obtain Cutthroat Trout Salmo clarki clarki 2 2.5 – 10 cm samples of ≥ Three Spine Gasterosteus aculeatus 2 5g; individual Stickleback minnows Whitespotted Hexogrammos stelleri 2 ranged from Greenling approx. Starry Flounder Platichthys stellatus 1 1 – 20g

Trophic Common Name Latin Name Sample Mean Length Mean Weight Sampling / Collection Methods Group Size (Range) (cm) (Range) (g) Forage Fish Pacific Herring Clupea harengus pallasi 2 11 - 18 45 – 160 Herring gill nets – 1 inch mesh size Cont’d Surf Smelt Hypomesus pretiosus 1 15 30 pretiosus Northern Engraulis mordax 1 13 28 Herring gill nets – 1 inch mesh size Anchovy mordax Pile Perch Rhacochilus vacca 3 14.1 54 Beach seine net – ¼ inch mesh size AND (13.5 – 15.0) (49 – 60) Herring gill nets – 2 inch mesh size Striped Seaperch Embiotoca lateralis 9 14.2 73 (12.5 – 17.5) (49 – 174) Predatory Pacific Staghorn Leptocottus armatus 9 17.4 106 Stainless steel prawn traps & bait Fish Sculpin (12.0 – 29.5) (22 – 344) English Sole Pleuronectes ventulus 1 15 74 Sinking gill net – 2 inch mesh size Starry Flounder2 Platichthys stellatus 1 15 (11 – 22) NR Whitespotted Hexogrammos stelleri 9 20 126 Stainless steel prawn traps & bait Greenling (18.5 – 21.5) ( 100 – 141) Spiny Dogfish Squalus Acanthias 13 82 (61 – 104) ca. 2000 Long-line fishing Marine bird Surf Scoters Melanitta perspicillata 10 NA NR Collected by the Canadian Wildlife Service 1Plankton sample was a composite of phytoplankton and zooplankton, as well as other pelagic invertebrates and algae; 2The starry flounder sample was pooled from 3 individuals; NA = not applicable; NR = not reported / recorded.

Figure 2.3. Generalized Trophic Linkages Between Eighteen Marine Organisms Collected from False Creek Harbour and the Species Trophic Positions (see Section 2.5.5).

2.2. Analytical Methods for Determining Phthalate Ester Concentrations in Environmental Samples

2.2.1. Materials Standards of the individual phthalates: dimethyl phthalate (DMP), diethyl phthalate

(DEP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP) and di(2-ethylhexyl) phthalate (DEHP) were purchased from Aldrich (Milwaukee, WI) and di-n-octyl phthalate

(DnOP) from American Biorganics (Niagara Falls, NY). Standards of phthalate isomeric mixtures (C6H4(COOR)2: R = C6 to C10): JAYFLEX DHP (mixture of C6 isomers),

JAYFLEX77 (mixture of C7 isomers) and Diisodecyl phthalate (mixture of C10 isomers) were obtained from Exxon Chemical (New Milford, CT). Diisooctyl phthalate (mixture of

C8 isomers) was purchased from Aldrich, and diisononyl phthalate (mixture of C9 isomers) was obtained from Aritech Chemical (Pittsburgh, PA). The isotope-labeled compounds: d4-

DEP, d4-DBP and d4-BBP used as method internal standards (IS); and d4-DEP and d4-BBP used as method performance standards (PS), were purchased from Cambridge Isotope

Laboratories (Andover, MA). Individual standard stock solutions were prepared at various concentrations in toluene and the spiking solutions were prepared in acetone. The calibration solutions were diluted from the stock solutions with methanol. Isotope-labeled PCBs were purchased from Cambridge Isotope Laboratories and the compounds: 13C-PCB 28, 13C-PCB

13 13 13 13 13 13 105, C-PCB 118, C-PCB 156, C-PCB 15, d5-PCB 38, C-PCB 77, C-PCB 126, C-

PCB 169, 13C-PCB 52, 13C-PCB 101, 13C-PCB 128, 13C-PCB 180, 13C-PCB 194, 13C-PCB

208, and 13C-PCB 209 were used as internal standards (IS); and 13C-PCB 111 was used as a performance / external standard (PS). The PCB-labeled stock solutions were prepared in n- nonane, and were diluted in toluene to prepare spiking solutions for the biota and sediment

17 samples, or in methanol for spiking water samples. All solutions were kept at 4oC in the dark. Solvents were “distilled-in-glass” grade (Caledon, ON, Canada) and reagent water was high-purity HPLC grade (Burdick and Jackson, MI). Alumina (Neutral) was purchased from ICN Biomedicals (Germany). Sodium acetate and anhydrous sodium sulfate (granular) were purchased from Aldrich.

2.2.2. Preparation of Glassware and Reagents Since, solvents were found to be the dominant source of background PE contamination, all solvents used were doubly distilled. Laboratory glassware was detergent washed, rinsed with water, then with doubly-distilled acetone, hexane, iso-octane, and dichloromethane, respectively, baked at 400oC for at least 10 hours and stored in clean aluminum foil. Mortars and pestles were cleaned using the same procedure as that for glassware but were baked at 150 o C for 10 hours. As part of the QA/QC protocol, solvent rinses were collected, processed in the same manner as real samples, and then analyzed by

GC/MS to ensure that background contamination levels of phthalates did not exceed the machine detection limit. Alumina and sodium sulfate were baked at 200o C and 450o C, respectively, for at least 24 hours, cooled and stored in a desiccator. Other materials such as

Teflon stoppers, GC septa and caps of sample vials, which decompose at elevated temperatures, were washed extensively with 1:1 dichloromethane/hexane (DCM/Hex).

2.2.3. Extraction and Cleanup of Sediment and Biota Samples The sediment and biota sample extraction procedure is summarized in Figure 2.5.

Approximately 2 g of sediment or 5 g biota sample was weighed and spiked with 100 ng of

PE surrogate internal standards (i.e., DMP-d4, DBP-d4, and DnOP-d4), and 25 μl of prepared

Non-ortho PCB internal standard (i.e., 13C-PCB 28, 13C-PCB 105, 13C-PCB 118, and 13C-

18 13 PCB 156), 25 μl of prepared Mono-ortho PCB internal standard (i.e., C-PCB 15, d5-PCB

38, 13C-PCB 77, 13C-PCB 126, and 13C-PCB 169), and 25 μl of prepared Di-ortho PCB internal standard (i.e., 13C-PCB 52, 13C-PCB 101, 13C-PCB 128, 13C-PCB 180, 13C-PCB

194, 13C-PCB 208, and 13C-PCB 209) (Table 2.2). The sample was then blended with 15 to

20 g of pre-baked sodium sulfate, and ground with mortar and pestle to a free-flowing powder. The homogenate was placed in a flask, extracted with 50 mL of 1:1 (v/v)

DCM/Hex in a Branson 5210 ultrasonic water-bath (Branson Ultrasonics Co., CT) for 10 min, and shaken on a shaker table (Eberbach Co., MI) for 10 min. Once the suspended particles settled, the supernatant was removed. The extraction was repeated twice more with fresh solvent. The combined extracts were concentrated to ca. 5 mL under a gentle stream of high-purity nitrogen. The concentrate was quantitatively transferred onto a 350 mm x 10 mm i.d. glass column packed with 15 g deactivated alumina (activated with 15% HPLC water, w/w) and capped with 1-2 cm of anhydrous Na2SO4.

To prepare samples for GC/MS analysis, the column was eluted with three consecutive 30 ml fractions of (1) hexane; (2) 1:9 DCM/Hex; and (3) 1:1 DCM/Hex. The first fraction, containing the polychlorinated biphenyls (PCBs) in hexane was evaporated to near dryness under a stream of nitrogen and then re-dissolved in 5 mL 1:1 DCM/Hex. The concentrate was eluted over an acid-base silica column with 60 mL of 1:1 DCM/Hex. The eluent was then evaporated to near dryness and re-dissolved in 5 mL of hexane. This concentrate was quantitatively transferred to a dry alumina column, which was eluted with

(1) 25 mL of hexane, which was discarded, and (2) 60 mL of 1:1 DCM/Hex. The eluent from the second fraction was blown down to 100 μL under a gentle stream of nitrogen, and then spiked with 30 μL of the PCB recovery standard solution (i.e., 13C-PCB 111, Table

19 2.2), and analyzed by GC-High Resolution Mass Spectrometer (GC/HRMS). The analytical methods and results for the PCBs will be reported in Gobas and Maldonado (in preparation).

The third fraction eluent (in the 1:1 DCM/Hexane), contained the phthalate esters. It was concentrated to ca. 100 μL under a stream of nitrogen and spiked with 50 ng of the phthalate recovery standards (i.e., DEP-d4 and BBP-d4, Table 2.2) before GC-MS/LRMS analysis.

After running the sample on the GC-MS, the extract was then evaporated to near dryness, reconstituted in 300μL of doubly distilled methanol, and analyzed by LC/ESI-MS.

Table 2.2. Composition of Phthalate Ester (PE) and Polychlorinated Biphenyl (PCB) Standards, and Amounts (ng) Added to Sediment and Biota Samples. Standard Compounds Amount (ng) of Each Compound PE Internal DMP-d4, DBP-d4, DnOP-d4 100 PE External / Recovery DEP-d4, BBP-d4 50 PCB non ortho Internal 13C-PCB 28, 13C-PCB 105, 13C-PCB 118, ca. 1 Standard 13C-PCB 156 13 13 13 PCB mono ortho Internal C-PCB 15, d5-PCB 38, C-PCB 77, C- ca. 1 Standard PCB 126, 13C-PCB 169 PCB di ortho Internal 13C-PCB 52, 13C-PCB 101, 13C-PCB 128, ca. 1 Standard 13C-PCB 180, 13C-PCB 194, 13C-PCB 208, 13C-PCB 209 PCB External / Recovery 13C-PCB 111 ca. 2 Standard

2.2.4. Extraction and Cleanup of Seawater Samples An overview of the procedure for extraction of the water samples is presented in

Figure 2.5. The water extraction apparatus consisted of an FMI valveless pump, which pumped water at a flow rate of 8-10 ml/min through a 47mm glass fiber filter (0.45μm diameter pore size, from Gelman Laboratory, Pall Corporation, Ann Arbor, Michigan), and two independent 47mm Octadecyl (C18) extraction disks, which were housed in 47mm

20 stainless steel in-line filter holders (from Gelman Laboratory, Pall Corporation, Ann Arbor,

Michigan) (Figure 2.4). The C18 disks were from 3M (St. Paul, MN), and the typical composition is 90 % octadecyl bonded silica particles and 10% matrix PTFE by weight

(Hagen et al. 1990). Extensive cleaning of these discs prior to extraction by subsequent

15min sonications in iso-octane, doubly distilled toluene, and 1:1 DCM/hexane was required to remove phthalate ester residues from the commercial membranes. The final extract from the sonications was checked by GC to confirm that residual phthalate levels were negligible.

Porcelain lab pump

Inflow

Stainless steel filter holders containing:

Glass fibre filter

C18 Empore extraction disks

1L water sample in glass bottle

Outflow Figure 2.4. Water Extraction Apparatus Consisting of FMI Valveless Laboratory Pump and Three 47mm Stainless Steel In-line Filter Holders Housing a Glass Fibre Filter (0.45μm diameter pore size) in Holder #1, and an Octadecyl (C18) Empore Extraction Disk in Holders #2 and #3.

21 After filtration of the 1L seawater sample (to which the internal standards had been added) at the Simon Fraser laboratory, the glass fibre filter and C18 disks were removed from the system and collected separately in glass vials containing 15 mL 1:1 DCM/Hex. These vials were refrigerated at 4°C and transferred to the IOS lab. The filter and disks were then extracted independently by 3 subsequent 5 min sonications with 20mL 1:1 DCM/hexane.

These extracts were combined and concentrated to 3-5mL under a gentle stream of high purity nitrogen, which was then quantitatively transferred to a neutral alumina column for cleanup. The alumina column was packed with 15g deactivated alumina (15% H20, w/w), topped with a 2cm layer of anhydrous Na2SO4 . Prior to introducing the extract, 15-20mL doubly distilled hexane was run through the column. The sample was then loaded on the column, which was then eluted with 3 consecutive 30mL fractions of (1) hexane, (2) 1:9

DCM/Hex, and (3) 1:1 DCM/Hex. The third fraction was collected and concentrated to

100μL and spiked with 50ng of the recovery standards (i.e., DEP-d4 and BBP-d4). The sample vial was capped with clean aluminum foil lined septa and analyzed by GC-MS. After

GC-MS analysis, the samples were evaporated to near dryness under a gentle stream of high purity nitrogen, re-dissolved in 300μL of doubly distilled methanol, and then analyzed by

LC/ESI-MS.

2.2.5. Quantification of Suspended Particulate Matter in the Seawater Samples

Suspended particulate matter was found on both the glass fiber filter and C18 extraction disk(s). Particulate matter amounts were quantified by pumping the remaining 3L of each seawater sample through the filtration system (at SFU) and subtracting the pre- filtered dry weights from the post-filtered dry weights of the glass fibre filter and C18 disks.

22 Particulate matter present on the glass fibre filter was greater than 0.45μm in diameter and was operationally defined “large diameter suspended matter” (LDSM). The particulate matter measured on the C18 disk was fine-grain material and was operationally defined as

“small diameter suspended matter” (SDSM).

Figure 2.5. Summary of Sediment, Biota and Water Sample Extraction Procedures for Phthalate Ester Analysis (Polychlorinated Biphenyls were Extracted Concurrently).

2.2.6. GC/MS Analysis of Environmental Samples Low-resolution gas chromatography with detection by mass spectrometry (GC/MS) was used for the quantification of the individual phthalate esters (i.e., DMP, DEP, DiBP,

DnBP, BBP, DEHP, DnOP, DnNP) in the marine samples. GC/MS analysis was carried out on a Finnigan Voyager GC/MS system (Manchester, UK) at the IOS laboratory. The mass spectrometer was operated at 70 eV in the EI mode with a resolution of approximately 1000.

Data were acquired in the selected ion recording mode (m/z 163 for DMP and m/z 149 for all other phthalates) and processed using Masslab software (version 1.4). The dwell time was 100 ms, with a delay time of 10 ms. The mass spectrometer was coupled to a Finnigan

8000 Series gas chromatograph. A J & W DB-5 fused silica capillary column (30 m x 0.25 mm, 0.1 μm film thickness) was used for separation. The injection port, GC/MS interface and ion source temperature were kept at a constant temperature of 250oC. Splitless injections of 1 μL were analyzed by programming the column temperature to go from 100oC

(held for 1 min) to 180 at 5oC/min, then to 240oC at 10oC/min, and to 280oC at 10oC/ min

(held for 10 min). Helium was used as the carrier gas at a flow rate of 1 mL/min.

2.2.7. LC/ESI-MS Analysis of Environmental Samples Liquid chromatography with electron spray ionization was applied at the IOS laboratory to quantify the concentrations of isomeric commercial mixtures of phthalate esters in the marine samples (i.e., C6, C7, c8, C9, and C10) (see Lin et al. in preparation).

The eluent was delivered by a Beckman Model 126 programmable solvent system controlled by a Beckman System Gold software (version 8.1) (Beckman, Fullerton, CA).

Chromatographic separations were performed on a 250mm x 2mm I.D. stainless steel C8 analytical column packed with 5μm Spherclone (Phenomenex, Torrance, CA). An OPTI-

25 SOLV Mini-Filter (Chromatographic Specialties Inc., ON) was used to protect the analytical column. For the determination of individual phthalates, gradient elution was applied using an eluent that contained mobile phase A (60:40 methanol/water with 0.5mM sodium acetate) and mobile phase B (pure methanol with 0.5mM sodium acetate). The solvent composition was held for 5 min at 60% A, and then linearly programmed to 100% A at 12 min, and then to 30 % A in 33 min. For the determination of phthalate ester isomeric mixtures, the eluent used was 90:10 methanol/water with 0.5mM sodium acetate, which was held constant throughout the analysis. The injection volume was 3μL, and the mobile phase flow rate was

0.22mL/min. To be compatible with ESI, a splitter was used to feed about 50μL/min of eluent into the sprayer. The flow-split ratio was regulated by adjusting the length and diameter of two capillary tubes.

Mass analysis was performed using a VG Quattro triple-quadrupole mass spectrometer equipped with a pneumatically assisted electrospray source (Micromass,

Manchester, UK). The source temperature was 150o C, and nitrogen was used as the bath and nebulizing gas (250, 16L/hr, respectively). Typical electrospray ionization conditions were as follows: electrospray capillary voltage, 3.7kV; high-voltage lens (counter electrode),

150V; skimmer cone voltage, 27V; focus (second skimmer) voltage, 20V. The tuning conditions were optimized by performing flow-injection analysis of a solution of stable isotope-labeled benzyl butyl phthalate (ring-d4). The mass spectrometer was operated in the positive ion mode. Mass spectra were scanned in the m/z range of 50 to 500 at the rate of

5s/scan, with an inter-scan delay of 10ms. A dwell time of 200ms per Dalton was used for selected ion monitoring LC-MS experiments. For quantitative analysis, the mass spectrometer was operated under single ion recording (SIR) mode, and was monitored for

26 m/z of 357, 385, 413, 441 and 469 for C6, C7, C8, C9 and C10, respectively as well as m/z

417 for the internal standard DnOP-d4. Data were processed using the Masslynx software

(version 2.1). Peak areas were obtained from the Masslynx data system by interactive processing, where the peak baselines were operator defined.

2.2.8. Optimization of ESI-MS Parameters Optimization of ESI-MS parameters was carried out by flow injection analysis (FIA) experiments. For FIA/ESI-MS, 20μL of phthalate standard solution was directly injected into the flow of the mobile phase at a flow rate of 20μL/min. The MS was operated in full scan mode under positive ion mode covering the mass range m/z 50 to 500. In negative ion mode, all phthalates tested did not show detectable signals. After preliminary tuning and signal optimization with FIA, the final optimization was accomplished with the LC column in place because, under chromatographic conditions, the system performance is compromised by i) the presence and condition of the LC column, ii) ionic strength additives, and iii) variable solvent compositions from gradient elution. The quantitative linearity of the

LC/ESI-MS response was tested for the concentration ranges of 0.0028 to 42.8 ng/μL for the individual phthalates, and 0.0428 to 55.1 ng/μL for phthalate ester isomeric mixtures.

2.2.9. LC/ESI-MS/MS Analysis of Environmental Samples To investigate the potential co-elution of sample matrix interferences with the phthalate ester isomers, tandem mass spectrometry (MS-MS) (at the IOS lab) was used for confirmation. The VG Quattro MS machine was operated in multiple reaction monitor

(MRM) mode to produce collision-induced dissociation (CID). To optimize the response

+ + + + + under MS-MS conditions, different modifiers (i.e., Na , Li , K , H , and NH4 ), concentrations of modifiers, and analytical/ monitoring conditions (i.e., collision energies,

27 and the collision gas pressures) were investigated and will be reported on in Ikonomou et al., in preparation. Lithium (Li+) was used as a solvent modifier because it had a higher overall sensitivity, and produced ESI-MS/MS spectra with several major daughter ions, and higher daughter to parent ion ratios, relative to the other molecular adducts. Argon was used as collision gas, with a pressure of about 2·10-4 mbar in the analyzer vacuum. Typical conditions used for lithiated ions were: collision energy 75 eV, cone voltage 33 V, capillary

4.23 kV, HV lens 320V. Under MS-MS conditions, the Li+ adduct produced two major common daughter ions, i.e., m/z 155 and 173, from the phthalate ester isomeric mixtures

(C6, C7, C8, C9, and C10). Specific ions were also formed for each isomer: diisodecyl

(C10) ions with m/z 453 (parent), 313 and 165 (daughters); diisononyl (C9) ions with m/z

425 (parent), 299, and 151 (daughters); diisooctyl (C8) ions with m/z 397 (parent), 285 and

137 (daughters); Jayflex 77 (C7) ions with m/z 369 (parent), 271 and 123 (daughters); and

Jayflex DHP (C6) ions with m/z 341 (parent), 257 and 109 (daughters).

2.2.10. MS Calibration, Recovery and Procedural Blanks Prior to sample analysis, calibration curves were constructed to verify the linearity of the MS response for all native phthalate esters covering the concentration range of 0.3pg/μL to 2000pg/μL (GC/MS), and 7pg/μL to 4000pg/μL (LC-ESI/MS). Machine detection limits were assessed by analyzing the lowest concentration standard solution (i.e., 0.3 pg/μL for

GC/MS, and 7pg/μL for LC-ESI/MS), and repeating this analysis periodically.

Additionally, a calibration standard was run at the beginning and end of each batch of sample runs. For GC-MS analysis, this standard calibration solution contained the individual phthalate test chemicals at concentrations of ca. 100pg/μL, as well as the internal standards and recovery standards at concentrations of ca. 500pg/μL. The LC-ESI/MS batch

28 calibration solution contained both the individual test chemicals and isomeric mixtures at concentrations of ca. 400 – 900pg/μL, and the internal and recovery standards at concentrations of ca. 250pg/μL.

To determine the recovery of the test chemicals throughout the extraction and clean up process, each sample and blank was spiked with 100ng of the internal standards (i.e.,

DMP-d4, DBP-d4 and DnOP-d4) prior to extraction. Recovered amounts were quantified by mass spectrometry, and used to correct the data for chemical loss and variance in machine sensitivity. 50ng of each external/ recovery standards (i.e., DEP-d4 and BBP-d4) were added prior to machine analysis to correct for variance in the sample injection and the sensitivity of the MS detector. Procedural blanks for the sediment and biota consisted of 10-20g of pre- baked sodium sulfate; those for the seawater consisted of 1L of well water.

2.2.11. Quantitation of Phthalate Esters in Environmental Samples Quantification was achieved by generation of relative response factors (RRFs) for each analyte, which relate peak area-to-mass ratios for two compounds, “i" and “j” (e.g., the internal standard and the recovery standard, or the test analyte and the internal standard)

(Equation 2.1):

RRFi/j = (Peak Areai / Massi) (2.1) (Peak Areaj / Massj)

Relative response factors were determined from analyzing a standard calibration solution

(with known amounts each analyte) before and after batch analysis, and using the mean of these two calibration runs for quantitation. RRFs for the recovery standards (RS) (DEP-d4 and BBP-d4) in the calibration solution were set to a value of one. RRFs for the internal standards (IS) (i.e., DMP-d4, DBP-d4, and DnOP-d4) in the calibration solution were determined from peak area-to-mass ratios with the recovery standard (RS) that was most

29 similar in molecular weight (i.e., DMP-d4 from DEP-d4; and DBP-d4 and DnOP-d4 from

BBP-d4) (i.e., RRFIS/RS). Relative response factors for the native phthalates (PE) in the calibration solution were determined from peak area-to-mass ratios with the internal standard most similar in molecular weight (i.e., DMP and DEP from DMP-d4; DiBP, DBP,

BBP, C6 and C7 from DBP-d4; and DEHP, DnOP, DnNP, C8, C9, and C10 from DnOP-d4)

(i.e., RRFPE/IS).

These relative response factors derived from the standard calibration solution were then applied to quantify internal standard recoveries and amounts of phthalate esters in the environmental samples and procedural blanks. The percent recovery was determined by quantification of internal standard amounts (i.e., DMP-d4, DBP-d4 and DnOP-d4) in the marine samples and blanks (Equation 2.2a):

MassIS = (Peak AreaIS / RRFIS/RS ) (2.2a) (Peak AreaRS / Mass SpikedRS)

RecoveryIS = MassIS Mass SpikedIS

To quantify the native phthalate ester amounts in each sample and blank, the RRFPE/IS was then used (Equation 2.2b):

MassPE = (Peak AreaPE / RRFPE/IS ) (2.2b) (Peak AreaIS / Mass SpikedIS)

2.2.12. Quantification of Diisodecyl Phthalate (C10) in Biota Samples While LC/ESI-MS/MS confirmation revealed no interferences for C6-C9 phthalate isomers, a significant interference contributed the C10 response in the biological tissue samples. As a result, MS-MS confirmation using Li+ was required for quantification of diisodecyl phthalate (C10) in the biological samples. Approximately 50% of the biota

30 samples were confirmed using LC/ESI-MS/MS. The ratio of C10 to the total peak area (i.e.,

C10 + interference) was determined for these samples and ranged from approximately 70% of the mass response for green algae and plankton samples to approximately 0.1% in fish tissue samples (Table 2.3). To estimate the C10 concentration in samples that were not confirmed with MS-MS, the mean fraction of C10 in the total peak area for each specific species was applied to the unconfirmed LC-MS concentration data (which included C10 plus the interference). These concentration data are reported in Section 3.4 (Biota

Concentration Results).

Table 2.3. Mean Percentage of C10 in the Total Peak (C10 + Interference), the Coefficient of Variation (%), Sample size (n), and Number of Samples with Non-Detect C10 Concentrations for Biota Samples Confirmed using LC/ESI-MS/MS Species Mean % of C10 Coefficient of Samples ND1 Samples in the Total Peak Variation (%) Confirmed (n) for C10 Green Algae 72.7 18.8 3 0 Brown Algae 4.7 2 0 Plankton 65.4 17.5 5 1 Mussels 0.9 47.0 4 0 Oysters 2.0 55.1 6 0 M. Clams 1.4 5.2 2 0 G. Clams 5.0 34.2 3 0 Starfish 0.2 2 0 D. Crabs 0.3 26.9 4 1 Minnows 2.6 94.4 7 4 P. Perch ND 2 2 S. Perch 33.4 74.1 7 3 Forage Fish ND 2 2 Sculpin 0.5 5 3 Greenling 0.4 8.3 6 3 Dogfish - Liver 0.1 27.2 5 3 Dogfish - Muscle 0.6 12.9 5 3 Dogfish - Embryo 0.2 70.7 2 0 S. Scoter (Bird) 2.5 70.2 9 0 1Number of samples with “Non-Detectable” (ND) concentrations of C10.

31 2.3. Quality Assurance and Control of Data (QA/QC)

After quantitation, data quality was evaluated. Each sample was corrected for background contamination of phthalate esters and chemical loss during sample extraction, and then screened against method detection limits (MDLs). Certain tables and figures for this section are presented in Appendix D “Data QA/QC”.

2.3.1. Sediment & Biota Concentration Data

2.3.1.1. Sodium Sulfate Blanks for Sediment & Biota Sample Analysis To assess the background contamination of phthalate esters, two or three sodium sulfate blanks were included in each batch of sediment and biota samples (4-6 samples). All samples were blank-corrected by subtracting the mean concentration of phthalate esters in the blanks from each sample in the batch, prior to recovery correction. (In cases of batches with one high blank, the highest blank was used for the correction. This was necessary for only four out of thirty-five biota batches).

For GC-MS analysis, mean concentrations of phthalate esters in the sodium sulfate blank ranged from 0.07 ng/g for DOP to 5.05 ng/g for DBP for biota analysis (n=85), and from 0.24 to 10.06 ng/g for DMP and DBP, respectively, for sediment analysis (n=20). For

LC-ESI/MS, the mean concentrations of phthalate ester isomers in the blanks ranged from

0.12 to 3.46 ng/g (biota blanks, n=46), and from 0.28 to 8.62 ng/g (sediment blanks, n=14) for C6 and C8, respectively, (Figure 2.6 and Table D.2.4 in Appendix D).

32 100

1 Sediment Biota

0.1 Concentration (ng/g NaSO4) Concentration (ng/g

0.01

P P P P P P P P 6 7 8 9 0 1 M E IB B B H O N C C C C E C D D D D B D D D Phthalate Ester Figure 2.6. Mean Phthalate Ester Concentrations (ng/g) in Sodium Sulfate Procedural Blanks for Sediment and Biota Analysis. Error bars represent one standard deviation.

2.3.1.2. Method Detection Limits for Sediment & Biota Concentration Data To ensure that background contamination did not contribute to the reported environmental concentrations, method detection limits (MDLs) were determined. MDLs were defined as 3 standard deviations above the mean blank concentration. (When data are blank corrected, the MDL is simply equal to 3 standard deviations). The MDLs were determined on a per-batch basis for the biota samples, and by using all the procedural blanks for sediment samples (n=18), since sediment blanks were consistent between batches. Batch

MDLs were used for the biota data because of inter-batch variability in the blanks (i.e., background contamination was usually reduced in later batches). Inter-batch variability in the blanks was an issue for the biota data because environmental concentrations of phthalate

33 esters were typically lower in the biota samples, relative to the sediment samples. The batch-based MDL method is demonstrated for eight representative biota batches in Figure

D.2.7 (Appendix D). Figure D.2.7 shows that, with the exception of DMP, DEP, DiBP, and

C6, there is variability in the blank concentrations between batches, particularly for DnBP,

DEHP, and C8.

The sediment and biota MDLs are presented in Table 2.5. Since the biota MDLs were calculated on a per-batch basis, the mean variability (i.e., 3 standard deviations) and the mean MDLs (i.e., mean blank concentrations + mean 3 standard deviations), for 35 biota batches, are reported in the table. All sediment and biota samples were compared to the

MDL. The concentration data is presented with the MDLs in Figures D.2.8 (Sediment), and

D.2.9 (Biota) (Appendix D). (The data in these figures are blank-corrected, so the MDLs are equivalent to 3 standard deviations). Concentrations that were greater than the MDL were used in further analysis and reporting. Concentrations that were greater than the average blank level, but did not meet the MDL, were always excluded for the sediment data, and usually excluded for the biota data. Biota concentrations that did not meet the batch MDL, but were within the range of data from other batches that met the MDL for that particular congener and species, were included in the analysis (see Figure D.2.9). Again, because the biota MDLs were calculated on a per-batch basis, the 10th, 50th, and 90th percentiles of the batch MDLs are presented in Figure D.2.9.

34 Table 2.5. Mean Concentrations (ng/g) of Phthalate Esters in Sodium Sulfate Procedural Blanks for Biota and Sediment Analysis, 3 Standard Deviations of the Blanks, and Method Detection Limits Defined as the Mean Blank Concentration + 3 Standard Deviations. Phthalate Biota Analysis Sediment Analysis Ester Mean 3 Stdev1 MDL1 Mean 3 Stdev MDL GC-MS DMP 0.11 0.04 0.15 0.24 0.44 0.68 DEP 1.13 0.38 1.51 2.99 4.75 7.74 DIBP 0.33 0.14 0.47 0.54 0.54 1.08 DBP 5.05 1.77 6.82 10.1 12.2 22.3 BBP 0.8 0.31 1.11 2.27 4.8 6.07 DEHP 2.14 0.92 3.06 8.83 15.6 24.4 DOP 0.07 0.02 0.09 0.8 2.17 2.97 DNP 0.08 0.02 0.1 0.5 1.07 1.57 LC-ESI/MS C6 0.12 0.04 0.16 0.28 0.28 0.56 C7 0.39 0.14 0.53 0.79 1.92 2.71 C8 3.46 3.02 6.48 8.62 32.5 41.1 C9 0.85 0.19 1.04 1.6 2.83 4.43 C10 1.19 0.4 1.59 1.54 3.08 4.62 1The mean “3 standard deviations” of the blanks in 35 biota batches is presented and used for determining the mean MDL1.

2.3.1.3. Recovery of Internal Standards in Sediment & Biota Samples To correct for chemical loss during the extraction procedure, and changes in machine sensitivity, deuterated internal standards (IS) (i.e., DMP-d4, DBP-d4, DOP-d4) were added to all samples prior to extraction. The fraction of IS recovered gives an indication of the efficiency of extraction. The recoveries for the sediment and biota samples are presented in

Table 2.6. Mean IS recoveries from spiked False Creek sediments ranged from 82 to 95%

(GC/MS) and from 79 to 101% (LC-ESI/MS) for DMP-d4 and DOP-d4 respectively. Those for spiked sodium sulfate sediment blanks ranged from 72 to 78% (GC-MS), and 80 to

100% (LC-ESI/MS) for DMP-d4 and DOP-d4 respectively. Mean IS recoveries in biota

35 samples were 84, 91, and 71% (GC/MS), and 76, 99 and 90% (LC-ESI/MS), and in sodium sulfate blanks were 82, 89, and 88% (GC/MS) and 80, 96, 100% (LC-ESI/MS). The relatively low recovery for DOP-d4 in some biological samples analyzed by GC/MS (e.g., dogfish liver and crab hepatopancreas) coincided with high lipid contents in those tissues. If the recovery of any surrogate standard (i.e., IS) was outside the range of 50 to 130%, the sample was re-processed and reanalyzed, or the data were excluded from further analysis.

Table 2.6. Mean (+/- standard deviation) Recoveries of Internal Standards from Spiked False Creek Sediment and Biota Samples and Sodium Sulfate Blanks (%) 1 Media Analysis Material DMP-d4 DBP-d4 DOP-d4 Sediment GC/MS False Creek 82 +/- 12 89 +/- 12 95 +/- 19 Na2SO4 Blanks 72 +/- 13 73 +/- 16 78 +/-21 LC-ESI/MS False Creek 79 +/- 21 102 +/- 23 101 +/- 20 Na2SO4 Blanks 80 +/- 13 96 +/- 7 100 +/- 13 Biota GC/MS False Creek 84 +/- 15 91 +/- 15 71 +/- 23a Na2SO4 Blanks 82 +/- 10 89 +/- 12 88 +/- 13 LC-ESI/MS False Creek 76 +/- 18 99 +/- 9 90 +/- 24 Na2SO4 Blanks 80 +/- 13 96 +/- 7 100 +/- 13 1DMP-d4 recoveries were not used for LC-ESI/MS for C6-C10 isomers

2.3.2. Seawater Concentration Data

2.3.2.1. Recovery of Internal Standards in Seawater Samples Total water concentrations were determined by adding the measured amounts on the glass fibre filter and C18 extraction disks, and then correcting this total concentration for sample recovery. To determine the recovery of the test chemicals throughout the extraction and clean up process, water samples were spiked with internal standards (i.e., DMP-d4,

DBP-d4 and DnOP-d4) approximately 1 hour prior to filtration; external standards (i.e., DEP- d4 and BBP-d4) were added to sample extracts prior to machine injection. Total water

36 recoveries were determined by comparing the observed amounts of the internal standards on both the glass fiber and C18 extraction disks to the amount of chemical spiked. Mean internal standard recoveries were 70, 86 and 37% (GC/MS) and 54, 93 and 50% (LC-ESI/MS) in seawater, and 70, 79 and 48% (GC/MS) and 58, 86, and 69% (LC-ESI/MS) in well water

(Table 2.7, Figure 2.11). The apparent drop in recovery from 80% for DBP to 40% for DOP agreed with similar observations by Holadova and Hajslova (1995) and is believed to reflect increased adsorption to the glass wall of the bottle due to the increase in KOW. Congeners for which the observed recoveries were lower than 50% (DMP to BBP, and C6 to C7) were excluded from the data set. Due to consistently lower recoveries for the higher molecular weight phthalates (i.e., DEHP to DnNP, and C8 to C10), data for these PEs with recoveries below 30% were excluded.

Table 2.7. Mean (+/- standard deviation) Internal Standard Recoveries for False Creek Seawater Samples and Well Water Blanks (%)

Media Analysis Material DMP-d4 DBP-d4 DOP-d4 Water GC/MS False Creek 70 +/- 20 86 +/- 28 37 +/- 12 Well Water 70 +/- 32 79 +/- 36 48 +/- 22 LC-ESI/MS False Creek 54 +/- 16* 93 +/- 33 50 +/- 16 Well Water 58 +/- 27* 86 +/- 38 69 +/- 31 *DMP-d4 recoveries were not used for LC-ESI/MS for C6-C10 isomers

37 Well Water Blanks False Creek Seawater 120%

100%

80%

60%

C18 C18 Recovery (%) Recovery 40% C18 C18 C18 C18 20% GF GF GF GF 0% DMP-d4 DnBP-d4 DnOP-d4 Internal Standard Figure 2.11. Mean Total Recoveries (%) of Internal Standards in Spiked Well Water Blanks and False Creek Seawater Samples using GC/MS Analysis. Bars indicate fractions on the

Glass Fibre Filter (GF) and C18 Extraction Disks (C18). Error bars indicate one standard deviation.

Recoveries of the internal standards in the water appeared to follow a bilinear relationship with Kow. Hence, a bilinear relationship was applied to determine the recovery of each phthalate ester in each sample. The linear relationship, recovery (%) = mi * log

KOW + bi, was used to determine the recoveries of phthalates with log KOW’s between 1.61 and 4.45, where the slope (mi), and y-intercept (bi) were determined from the recoveries of the internal standards in each sample. A second relationship was used to determine recoveries for PEs with a log KOW between 4.45 and > 8.06. For the isomeric mixtures, the linear relationships were based on the number of carbons (C) on each ester chain (i.e., recovery (%) = mi * C + bi). The first linear relationship was used to determine recoveries

38 for phthalates with 1 to 4 carbons on the ester chains; the second was used to determine recoveries for phthalates with ester chains of 5 to 10 carbons.

2.3.2.2. Background Contamination of Seawater Samples Each sample was also screened for contamination, where concentrations of a particular congener in a sample were 25 to >100 times greater than those in the other replicate samples from the same station. If both the glass fibre filter (GF) and C18 extraction disks (C18) were contaminated for a particular congener, then that concentration was discarded. If only one of the GF filter or the C18 disk was contaminated for a particular congener, then a total concentration was determined from the chemical concentration on the uncontaminated-disk. This calculation was based on the partitioning behaviour of the substance between the two filter types.

2.3.2.3. Well Water Blanks for Seawater Sample Analysis Water blanks consisted of 1L of well water. They were extracted at the same time, and following the same procedures as the seawater samples (in batches of two or three blanks per three seawater samples). After recovery correction, each water sample was then blank-corrected by subtracting the mean response of the blanks in the batch from the seawater observations. Mean phthalate ester concentrations in well water blanks ranged from 2.16 ng/L for DOP to 128 ng/L for DBP, for GC/MS analysis, and from 3.50 ng/L for

C6 to 902 ng/L for C8 for LC/MS analysis (Figure 2.12 and Table D.2.8 in Appendix D).

39 1000

100

1 Concentration (ng/L) Concentration

0.1

P P P P P P P P 6 7 8 9 0 1 M E IB B B H O N C C C C B E C D D D D D D D Phthalate Ester Figure 2.12. Mean Concentrations (ng/L) of Phthalate Esters in Well Water Blanks. Error bars represent one standard deviation.

2.3.2.4. Method Detection Limits for Seawater Concentration Data Method detection limits (MDLs) for the water data were defined as 3 standard deviations above the mean phthalate ester concentrations in the well water blanks. The

MDLs were determined on a per-batch basis due to inter-batch variability in the blanks. The uncorrected sample concentrations (i.e., recovery corrected, but not blank corrected) are compared to the MDLs in Figure D.2.13 (Appendix D). Concentrations that were greater than the MDL were used in further analysis and reporting. Concentrations less than the

MDL were excluded from the data set. Table 2.8 presents the minimum and maximum method detection limits of phthalate esters in 4 batches of water samples. For DMP, DEP,

DiBP, DBP, DEHP, DOP and C10, the range of observed method detection limits was relatively small, indicating good reproducibility between the analyses. However, for BBP,

40 DNP and C6, C7, C8 and C9, there were substantial differences between the MDLs among batches of water samples due to the introduction of background contaminants throughout the chemical extraction process.

Table 2.9. Minimum and Maximum Method Detection Limits (MDLs) in ng/L among 4 Batches of Water Samples. MDLs represent the mean PE concentration in the batch blanks + 3 standard deviations. Phthalate Ester Minimum MDL Maximum MDL DMP 3.32 4.32 DEP 39.3 51.6 DIBP 6.39 7.9 DBP 177 221 BBP 6.56 44.4 DEHP 397 544 DOP 6.01 15.3 DNP 4.31 34.9 C6 4.7 25.9 C7 8.3 61.2 C8 327 1,060 C9 199 534 C10 50 99.3

2.3.2.5. Determination of the “Total”, “C18”, and “Freely Dissolved” Concentrations in the Seawater Samples, and the Chemical Phases that they Represent

The chemical in the water phase can be divided into different fractions. It may occur in the freely dissolved form, or associated with particulate matter. The particulate phase of the water contains suspended material of varying sizes. For the purpose of this study, we have divided this fraction into “large diameter suspended matter” (LDMS) (or particulate organic carbon), operationally defined as particles > 0.45μm in diameter, and “small

41 diameter suspended matter” (SDSM) (or dissolved organic carbon), operationally defined as solids < 0.45μm in diameter (Figure 2.14). Based on these different fractions of chemical in the water, three concentrations were derived in this study: (1) the “total water concentration”

CW(tot) (ng/L), (2) the “C18 water concentration” CW(C18) (ng/L), and (3) the “freely dissolved water concentration” CW(FD) (ng/L). The total water concentration was measured from combining the observed PE amounts on the glass fibre filter, MGF (ng), and C18 extraction disks, MC18 (ng), and represents all fractions or forms of the chemical in the water medium

(i.e., chemical bound to large diameter suspended matter (LDSM), and to small diameter suspended matter (SDSM), and freely dissolved chemical), i.e.,

CW(tot) = (MGF + MC18) / VW (2.3)

Where VW (L) is the volume of filtered sample water.

The C18 water concentration represents the observed PE amounts on the C18 extraction disks, and was determined as:

CW(C18) = CW(tot)·fC18 (2.4)

Where fC18 is the mean fraction of total chemical concentration detected on the C18 disks.

The C18 concentration includes two fractions of the chemical: dissolved organic-bound (i.e., chemical bound to SDSM), and freely dissolved chemical, since the large diameter suspended matter has been removed by the glass fibre filter. The “freely dissolved water” concentration was estimated by fitting observed concentrations on the glass fibre and C18 extraction disks to a three phase partitioning model, as described in Section 3.2.

42 Total Water Concentration

Particulate Freely Organic Dissolved Carbon Dissolved Organic Carbon

Freely Dissolved C18 Water Water Concentration Concentration (Model Estimated)

Figure 2.14. Illustration of the Particulate Organic Carbon (POC) – Bound Chemical (Large Diameter Suspended Matter “LDSM”), Dissolved Organic Carbon (DOC) – Bound Chemical (Small Diameter Suspended Matter “SDSM”), and the Freely Dissolved Chemical Fraction in the Water Phase and the Three Water Concentrations Reported in the Study.

2.3.3. Summary of the Sediment, Biota and Seawater Data Quality Table 2.10 gives an indication of the overall quality of all the data for both the individual phthalate congeners and the isomeric mixtures. Generally, for the biota and sediment data, more than 85% of the data meet the MDL screening requirements and provide reportable concentrations. However, the quality of the water data was generally lower, and varied between congeners. Specifically, the fraction of observed concentrations exceeding the MDLs ranged between 100% for DMP to as low as 17% for C8. Low ambient concentrations in certain samples and congeners, making background contamination a more significant factor, is one cause for the low proportion of values exceeding the MDL. A second factor causing a low proportion of the samples to exceed the MDL was the variation

43 in the MDL between batches of samples, allowing water concentrations to exceed the MDL in certain batches but not in others. The variation in the MDL is mainly due to differences in the levels of background contamination between batches, as well as variability in these background levels in the blanks within a batch.

Table 2.10. Number of Samples with Detectable Concentrations above the Method Detection Limits (MDLs). Media: Sediment Water Biota n=17 GC ; n=13 LC n=12 n=155 GC ; n=141 LC Data No. Samples > MDL No. Samples > MDL No. Samples > MDL Points: Samples (%) Samples (%) Samples (%) Detected Detected Detected DMP 17 17 (100) 12 12 (100) 150 147 (96) DEP 17 15 (88) 12 11 (92) 154 150 (95) DiBP 17 17 (100) 12 8 (67) 152 146 (89) DBP 17 17 (100) 12 7 (58) 154 150 (88) BBP 17 17 (100) 12 11 (92) 150 149 (95) DEHP 17 17 (100) 12 4 (33) 140 137 (90) DnOP 17 17 (100) 12 5 (42) 109 101 (88) DNP 17 17 (100) 12 4 (33) 84 78 (87) C6 13 11 (85) 12 5 (42) 81 80 (99) C7 13 12 (92) 12 5 (42) 82 76 (93) C8 13 12 (92) 12 2 (17) 128 120 (94) C9 13 12 (92) 12 3 (25) 76 68 (89) C10 13 12 (92) 12 2 (17) 56/812 51 (91) 1Certain biota data were excluded to low recoveries, interferences, and background contamination for DEHP, DnOP, DnNP, C6, C7, C8 and C9; 2An interference co-eluted with C10 in the biota samples for LC-ESI/MS analysis, MS-MS confirmation using a Li+ adduct was conducted on 81 biota samples.

44 2.4. Measurements of Organic Carbon & Lipid Contents in Sediment and Biota Samples

2.4.1. Organic Carbon Content Analysis

2.4.1.1. Sediment Samples Organic carbon content analysis in the marine samples was conducted at IOS and the methodology follow Van Iperen and Helder (1985). Approximately 500mg of dried sediment was acidified in a clean crucible with 1N HCl to remove carbonates prior to

Carbon/Nitrogen analysis. The acidified sample was then dried in an oven at 70ºC for 2 hours followed by another 2 hours at 105ºC. The sample was then allowed to hydrate for

2.5 hours, prior to analysis. Subsamples of approximately 8–10mg were weighed into tin cups for Carbon/Nitrogen analysis on a Leemans 440 Elemental Analyser. Acetanilide standards, containing 71.09% Carbon and 10.36% Nitrogen, were included in the sediment batches and sample duplicates were analyzed (pooled standard deviation for sample duplicates was 0.23%, where n = 3 sample pairs). Organic carbon content was expressed on a dry weight basis as g OC/ g dry sediment.

2.4.1.2. Algae Green and brown algae samples were rinsed with double-milli-q (dmq) water to remove sand, shell fragments and other inorganic substances that might contribute to the total organic carbon content (TOC) measurement. Algae samples were dried overnight at

60°C to achieve a stable weight, homogenized, and then subsampled for TOC measurement.

A 2-3 mg sample was then analyzed in a Leeman's Elemental Analyzer, which was

45 calibrated with acetanilide. Organic carbon content was expressed on a dry weight basis as g

OC/ g wet algae. Moisture contents were determined in unwashed algae samples.

2.4.1.3. Plankton Plankton samples were homogenized and subsampled with a clean spoon. Large wood pieces, leaves, and non-planktonic material were removed, however, small pieces of algae were included. The cleaned sample was filtered with double-milli-q water on acid- washed, combusted 47mm - 0.8 μm nucleopore filters to remove salts. Samples were then oven dried at 60°C until their weight was stable. Material was then further cleaned by sieving it through a 1000μm mesh to remove more debris. The dried particulate material was then homogenized with a mortar and pestle, transferred to a clean vial and acidified with 4% HCl to remove inorganic carbon (i.e., CaC03). The homogenate was then transferred to a combusted 25 mm nuclepore filter, rinsed three times with dmq water to remove the acid, and oven dried at 60°C to a stable weight. A 2 - 3 mg sample was analyzed in Leeman's Elemental Analyzer, which was standardized with acetanilide. Organic carbon content was expressed on a wet weight basis as g OC/ g wet plankton.

2.4.1.4. Particulate Matter Suspended solids were collected on the 47-mm diameter; 0.45-μm pore size, glass fibre filters. The samples were fumed with concentrated HCl, to remove inorganic carbon, and analyzed on the Leeman’s Elemental Analyzer. Organic carbon content was expressed on a wet weight basis as g OC/ g wet particulate matter.

46 2.4.2. Lipid Content Determination Lipid contents in the biota samples were measured at the IOS laboratory. For each biota sample, 5g of wet tissue (muscle, or whole body) was weighed on an aluminum boat and then transferred to a glass mortar where it was homogenized with 100g anhydrous sodium sulfate. The homogenate was transferred to a 30cm x 30cm glass column, which was packed with glass wool at the tip and a Turbovap was placed under the column. To remove any remaining sample material, the aluminum weigh boat, mortar and pestle, funnel, and spatula were rinsed three times with 1:1 DCM/Hexane. The column was then eluted with 100mL 1:1 DCM/Hexane. The extract was then reduced to 1mL in the Turbovap and quantitatively transferred with 1:1 DCM/Hex to another pre-weighed aluminum weigh boat.

The weigh boat and solvent were dried for several hours at 40oC in a vented oven, and then cooled completely in a desiccator. The sample weight was determined, and lipid content was calculated on a wet weight basis.

2.5. Data Analysis and Normalizations

2.5.1. Analysis of Concentration Distributions All concentration data were tested for normality using Kolmogorov-Smirnov and

Shapiro-Wilk normality tests. The results of these tests are reported in Tables E.2.1 to

E.2.17 of Appendix E. In general, both the environmental concentrations and the blank concentrations for all media and species were log-normally distributed. Therefore, data were log transformed; geometric means and upper and lower standard deviations are reported.

47 2.5.2. Sediment Organic Carbon Normalization

Sediment concentrations were measured on a dry weight basis, Cdry (ng/g dry sediment). Organic carbon (OC) contents were measured for each individual sediment sample (Table G.1, Appendix G), and organic carbon normalized concentrations COC (ng/g organic carbon), were computed on a sample-specific basis, as:

COC = Cdry / φOC (2.5)

Where φOC is the fraction of organic carbon in the dry sediment material (g OC / g dry sediment). In False Creek, the average organic carbon content of the sediments was 2.80%

(± 0.31%, n=12).

2.5.3. Biota Lipid Normalizations Lipid contents were analyzed for each individual biota sample (Table G.3 of

Appendix G) and samples were lipid normalized on a sample specific basis. Lipid normalized concentrations for the biota, Clipid (ng/g lipid tissue), with the exception of the plankton and algae, were calculated as:

Clipid = Cwet / L (2.6)

Where Cwet (ng/g wet tissue) is the wet weight biota concentration and L is the lipid fraction of the sampled tissue (g lipid / g wet tissue). The mean lipid contents for the species collected from False Creek are reported in Table 2.11.

Plankton and algae samples were lipid and organic carbon normalized following:

Clipid = Cwet / [L + (0.35 * φOC)] (2.7)

Where φOC is the fraction of non-lipid organic carbon in the wet sample, (g OC / g wet sample); and 0.35 is a proportionality constant recommended by Seth and others (1999) to relate the sorbing properties of organic carbon to those of octanol. The rationale for

48 including the organic carbon contents in the normalization of the plankton and algae samples is that these organisms possess low lipid contents, and relatively high organic carbon contents (Table 2.11). Organic carbon serves as the organisms energy and carbon source, and due to its relatively high content, is likely to serve as an important site for chemical accumulation. The normalization of plankton and algae data is further discussed in the

Section 3.4 (Biota Discussion).

Table 2.11. Mean Lipid Contents (%, g lipid/ g wet tissue) and Organic Carbon Contents (% dry weight and % wet weight) (± Standard Deviation) in Biota Tissues that were Analyzed for Phthalate Esters. Species Tissue Type Mean Lipid Mean Organic Content (± Carbon Content Stdev) (%) (± Stdev) (%) Plankton Whole Organism 0.09% (± 0.02) 40% (± 9)dry; 0.6% (± 0.2) wet Green Algae Whole Organism 0.20% (± 0.10) 34% (± 3) dry; 6.1% (± 1.5) wet Brown Algae Whole Organism 0.08% (± 0.02) 36% (± 3) dry; 6.3% (± 5.3) wet Geoduck Clams Whole Organism 0.7 (± 0.2) NA Manila Clams Whole Organism 1.2 (± 0.2) NA Blue Mussels Whole Organism 1.3 (± 0.1) NA Pacific Oysters Whole Organism 2.1 (± 0.6) NA Dungeness Crab Hepatopancreas 8.0 (± 6.0) NA Purple Seastar Stomach Section 2.5 – 18 NA Minnows Whole Body 2.1 (± 1.0) NA Striped Seaperch Muscle 0.17 (± 0.09) NA Pile Perch Muscle 0.7 (± 0.9) NA Forage Fish Muscle 3.2 (± 1.3) NA Whitespotted Greenling Muscle 0.6 (± 0.4) NA Pacific Staghorn Sculpin Muscle 0.3 (± 0.1) NA English Sole Muscle 0.5 NA Spiny Dogfish Whole Embryos 6 – 28 NA Spiny Dogfish Liver 62 (± 10) NA Spiny Dogfish Muscle 8.3 (± 3.9) NA Surf Scoter Liver 2.2 (± 0.6) NA

2.5.4. Fugacity Calculations In addition to wet/dry weight and lipid/organic carbon weight concentrations, the data are expressed in terms of fugacities such that the phthalate ester levels in the various media and species can be compared on a common or “normalized” basis. Fugacity, f (Pa), is related to concentration, C (mol/m3), through the linear relationship: f = C / Z; where Z is the fugacity capacity of the medium (Pa m3 / mol) (see section 1.3). Fugacity capacities (Z) were determined as:

Water ZW = 1/H (2.8)

Sediment ZSED = KP * ρ * Zw ZSED = 0.35 * φOC * KOW * ρ * (1/H) (2.9)

Algae and Plankton ZGA/PK = (L * KOW * ρ * Zw) + (φOC * 0.35 * KOW * ρ * Zw) + (W * Zw) = (L * KOW * ρ * (1/H)) + (φOC * 0.35 * KOW * ρ * (1/H)) + (W * (1/H)) (2.10)

Benthic Invertebrates, Fish and Birds ZBIO = Kb * ρ * Zw = L * KOW * ρ * (1/H) (2.11)

Chemical fugacities in the various media were calculated following equations 2.12 to

2.15 (i.e., f = C/Z). In the equations, a proportionality constant of 0.35 was used to relate the sorptive capacity of organic carbon to that of octanol, (Seth et al. 1999, and Mackay 1991).

For the water, three chemical concentrations were determined based on the different chemical fractions in the water (Figure 2.14). Therefore, three chemical fugacities in the water phase are presented: “fW(tot)” (based on the total water concentration); “fW(C18)” (based

50 on the C18 water concentration) and “fW(FD)” (based on the freely dissolved water concentration).

Water fW = CW / (1/H) (2.12)

Sediment fsed = Csed / [0.35 * φOC * KOW * ρ * (1/H)] (2.13)

Algae and Plankton fGA/PK = CGA/PK / [L * KOW * (1/H)) + (φOC * 0.35 * KOW * (1/H)) + (W * (1/H))] (2.14)

Benthic Invertebrates, Fish and Bird Tissues fBIO = CBIO / [L * KOW * ρ * (1/H)] (2.15) where H = Henry’s Law constant (mol / Pa m3) (Table 1.3) Kp = particle – water partition coefficient Kb = biota – water partition coefficient ρ = density (kg/L) (sediment = 1.5 kg/L; biota = 1.0 kg/L) 3 Zw = fugacity capacity of water (Pa m / mol) (Table 1.3) φOC = fraction of organic carbon in sediment / organism L = fraction of lipid in tissue W = moisture content (water fraction) of organism KOW = octanol – water partition coefficient (Table 1.3)

2.5.5. Trophic Position Calculation Marine food webs tend to be complex and characterized by numerous linkages between species. To explore the movement of phthalate esters through the marine food web, it was necessary to determine the dietary interactions and quantify the relative trophic positions of the species collected for the study. A literature review was conducted on the species collected in this study, as well as other key species in the Georgia Basin/

Southwestern British Columbia food web (see Appendix B, Pauly and Christensen 1996,

Butler 1964, 1980, Cass et al. 1990, Dygert 1990, Forrester 1969, Hart 1973, Jamieson and

Francis 1986, Jones 1976, Ketchen 1996, Levy 1985, Miller 1967, Murie 1995, Nybakken

1997, Onate 1991, Pratasik 1993, Richards 1987, Ricketts et al. 1985, Robles 1987, Starr et

51 al. 1990, Taylor 1964, Vermeer and Ydenburg 1989). Table 2.12 summarizes the prey items of these species, and their relative dietary proportions. Trophic positions were calculated according to Equation 2.16, based on Vander Zanden and Rasmussen (1996), and are listed in Table 2.13. The general trophic linkages of the study species, and their trophic positions, were presented in Figure 2.3.

TPpredator = ( ∑ TPprey * pprey ) + 1 (2.16) = (TP1 * p1) + (TP2 * p2) + (TPi * pi) + 1

Where “TP” is the trophic position of the predator or prey, and “pi” is the proportion of prey item i in the diet of the predator. The dietary matrix used for the calculation of trophic position is presented in Tables C.2.1 and C.2.2 of Appendix C. Species at the base of the food chain were assigned trophic positions according to Vander Zanden and Rasmussen

(1996). Additionally, certain organisms were lumped together into trophic guilds for the purpose of the calculation.

52 Table 2.12. Latin Name, Common Name, Trophic Position, Prey Items and their

Dietary Proportions, and Predators of Key Resident Marine Species in the Georgia

Basin Ecosystem.

Species Species TP Prey Species and their Predators Comments/ Latin Name Common Dietary Proportions References Name Squalus Spiny 4.07 Herring 0.22 Seals Jones 1976 acanthias Dogfish Euphausiids 0.14 Sea Lions Plankton (Zooplankton) 0.10 Shrimp 0.08 Crabs 0.07 Hake 0.07 Flatfish 0.06 Eulachon/Smelt 0.06 Octupus 0.03 Other fish 0.12 Other Invertebrates 0.05 Hexo- White- 3.81 Polychaetes (Benthic Inverts) ~ 0.45 Waterbirds Hart 1973 grammos spotted Crustaceans – Crabs ~ 0.10 Large fish stelleri Greenling Shrimps & Euphausiids ~ 0.19 Pelagic Invertebrates ~ 0.10 Small Forage Fish ~ 0.10 Pacific Herring ~ 0.05 Flatfish ~ 0.01 Parophrys English 3.74 Polychaetes (Benthic Inverts) 0.45 Bottom fish - benthic feeder vetulus Sole Brittle Stars (& Seastars) 0.20 Waterbirds - feeding stops Clams 0.20 during winter Sandlance (Sm. Forage Fish) 0.02 Clam siphons (Benthic Inverts) 0.02 Forrester 1969 Shrimp (& Euphausiids) 0.02 Amphipods (Benthic Inverts) 0.07

Small Crabs 0.02 Cancer Dungeness 3.55 Clams ~ 0.65 Octupus - carnivore magister Crab Other Bivalves ~ 0.10 Dogfish/shark Shrimp ~ 0.10 Halibut Pauly and Crustaceans (Pelagic Inverts) ~ 0.05 Sculpins Christensen Polychaetes (Benthic Inverts) ~ 0.05 Flounders 1996 Fish (Sm. Forage Fish) ~ 0.05 Rockfish Waterbirds Seals Platichthys Starry 3.54 Nemertean & Priapulid worms 0.42 Water birds - benthic feeder stellatus Flounder (Benthic Inverts) Bottom fish - January to Polychaetes (Benthic Inverts) 0.28 June feeding Clams 0.23 stops Small crabs 0.04 Miller 1967

53 Species Species TP Prey Species and their Predators Comments/ Latin Name Common Dietary Proportions References Name …Starry Brittle stars (& Seastars) 0.01 Flounder Amphipods (Benthic Inverts) 0.01 Mysids (Pelagic Inverts) 0.01 Leptocottus Pacific 3.51 Amphipods (Benthic Inverts) ~ 0.28 Waterbirds Pauly & armatus Staghorn Nereid worms (Benthic Inverts) ~ 0.17 Large fish Christensen Sculpin Anchovies/Small Forage Fish ~ 0.20 1996; Lingcod Larvae & Eggs ~ 0.05 Wang 1986 Pelagic Invertebrates ~ 0.25 Melanitta Surf Scoter 3.49 Mussels 0.88 Vermeer & perspi- Other Bivalves 0.10 Ydenberg 1989 cillata Seastars 0.02 Pisaster Purple 3.47 Mussels ~ 0.80 Waterbirds - voracious ochraceus seastar Clams (& Oysters) ~ 0.16 Seals predator Snails ~ 0.02 Sea lions Chitons ~ 0.01 Ricketts et al. Barnacles ~ 0.01 1985 Limpets < 0.01 Sea anemones < 0.01 Cancer Juvenile 3.37 Crustaceans ~ 0.40 Fish Pauly and magister Dungeness Worms ~ 0.40 Starfish Christensen Crabs Shrimps ~ 0.10 1996 (Small Mollusks ~ 0.05 Crabs) Minnows / Larval Fish ~ 0.05 Clupea Pacific 3.32 Euphausiids (& Shrimps) ~ 0.10 Gulls - may filter harengus Herring Amphipods (Benthic Inverts) ~ 0.10 Diving ducks feed when pallasi Copepods (Zooplankton) ~ 0.25 Salmon other food is Cladocerans (Zooplankton) ~ 0.25 Dogfish not available Decapods (Zooplankton) ~ 0.18 Sharks - ceases feeding Barnacles (Pelagic Inverts) Lingcod in winter prior Polychaetes (Benthic Inverts) Seals to spawning

Clam larvae (Pelagic Inverts) Sea lions Hart 1973 Shrimps (& Euphausiids) Whales Crabs (small) ~ 0.03 Eulochons (Sm. Forage Fish) ~ 0.07 Starry Flounder (Flatfish) ~ 0.02 Ronquil (Lg. Fish) < 0.01 Sandlance (Sm. Forage Fish) Hake (Lg. Fish) Sculpins (Lg. Fish) Rockfish (Lg. Fish) Hypomesus Surf Smelt 3.18 Copepods (Zooplankton) ~ 0.70 Sculpins Pauly and pretiosus Amphipods (Benthic Inverts) ~ 0.10 Starry Flounder Christensen pretiosus Euphausiids (& Shrimps) ~ 0.10 Surfperch 1996 Comb jellies (Pelagic Inverts) ~ 0.05 Large fish Larval fish (& Minnows) ~ 0.03 Waterbirds

54 Species Species TP Prey Species and their Predators Comments/ Latin Name Common Dietary Proportions References Name Pandalus Pink Shrimp 3.16 Mysids (Pelagic Inverts) ~ 0.60 Dogfish Butler 1980 borealis Amphipods (Benthic Inverts) ~ 0.20 Cod Other Crustaceans (Pelagic ~ 0.10 Turbot Polychaetes (Benthic Inverts) ~ 0.10 Hake Embiotoca Striped 3.05 Amphipods (Benthic Inverts) ~ 0.10 Larger fish Hart 1973 lateralis Seaperch Shrimps (& Euphausiids) ~ 0.10 Algae ~ 0.15 Worms (Benthic Inverts) Mussels ~ 0.05 Herring eggs (Larval Fish) ~ 0.02 Pelagic Invertebrates ~ 0.58 Panope Geoduck 2.53 Phytoplankton ~ 0.60 Sea stars - suspension abrupta Clams Zooplankton ~ 0.15 Crabs feeder Detritus ~ 0.25 Fish, Birds Pauly ...1996 Mytilus Blue 2.48 Phytoplankton ~ 0.60 Diving ducks - filter-feeder edulis Mussels Zooplankton ~ 0.25 Sea stars Bacteria Crabs Jamieson & Detritus ~ 0.15 Snails Urchins Francis 1986 Crassostrea Pacific 2.48 Diatoms (Phytoplankton) ~ 0.60 Sea stars - suspension gigas Oyster Detritus ~ 0.15 Oyster drills feeder Zooplankton ~ 0.25 Ctenophores Pauly…1996 Tapes Manila 2.40 Phytoplankton ~ 0.70 Water birds - suspension philippin- Clams Zooplankton ~ 0.10 feeder arum Detritus ~ 0.20 Pauly…1996 Cymato- Shiner 2.33 Small crustaceans (Pelagic ~ 0.25 Fish Wang 1986; gaster Surfperch Copepods (Zooplankton) Pauly & aggregeta (Minnows) Amphipods (Benthic Inverts) ~ 0.05 Christensen Algae ~ 0.10 1996 Phytoplankton ~ 0.60

55 Table 2.13. Summary of Trophic Positions for Species Collected from False Creek Species Trophic Position Spiny Dogfish 4.07 Whitespotted Greenling 3.81 English Sole 3.74 Starry Flounder 3.54 “Sole” (Starry Flounder + English Sole) 3.64 Dungeness Crab 3.55 Staghorn Sculpin 3.51 Surf Scoter 3.49 Starfish 3.47 Pacific Herring 3.32 Surf Smelt 3.18 “Forage Fish” (Surf Smelt + Pacific Herring) 3.25 Striped Seaperch 3.05 Pile Perch 3.05 Geoduck Clams 2.53 Blue Mussels 2.48 Pacific Oyster 2.48 Manila Clams 2.40 Minnows 2.33 “Plankton” (Phytoplankton + Zooplankton) 1.00 Algae 1.00

56 3. RESULTS & DISCUSSION

Overview: The results section is divided into six parts describing (i) the concentrations

of phthalate esters and corresponding fugacities in the sediment (Section 3.1), (ii) seawater (Section 3.2), and (iii) marine species (Section 3.4), and (iv) the distribution of

phthalate esters between the sediment and seawater (Section 3.3), (v) the biota and

seawater (i.e., bioaccumulation factors) (Section 3.5), and (vi) the biota and sediment

(i.e., biota-sediment accumulation factors) (Section 3.6). Data tables of mean phthalate

ester concentrations and fugacities in the various media, bioaccumulation and biota-

sediment accumulation factors, and summaries of reported phthalate ester concentrations

in various locations throughout the world are presented in Appendix F. The original

concentration data, including recoveries and supporting measurements (i.e., lipid and/or

organic carbon contents), are presented for each sample in Appendix G.

3.1. Sediment Concentrations of Phthalate Esters

3.1.1. Concentration Summary All phthalate ester congeners and isomeric mixtures were detected in the ppb to

ppm range in False Creek Harbour sediments. The observed phthalate ester

concentrations in the sediments are presented in Table F.3.1 (Appendix F), and Figures

3.0 and 3.1. The results are expressed in terms of dry weight concentrations, Cdry (ng/g

dry sediment), organic carbon normalized concentrations, COC (ng/g organic carbon)

(Equation 2.5), and corresponding fugacities (Pa) (Equation 2.13).

57 Average sediment concentrations of the individual phthalates ranged from 4.0 ng/g dry wt. for DiBP to 2,090 ng/g dry wt. for DEHP. For the isomeric mixtures, average concentrations ranged from 6.7 to 2,100 ng/g dry sediment for C6 and C8, respectively (Table F.3.1 and Figure 3.0). DEHP and C8 isomers (including DEHP) were present in the highest concentrations (2,100 ng/g dry weight). Sediments also contained significant levels of DBP (114 ng/g dry), C9 (483 ng/g dry) and C10 isomers (385 ng/g dry). In terms of total phthalate esters, determined as the sum of concentrations of DMP,

DEP, DiBP, DBP, BBP, C6, C7, DEHP, DnOP, C9 and C10, levels in the sediments were approximately 3,270 ng/g dry wt. In False Creek, the average organic carbon content of the sediments was 2.80% (± 0.31%, n=12). Organic carbon normalized concentrations ranged from 137 to 75,200 ng/g OC for DiBP and C8, respectively (Table F.3.1, Figure

3.0).

Fugacities in the sediments ranged from 0.10 nPa for DnNP to 3,120 nPa for

DMP, and from 0.07 to 15.5 nPa for C10 and C8 isomers, respectively (Table F.3.1,

Figure 3.1). Phthalate fugacities in the sediments were relatively low for the high molecular weight compounds, and higher for the low molecular weight PEs (Figure 3.1).

Figure 3.1B compares the concentration and fugacity profiles for phthalate esters in False

Creek Harbour sediments. It reveals that although the low molecular weight PEs (e.g.,

DMP, DEP, and DBP) are present at relatively low concentrations, they present the highest fugacities in the sediments. This is in contrast to the high molecular weight PEs

(e.g., C8, C9, and C10), which are present at the highest concentrations but correspond to the lowest fugacities in the sediment matrix.

58 10000 1000000 A) B)

100000 1000

10000

100

1000 Concentration (ng/g OC) (ng/g Concentration

Concentration (ng/g dry wt.) dry (ng/g Concentration 10 100

1 10

P P 0 B C6 C7 C8 C9 C6 C7 C8 C9 DEP DBP BBP DOP DNP C10 DMP DE DBP BBP DOP DNP C1 DMP DI DIBP DEHP DEHP Phthalate Ester Phthalate Ester Figure 3.1. Phthalate Ester Concentrations in False Creek Harbour Sediments, Expressed on a Dry Weight Basis (ng/g dry sediment) (A), and on an Organic Carbon Normalized Basis (ng/g organic carbon) (B).

C) D) Organic Carbon Fugacity 10000 1000000 10000

1000 100000 1000

100 10000 100 Fugacity (nPa)

10 1000 10

Fugacity (nPa) Fugacity 1 100 1 Concentration (ng/g OC) (ng/g Concentration 0.1 10 0.1

0.01 1 0.01 P P P 7 8 IBP H C6 C C C9 10 P DMP DEP D DB BBP E DO DNP C M IBP BP C6 C7 C8 C9 D D DEP DBP B DOP DNP C10 D DEHP Phthalate Ester Phthalate Ester Figure 3.1. Phthalate Ester Fugacities (nPa) in False Creek Harbour Sediments (C), and Comparison of Phthalate Ester Concentration (ng/n OC) and Fugacity (nPa) Profiles in False Creek Harbour Sediments (D).

3.1.2. Spatial Variability An Analysis of Variance (ANOVA) was used to determine whether there were statistically significant differences in the concentrations of phthalate esters between the four sediment stations within False Creek. The results indicate that the “North Central” sampling station had statistically significantly lower levels of certain phthalate esters, particularly the larger molecular weight PEs (i.e., BBP, DEHP, DnOP, C9, and C10), relative to the other stations, particularly “East Basin” (Figure 3.2, Appendix E). This difference in concentration is likely due to greater tidal flushing near the mouth of the embayment (North

Central), or reduced flushing in the more inland and protected sections of the harbour (e.g.,

East Basin). These high molecular weight substances have relatively long half-lives in sediments, and are likely to persist with reduced mechanisms of removal. Also, there may be additional sources of these high molecular weight phthalates in the eastern section of

False Creek, from, for example, municipal and industrial outflows.

Overall, however, the sediment concentrations within False Creek were sufficiently homogenous to support combining all the data. Specifically, there were no statistically significant differences between the sediment sampling stations for eight of the thirteen phthalate esters, and for the substances that did exhibit statistically significant spatial differences, concentrations between the low and high stations differed by only a factor of 2 to 3. Additionally, pooling the sediment data enables an assessment of the overall chemical distribution in the environment and movement through the food web.

Figure 3.2. Concentrations (ng/g OC) of Phthalate Esters in Four Sediment Stations in False Creek Harbour. Starred bars indicate statistically significant differences in concentration between 1 station and the other 3 (single star), or between 2 stations (double star).

3.2. Seawater Concentrations of Phthalate Esters

3.2.1. “Total” Seawater Concentration Summary Total water concentrations of phthalates in seawater were determined as the sum of the PE concentrations on the glass fibre filter and the C18 disks. These concentrations and their standard deviations are presented in Table F.3.3 (Appendix F) and Figure 3.3 and ranged from 3.5 ng/L for DMP and BBP to 275 ng/L for DEHP and C8 isomers. The concentrations were determined as the geometric mean of the observations exceeding the

MDL. While other methods exist to account for observations below the MDL, this method was selected because the main cause of samples not exceeding the MDL was background contamination, rather than variability in phthalate ester concentrations between the replicate water samples. A minimum of 4 observations above the MDL was considered to constitute a large enough sample size to determine the average water concentration. Hence, the water concentrations of C8 and C9 are only considered estimates. The total phthalate ester water concentration, determined as the sum of concentrations of DMP, DEP, DiBP, DBP, BBP,

C6, C7, DEHP, DnOP, C9, and C10 was 735 ng/L. Significant losses due to biodegradation have previously been reported to occur within a period of 3 to 17 days at 20ºC (Schouten et al. 1979, Walker et al. 1984, and Russell et al. 1985) but were reported to be negligible at

4ºC (Ritsema et al. 1989). Biodegradation losses were not expected to be a factor in this study because of the short pre-extraction period at 4ºC.

The concentration of DEHP in False Creek seawater (275 ng/L) was lower than the

USEPA adopted Maximum Acceptable Concentration of 6000 ng/L (USEPA 1991), and the

Canadian interim water quality guideline for freshwater of 16,000 ng/L (CCME 1999). The

63 concentration of DBP in False Creek seawater (110 ng/L) was also less than the Canadian interim water quality guideline for freshwater of 19,000 ng/L (CCME 1999).

1000 (2) (4) (11) (7) (4) (5) (3) (9) 100

(5) (5)

(8) 10 (12) (11) Total Concentration (ng/L) Concentration Total

P P BP HP OP N C6 C7 C8 C9 DMP DEP DB B C10 DiBP DE Dn Dn Phthalate Ester Figure 3.3. Total Concentrations (Mean ± Standard Deviations, ng/L) of Phthalate Esters in Seawater Samples from False Creek Harbour. (Number of samples for which water concentration exceeded the MDL, in brackets).

3.2.2. Spatial Variability For those congeners with sufficient data above the MDL, there were no statistically significant differences (ANOVA, p>0.05) between the geometric means of the water concentrations from the four sampling stations, indicating that the distribution of phthalate esters in the water was relatively homogeneous throughout the tested inlet.

64 3.2.3. Ratio of Seawater Concentrations to Aqueous Solubilities A comparison of the observed total seawater concentrations to the aqueous solubilities of phthalates (as reviewed by Cousins and Mackay 2000) indicates that while

DMP concentrations in seawater were only a minute fraction of DMPs solubility in water, the ratio of the water concentration (Cw) and the solubility (Sw), i.e. Cw/Sw, appears to increase with increasing log Kow to a maximum value of 60% for C10 (Figure 3.4). This suggests that the C10 concentration in seawater is approaching the maximum amount of C10 that can actually be dissolved in water. The notion of C10 phthalate esters approaching their

“saturation level” in water is highly unlikely but indicates that small amounts of suspended matter in the water column may play an overwhelming role in controlling the total water concentration. Thus, a second series of data is presented in Figure 3.4, showing ratios based on model-fitted freely dissolved water concentrations (see section 1.1.2.2), which illustrates that the ratio between the freely dissolved water concentration and the aqueous solubility for the high KOW phthalates is approximately 0.001%.

65 Total Freely Dissolved 1

0.01

0.0001

1E-06

Concentration / Solubility 1E-08

1E-10 0246810 Log Kow (Seawater)

Figure 3.4. Ratio of the Seawater Concentrations (CW, ng/L) to the Aqueous Solubilities

(SW, ng/L) of Phthalate Esters, for the Total Seawater Concentration and the Freely Dissolved Seawater Concentration, as a Function of the Octanol - Seawater Partition Coefficient.

3.2.4. Distribution of Phthalate Ester Internal Standards between the

Glass Fibre Filter and C18 Extraction Disks Table 3.5 and Figure 3.5 illustrate the distribution of the spiked internal standards between the glass fiber filter (representing particle-sorbed phthalates) and the C18 extraction disks (representing dissolved phthalates). It shows that the fraction of chemical on the C18 extraction disks falls with increasing Kow from 99% for DMP to 14% for DOP. This relationship is in general agreement with the two-phase sorption model for organic chemicals to suspended particulate matter, where the freely dissolved fraction (FDW, unitless) of the total water concentration can be expressed as:

66 FDW = 1/[1 + (α · KOW)] (3.3)

Where α is the product of the concentration of suspended matter (ϕSM, kg/L), the organic carbon content of the suspended matter (ϕOC, kg/kg), and a constant (ω, L/kg), which represents (i) the ratio between the organic carbon-water partition coefficient of suspended matter (KOC) and the octanol-water partition coefficient (KOW) (i.e., Koc/Kow), and (ii) the degree of chemical disequilibrium between the suspended organic matter and the seawater

(i.e., Observed KOC / Predicted KOC), i.e., α = ϕSM · ϕOC · ω. Fitting equation 3.3 to the observed data results in a value of 4.8 · 10-6 for α. The suspended matter concentration in the seawater at our test site, as determined by the mass of suspended matter measured on the glass fiber filters after filtration, was 1.47 (± 1.05) mg/L (n=12), and the organic carbon content was 40%, indicating that ω was approximately 8.1 (L/kg).

Table 3.5. Mean Fractions of Internal Standards on the Glass Fibre Filter (GF) and C18

Extraction Disks (C18) in Well Water Blanks and False Creek Seawater Samples (%).

Internal Log KOW Well Water Blanks False Creek Seawater Standard (Seawater) GF C18 GF C18 DMP-d4 1.80 0.14 (± 0.18) 99.86 (± 0.18) 0.05 (± 0.05) 99.95 (± 0.05) DBP-d4 4.58 13 (± 13) 87 (± 13) 15 (± 14) 85 (± 14) DnOP-d4 8.20 85 (± 13) 15 (± 13) 94 (± 4) 6 (± 4)

67 C18 FDW Model C18 FDW Model GF PB Model 1.2 1.2

1 1

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2 Fraction of Phthalate Ester Disk on C18

0 on GF C18 Ester / Phthalate of Fraction 0 024681012 024681012 Log Kow (Seawater) Log Kow (Seawater) Figure 3.5. Mean observed fractions (± standard deviation) of spiked phthalate ester internal standards on the Glass Fibre Filter and C18 Extraction Disks in False Creek Harbour seawater samples, and the model-fitted Freely Dissolved (FDW Model) and Particulate- Bound (PB Model) Fractions, determined from Equation 3.3.

3.2.5. Distribution of Seawater Borne Phthalate Esters between the

Glass Fibre Filter and C18 Extraction Disks Table 3.6 and Figure 3.6 illustrate the distribution of the seawater borne phthalates between the glass fiber filter and the C18 extraction disks. They show that the fraction of phthalates on the C18 extraction disks drops from 89 ± 4% (n=12) for DMP and 89 ± 10%

(n=12) for DEP to approximately 40% for DEHP and the other high KOW phthalate esters.

This relationship between the freely dissolved water fraction (FDW) and KOW, observed for the seawater borne phthalates, is not consistent at high KOW with the inverse relationship between FDW and Kow observed for the internal standards and expected from the two- phase sorption model in equation 3.3. The main reason for this discrepancy is that the C18 extraction disks do not only capture freely dissolved phthalates but also phthalates sorbed to

68 small diameter (i.e. less than 0.45 μm) particulate matter. This small diameter suspended matter (SDSM) was visible after extraction and was present at a concentration of 0.66 (±

0.28) mg/L (n=7). Phthalates captured on the C18 extraction disks, therefore, represent a combination of phthalates in both the freely dissolved form and SDSM-sorbed form. A three-phase sorption model describes this fraction (FC18) as:

FC18 = 1 + βSDSM·Kow / 1 + βSDSM·Kow + βLDSM·Kow (3.4)

Where βSDSM is the product of the concentration of small diameter suspended matter φSDSM

(kg/L), the organic carbon content φSDOC (kg/kg), and a constant ωSDSM (L/kg), which represents the ratio of KOC to KOW, and the degree of chemical disequilibrium between the small diameter suspended matter and the seawater, i.e., βSDSM = φSDSM · φSDOC · ωSDSM, and

βLDSM is the product of the concentration of large diameter suspended matter φLDSM (kg/L), the organic carbon content φLDOC (kg/kg), and a constant ωLDSM (L/kg) representing

KOC/KOW and Observed KOC / Predicted KOC, i.e., βLDSM = φLDSM · φLDOC · ωLDSM. Fitting this model to the data by minimizing the sum of squared deviations between observed and

-5 -5 predicted values resulted in a βLDSM of 2.0·10 and a βSDSM of 1.7·10 , illustrating that, of suspended-matter-bound phthalates, 55% was associated with large particles (>0.45 μm), and 45% with small diameter suspended matter. These fractions are in approximate agreement with the 69:31 ratio of the concentrations of LDSM, i.e. 1.47 (± 1.05) mg/L

(n=12), and SDSM, i.e. 0.66 (± 0.28) mg/L (n=7) suggesting that LDSM and SDSM exhibit comparable sorption capacities. The latter suggests that, as long as organic matter contents are equivalent, large and small diameter organic matter have a similar sorption affinity for phthalate esters. This is in contrast with many other findings for organic chemicals that suggest that “dissolved” organic matter exhibits only 10% of the sorption capacity of

69 particulate organic matter. The application of chemical spiking as a method for measuring sorption capacity may explain some of the discrepancy between the sorption capacity measured in this study to those in other studies. For example, fitting the measured C18-bound fractions of the internal standards (which were applied by spiking) to equation 3.4, results in

-6 -7 a βLDSM of 4.9·10 and a βSDSM of 2.7·10 , suggesting that SDSM has only 5% of the sorptive capacity of LDSM. The difference in sorption between the spiked internal standards and the seawater-borne phthalates is likely due to the difference in chemical sorption time, i.e., 1 hr for the internal standards and much longer periods for the seawater borne phthalates. Comparing the values for βLDSM and βSDSM between the spiked and water-borne phthalates suggests that, after 1 hr, LDSM has reached only 29% of its sorption potential, while SDSM has reached only 1.6% of its sorption potential. The slower sorption kinetics onto SDSM compared to LDSM may explain the apparent lower sorption capacity of SDSM in spiking studies.

Because of the inability of the C18 extraction disks to distinguish between freely dissolved and SDSM-sorbed phthalates, freely dissolved seawater concentrations can only be estimated by fitting the observed fractions of phthalate esters in the seawater to the 3- phase sorption model:

FDW = 1 /( 1 + βSDSM·Kow + βLDSM·Kow) (3.5)

-5 -5 Where βLDSM is 2.0·10 and βSDSM is 1.7·10 , resulting in estimates of the freely dissolved fraction (FDW) ranging from virtually 100% for DMP to 8·10-5% for C10 (Table 3.7). A breakdown of the composition of phthalate ester concentrations in seawater of False Creek is illustrated in Figure 3.7 and suggests that, for example, of the total DEHP aqueous concentration of 275 ng/L only 0.02% or 0.05 ng/L may be in the freely dissolved form.

70 Following the widely accepted hypothesis that only the freely dissolved chemical can be absorbed via the respiratory surface of most aquatic organisms (Black and McCarthy 1988,

Landrum et al. 1985, McCarthy and Jimenez 1985, Gobas and Zhang 1994, Gobas and

Russell 1991), implies that, for the high molecular weight phthalates, the actual water concentrations to which aquatic organisms are exposed via their respiratory surfaces, are much lower than the observed total water concentrations.

Table 3.6. Mean Observed Fractions (%) (± Standard Deviations) of Seawater Borne

Phthalate Esters on the Glass Fibre Filter (GF) and C18 Extraction Disks (C18) in Well Water Blanks and False Creek Seawater Samples.

Phthalate Log KOW Well Water Blanks False Creek Seawater Ester (Seawater) GF C18 GF C18 DMP 1.80 17 (± 6) 83 (± 6) 11 (± 4) 89 (± 4) DEP 2.77 35 (± 10) 65 (± 10) 11 (± 10) 89 (± 10) DiBP 4.58 39 (± 8) 61 (± 8) 29 (± 6) 71 (± 6) DBP 4.58 36 (± 7) 64 (± 7) 32 (± 8) 68 (± 8) BBP 5.03 44 (± 7) 56 (± 7) 49 (± 10) 51 (± 10) DEHP 8.20 61 (± 20) 39 (± 20) 55 (± 10) 45 (± 10) DOP 8.20 61 (± 28) 39 (± 28) 57 (± 19) 43 (± 19) DNP 9.11 61 (± 29) 39 (± 29) 60 (± 17) 40 (± 17) C6 6.69 54 (± 16) 46 (± 16) 47 (± 29) 53 (± 29) C7 7.44 53 (± 16) 47 (± 16) 55 (± 15) 45 (± 15) C8 8.20 58 (± 15) 42 (± 15) 53 (± 11) 47 (± 11) C9 9.11 53 (± 27) 47 (± 27) 53 (± 16) 47 (± 16) C10 10.6 57 (± 28) 43 (± 28) 66 (± 18) 33 (± 18)

71 Table 3.7. Mean Fractions (%) of Phthalate Esters Bound to Large and Small Diameter Suspended Matter (LDSM, SDSM) and Freely Dissolved in False Creek Harbour Seawater, Determined from the 3-Phase Sorption Model (Eqn 3.5).

Phthalate Log KOW LDSM-Bound SDSM-Bound Freely Dissolved Ester (Seawater) Fraction (%) Fraction (%) Fraction (%) DMP 1.80 0.13 0.10 99.8 DEP 2.77 1.15 0.96 97.9 DiBP 4.58 31.8 26.4 41.8 DBP 4.58 31.8 26.4 41.8 BBP 5.03 43.6 36.1 20.3 C6 6.69 54.4 45.1 0.554 C7 7.44 54.6 45.3 0.098 DEHP 8.20 54.7 45.3 0.017 DnOP 8.20 54.7 45.3 0.017 C8 8.20 54.7 45.3 0.017 DnNP 9.11 54.7 45.3 0.002 C9 9.11 54.7 45.3 0.002 C10 10.6 54.7 45.3 8·10-5

72 C18 FDW (2-phase model) C18 (3-phase model) FDW (3-phase model) 1.2

0.8

0.6 Fraction Fraction

0.4

0.2

0 024681012 Log Kow (Seawater) Figure 3.6. Mean observed fractions (± standard deviations) of seawater-borne phthalate esters on the C18 Extraction Disks in seawater samples from False Creek Harbour, the 2-phase model-fitted Freely Dissolved Fraction (Eqn. 3.3) and the 3-phase model-fitted C18 Fraction (SDSM-bound + FDW) (Eqn. 3.4) and Freely Dissolved Fraction (Eqn. 3.5).

Freely Dissolved SDSM Bound LDSM Bound Freely Dissolved SDSM Bound LDSM Bound 1 1

0.9 0.1 0.8

0.7 0.01

0.6 0.001 0.5 0.0001 Fraction Fraction 0.4

0.3 0.00001 0.2 0.000001 0.1

0 0.0000001

P 6 P P P P 7 8 9 C C7 C8 C9 B C6 C HP C C MP C10 DMP DE DB B E C10 D DE DiBP DBP BBP DiB D DnOP DnNP DEHPDnOP DnNP Phthalate Ester Phthalate Ester

Figure 3.7. Fraction of Phthalate Esters Bound to Large Diameter Suspended Matter (LDSM) (), Bound to Small Diameter Suspended Matter (), and Freely Dissolved () in False Creek Harbour Seawater, Determined from the 3-Phase Sorption Model (Eqn. 3.5). The y-axis on the right panel is expressed on a logarithmic scale.

3.2.6. Summary of the “Total”, “C18”, and “Freely Dissolved” Water Concentrations Three water concentrations were determined for phthalate esters in False Creek

Harbour seawater (Table F.3.8 in Appendix F, Figure 3.8). The “total” water concentration includes all forms of chemical in the water phase, i.e., bound to large and small diameter suspended matter, and freely dissolved. Total water concentrations ranged from 3.51 to 275 ng/L for DMP, and DEHP respectively. The “C18” water concentration includes chemical associated with small diameter suspended matter, and freely dissolved chemical. Mean C18 concentrations ranged from 3.14 ng/L for DMP to 124 ng/L for DEHP and 130ng/L for C8 isomers. “Freely dissolved” water concentrations ranged from 5.9·10-5 ng/L for C10 isomers to 123 ng/L for DEP.

Since the chemical substance must be in the freely dissolved form in order for it to be taken up by organisms through their respiratory membranes, the freely dissolved water concentration represents the actual phthalate ester levels in the water to which organisms are effectively exposed. Filtering the water and determining the chemical concentration on the

C18 disks was conducted in an attempt to experimentally measure the freely dissolved fraction of the substance in the water. However, the C18 disks captured both the freely dissolved substance and chemical associated with small diameter suspended matter in the water column. Measurements of suspended matter revealed significant amounts of small diameter suspended material in the water samples. Model calculations indicate that this

SDSM played a major role in controlling the distribution of the chemicals in the water by serving as sorptive material for the high KOW substances, in particular. The total water concentration gives an indication of the overall mass of the chemical substance in both the

75 water and study system in general. Table 3.8 reveals that, although some of the high KOW substances were present at relatively high concentration in the False Creek Harbour seawater

(e.g. DEHP, DnNP, C8, C9 and C10), the actual bioavailability of these substances, indicated by the freely dissolved water concentration, was extremely low.

Total (LDSM+SDSM+FD) C18 (SDSM+FD) Freely Dissolved (FD) 1000

100

0.1

0.01

Seawater Concentration (ng/L) 0.001

0.0001

0.00001

P P P P 6 8 9 0 B B B HP O C C7 C C 1 DEP i D B E C DMP D D Dn DnNP Phthalate Ester Figure 3.8. Mean Phthalate Ester Concentrations (± Standard Deviations, ng/L) in False Creek Harbour Seawater. “Total” concentrations include chemical bound to large and small diameter suspended matter (LDSM, SDSM) and freely dissolved chemical. “C18” concentrations include SDSM-bound and freely dissolved chemical. The third bar represents model estimates of the “Freely Dissolved” chemical concentration.

76 3.2.7. Chemical Fugacities in the Water Using the three concentrations of phthalate esters in False Creek Harbour seawater

(i.e., total, C18, and freely dissolved) (Figure 3.8), three fugacities were determined following Equation 2.12, i.e., fW = CW / (1/H) (Table F.3.9 in Appendix F, Figure 3.9).

“Total” and ‘C18” fugacities of phthalates in the water ranged from 0.16 nPa for DMP to

4,630 nPa for C9 isomers. Fugacities based on the “freely dissolved” water fraction ranged between 0.0034 for C10, and 220 nPa for DBP. As explained with the three water concentrations, the “freely dissolved” fugacity is believed to be the best measure of the actual chemical fugacity in the water to which the organisms are exposed, since only the freely dissolved chemical is bioavailable to the organisms for uptake via their respiratory surfaces. The difference between the three types of water is most significant for the high molecular weight phthalates (e.g., phthalates with ≥ 6 carbon chains), whose effective or freely dissolved fugacities in the water are relatively low (Figure 3.9).

77 Total (LDSM+SDSM+FD) C18 (SDSM+FD) Freely Dissolved (FD) 1E-5

1E-6

1E-7

1E-8

1E-9 Fugacity (Pa) Fugacity 1E-10

1E-11

1E-12

P P P P M EP iB B C6 C7 C8 C9 10 D D DB B C D DEHP DnOP DnNP Phthalate Ester

Figure 3.9. Mean “Total”, “C18”, and “Freely Dissolved” Fugacities (± Standard Deviations, Pa) in False Creek Harbour Seawater. “Total” fugacities include chemical bound to large and small diameter suspended matter (LDSM, SDSM) and freely dissolved chemical. “C18” fugacities include SDSM-bound and freely dissolved chemical. The third bar represents estimates of the fugacity based on “Freely Dissolved” concentrations.

3.3. Sediment - Water Distribution of Phthalate Esters

Figure 3.10 illustrates organic carbon normalized sediment-water distribution coefficients (KOC, L/kg OC) for phthalate esters as a function of KOW. The organic carbon content of the False Creek bottom sediments was 2.8 (± 0.31)% (n=12). Linear regression between log KOC (L/kg OC) (determined as the ratio of the sediment (Table 3.1) and “total” water concentration (Table 3.8)), and log KOW resulted in:

78 2 Log KOC = 0.063 (± 0.060) · log KOW + 4.53 (± 0.45) r = 0.08, n = 12 (3.6)

Equation 3.6 illustrates a statistically insignificant relationship (p > 0.05) between sediment- water partition coefficients and KOW, where standard deviations are reported for the y- intercept and slope. The distribution of phthalates between sediment and water does not appear to follow a Karickhoff style relationship where KOC is a linear function of KOW (Seth et al. 1999). Relationships similar to equation 3.6 have been observed for PCBs and other organochlorines and are not expected to be specific to phthalate esters (MacLean 1999). One of the causes for the deviation between the observed distribution coefficients and the reported equilibrium partition coefficients is expressing the sediment-water distribution coefficients based on the “total”, rather than the “freely dissolved” water concentration, in particular for the higher molecular weight phthalates. If the sediment-water distribution coefficients (KOC, L/kg OC) are expressed based on the estimated “freely dissolved” concentrations, the relationship improves substantially (p = 3.0·10-6, ± standard deviations for y-intercept and slope), i.e.

2 Log KOC = 0.823 (± 0.097) · log Kow + 2.07 (± 0.69) r = 0.87, n = 12 (3.7)

Comparing the observed sediment-water distribution coefficients (based on “freely dissolved” water concentrations, i.e., equation 3.7), to the expected sediment-water equilibrium partition coefficients (Seth et al. 1999), i.e., equation 3.8,

Log KOC = 1.0 · log Kow + log (0.35) (3.8)

= 1.0 · log Kow - 0.456 reveals that phthalate esters are at a chemical disequilibrium in False Creek Harbour sediments and water (Figure 3.10). The sediment-water distribution coefficient (KOC) of

DMP was 17,700 fold greater than its equilibrium based sediment-water partition

79 coefficient. The observed degree of disequilibrium appeared to drop with increasing KOW to a factor of 30 for DEP and to values ranging between 2.7 and 44 for all other phthalate esters

(Table 3.7). The surprisingly high degree of disequilibrium for DMP is unlikely to be due to experimental error as extraction recoveries were high, pre-analysis losses are expected to be low due to the short pre-extraction period, and evaporative losses were avoided (and DMP has a low Henry Law constant of DMP, i.e., 9.78.10-3 Pa.m3.mol-1). Also, because of DMPs low KOW, the total water concentration reflects virtually entirely freely dissolved chemical, thus reducing potential error involved in the estimation of the freely dissolved fraction.

The importance of the observed sediment-water disequilibria is that it can affect the exposure pathways of the organisms in the food web. High chemical concentrations in the sediments relative to those in the water can elevate the transfer of phthalates from sediments into organisms directly, through the ventilation of sediment pore-water, or indirectly, through dietary transfer via the benthic food-chain. In other words, sediment-water disequilibria increase the importance of the bottom sediments as a route of exposure.

80 Table 3.9. Observed and Predicted Sediment-Water Partition Coefficients (OBS KOC and PRED KOC, L/kg OC) based on the Freely Dissolved Water Concentration, and the Ratio between the Observed and Predicted Partition Coefficients.

PE OBS KOC PRED KOC Ratio OBS/PRED DMP 3.87 ·10+5 2.19 ·10+1 17,700 DEP 5.95 ·10+3 2.05 ·10+2 28.9 DiBP 6.35 ·10+4 1.33 ·10+4 4.79 DBP 7.99 ·10+4 1.33 ·10+4 6.03 BBP 1.64 ·10+6 3.74 ·10+4 43.9 DEHP 1.58 ·10+9 5.53 ·10+7 28.6 DnOP 8.20 ·10+8 5.53 ·10+7 14.8 DnNP 1.22 ·10+9 4.49 ·10+8 2.72 C6 4.66 ·10+6 1.71 ·10+6 2.73 C7 1.13 ·10+8 9.72 ·10+6 11.6 C8 1.78 ·10+9 5.53 ·10+7 32.3 C9 9.69 ·10+9 4.49 ·10+8 21.6 C10 2.45 ·10+11 1.24 ·10+10 19.8

Obs Koc (TOT) Obs Koc (FD) Pred Koc 12

4 Log Koc (L/kg OC) LogKoc (L/kg

0 024681012 Log Kow

Figure 3.10. Observed Sediment-Water Partition Coefficients (Log KOC, L/kg OC), based on the Total Water Concentration “TOT”, and the Freely Dissolved Water Concentration “FD”, and the Predicted Sediment-Water Equilibrium Coefficient (L/kg OC), based on Seth et al. 1999.

81 3.4. Biota Concentrations of Phthalate Esters

3.4.1. Biota Concentration Overview Concentrations of phthalate esters in False Creek biota samples are reported in terms of (i) wet weight concentrations (Tables F.3.10 and F.3.11 in Appendix F), (ii) lipid normalized concentrations (Tables F.3.12 and F.3.13 in Appendix F), and (iii) fugacities

(Tables F.3.14 and F.3.15 in Appendix F).

Average biota concentrations of the individual phthalates ranged from < 0.1 ng/g wet wt. for DnOP in Whitespotted Greenling and DnNP in Forage Fish to 310 ng/g wet wt. for

DEHP in Green Algae. For the isomeric mixtures, average concentrations ranged from < 0.1 ng/g wet tissue for C6 in Striped Seaperch and Pile Perch to 200 ng/g wet wt. for C8 in

Spiny Dogfish liver samples. Significant levels of DEHP (up to 310 ng/g wet wt.), C8 isomers (up to 200 ng/g wet wt.), C10 isomers (up to 72 ng/g wet wt.), C9 isomers (up to 71 ng/g wet wt.), and DBP (up to 60 ng/g wet wt.) were detected in certain marine species

(Tables F.3.10 and F.3.11 in Appendix F). Mean lipid and organic carbon contents in the biota species were presented in Table 2.11 (Section 2.5.3), and the lipid normalized concentrations of phthalate esters in the species ranged from 2.2 ng/g lipid (DnOP in dogfish liver) to 28,700 ng/g lipid (C8 in plankton) (Table F.3.12 and F.3.13 in Appendix F).

Fugacities ranged from 9.1 · 10-5 nPa for DnOP in Spiny Dogfish liver samples to

2,950 nPa for DBP in Plankton. Fugacities of the isomers ranged from 9.3 · 10-6 nPa for C10 in Spiny Dogfish embryo samples to 5.1 nPa for C6 in Surf Scoter liver samples. Phthalate fugacities in the biota were relatively low for the high molecular weight phthalates (i.e.,

DEHP, DnOP, DnNP, C8, C9, and C10), and higher for the low and intermediate molecular weight phthalates, particularly DBP and DEP (Tables 3.14 and 3.15).

82 3.4.2. Spatial Variability Figure 3.11 illustrates the concentration of phthalate esters at three biota sampling stations within False Creek. An Analysis of Variance (ANOVA) was used to determine whether there were statistically significant differences in the concentrations of phthalate esters in the biota between the three stations. The results indicate that, as in the sediment matrix, some of the less mobile species (i.e., green algae, plankton, geoduck clams, and pacific oysters) in the East Basin sampling station had higher levels of certain phthalate esters, particularly the larger molecular weight PEs (i.e., BBP, DEHP, DnOP, DNP, C7, C8,

C9, C10), compared to the organisms in the North Central and Marina stations (Figure 3.11,

Appendix D). This difference in concentration is likely due to reduced tidal flushing in the

East Basin section of the harbour, which is the most inland station. Also, the elevated levels of the high molecular weight phthalates in the sediment matrix of the East Basin station may act as a source for the benthic or sedentary organisms, resulting in elevated concentrations in these organisms. For the fish species (e.g., striped seaperch Figure 3.11), there was no evidence of spatial differences in concentration. To assess the general trends in chemical movement through the food web, the biota data from the three stations were pooled for analysis. However to address the spatial variability, biota-sediment accumulation factors for the benthic species have been calculated on a station-specific basis (see section 3.5 Biota-

Sediment Distribution).

83 North Central Marina East Basin 1000000 A) Plankton * 100000 *

10000 * * 1000

* 100

Concentration (ng/g lipid) (ng/g Concentration 10

P P 6 OP C C7 C8 C9 DM DEP BBP DNP C10 DiBP DnB DEHP Dn Phthalate Ester

North Central Marina East Basin 100000 * B) Green Algae

10000 * * * * 1000 * * 100

10 Concentration (ng/g lipid) (ng/g Concentration

6 7 8 9 0 MP BP OP C C C C D DEP nBP B EHP DNP C1 DiBP D D Dn Phthalate Ester

84 North Central Marina East Basin 100000 C) Geoduck Clams * * 10000 * * * * 1000 * *

100

Concentration (ng/g lipid) (ng/g Concentration 1

P P P P 7 8 9 MP E B B O NP C6 C C C D D BBP D Di Dn DEHP Dn Phthalate Ester

North Central Marina East Basin 100000 D) Pacific Oyster * * 10000

* 1000 * * * * 100 *

* 10 Concentration (ng/glipid)

P P 6 8 MP EP B BP C C7 C C9 D D n B nOP DNP DiBP D DEH D Phthalate Ester

85 North Central Marina East Basin 100000 E) Striped Seaperch

10000

1000

100

10 Concentration (ng/g lipid) (ng/g Concentration 1

P P P P M B H OP C6 C7 C8 C9 D DEP BB DNP Di DnBP DE Dn Phthalate Ester

Figure 3.11. Mean Lipid Concentrations (± Standard Deviations, ng/g lipid wt.) of Phthalate Esters in Marine Biota Samples from Three Sampling Stations (“NC” = North Central, “Ma” = Marina, and “EB” = East Basin) in False Creek Harbour. Species presented are: A) Plankton, B) Green Algae, C) Geoduck Clams, D) Pacific Oysters, and E) Striped Seaperch. Starred bars (*) indicate statistically significant differences in concentration between 1 station and the other 2 (single star per chemical), or between 2 specific stations (two stars per chemical).

3.4.3. Distribution of Phthalate Esters in Sediment, Seawater, and Biota and Chemical Transfer through the Food Web

3.4.3.1. Low Molecular Weight Phthalates For all biota samples, dimethyl phthalate concentrations were relatively low, ranging from 4 to 192 ng/g lipid wt. (0.2 to 2.5 ng/g wet wt.) (Figure 3.12). Fugacities of DMP in the organisms ranged between 3 and 104 nPa, and fell between those in the sediment (3,120

86 nPa) and the water (0.2 nPa), for which a substantial sediment-water chemical disequilibrium existed (i.e., 17,700 fold) (Figure 3.13). Diethyl phthalate concentrations in the marine biota were higher than those observed for DMP and ranged between 32 and 968 ng/g lipid wt. (1 and 19 ng/g wet wt.) (Figure 3.14). Fugacities in the organisms ranged between 6 and 181 nPa, and were also between those in the sediment (391 nPa) and water

(14 nPa) (Figure 3.15).

To assess whether there was significant evidence of either biomagnification or trophic dilution in the food chain, linear regression analysis of fugacity (f) as a function of trophic position (TP) was conducted. The results for all phthalate esters are summarized in

Table 3.17 (Section 3.4.5), where “p” values indicate whether the slope of the correlation is statistically significantly different from zero. A positive slope indicates biomagnification is occurring, and a negative slope provides evidence of trophic dilution. For the low molecular weight phthalates, the fugacities did not show a statistically significant correlation with trophic position (Table 3.17, Figure 3.16). Rather, the fugacities were relatively constant throughout the food chain (i.e., fprey ≅ fpredator). In terms of the overall environmental distribution of these low molecular weight phthalates, the fugacities of DMP in all the species, and DEP in the majority of species (i.e., approximately 70%) were significantly lower than the sediment fugacity (ANOVA, p<0.05) and significantly higher than the water fugacity (ANOVA, p<0.05) (Tables E.3.1, and E.3.2, Appendix E). In summary, the fugacities of these low molecular weight substances appear to decrease from the sediments, to the biota, to the water (i.e., fsediment > fprey ≅ fpredator > fwater).

87 DMP 10

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i p o i g g f l l o t o a h k l l a s a e e r h Fi u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i f l . . S . e g g S g . O . . r g B M o P G M S P D o o M G Fr S G D D D

1000

100

10 Concentration (ng/g lipid) (ng/g Concentration 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l a s e e r h a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.12. Concentrations of Dimethyl Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top), Lipid Weight (ng/g lipid wt.) (bottom).

88 DMP 1000

100

10 Fugacity (nPa)

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

10000

1000

100

10 Fugacity (nPa)

0.1

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( ( ( (

d r . . . n e e s s s h e s s h h h n s e g l r e i l L o a a c c s E w s t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l a s a e e r h F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.13. Fugacities (nPa) of Dimethyl Phthalate in Marine Biota (λ), Sediment (ν), and

Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

89 DEP 100

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

10000

1000

100

10 Concentration (ng/g lipid) (ng/g Concentration 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.14. Concentrations of Diethyl Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

90 1000 DEP

100

10 Fugacity (nPa)

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i p o i g g f l l o t o a h k l l a s a e e r h Fi u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D

1000

100

Fugacity (nPa) 10

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( ( ( ( (

d r . . . n e e s s s h e s s h h h n s e g l r e i l L o a a c c s E w s t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l a s a e e r h F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.15. Fugacities (nPa) of Diethyl Phthalate in Marine Biota (λ), Sediment (ν), and

Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

91 3 3 DMP DEP

2 2

1 1 Log Fugacity (nPa) Log Fugacity (nPa) Log Fugacity

0 0 012345 012345 Trophic Position Trophic Position Figure 3.16. Log Fugacity (nPa) Versus Trophic Position for Dimethyl Phthalate (left) and Diethyl Phthalate (right).

3.4.3.2. Intermediate Molecular Weight Phthalates Di-iso-butyl phthalate concentrations in the marine biota ranged from 7 to 229 ng/g lipid wt. (0.2 and 4.1 ng/g wet wt.), or from 1 to 29 nPa on a fugacity basis (Figures 3.17 and 3.18). Di-n-butyl phthalate levels in the organisms were relatively high with lipid-based concentrations ranging between 89 and 11,700 ng/g (3 and 60 ng/g wet wt.) and fugacities ranging between 11 and 1,460 nPa (Figures 3.19 and 3.20). Concentrations of butylbenzyl phthalate in the biota ranged between 15 and 1,400 ng/g lipid (0.7 and 30 ng/g wet wt.), or between 0.09 and 8.6 nPa on a fugacity basis (Figures 3.21 and 3.22).

In terms of the overall environmental distribution of DiBP, DBP, and BBP, the fugacities of these substances in the biota were approximately 1 – 2 orders of magnitude lower than the sediment fugacity, and were less than or equal to the fugacity in the water.

Although the fugacities of DiBP and DBP in several species were up to an order of magnitude lower than the water fugacity (freely dissolved), these differences were not statistically significant (ANOVA, p > 0.05) (Table E.3.2, Appendix E). The fugacities of

92 DiBP, DnBP, and BBP showed a slight decline with trophic position (Figure 3.18), yet regression analysis indicated that this negative correlation was not statistically significant

(Table 3.17). However, ANOVA tests revealed that the fugacities of these substances in the dogfish muscle and liver samples were statistically significantly lower than fugacities in some of the lower trophic species such as plankton, green algae, geoduck clams, striped seaperch, and staghorn sculpin (ANOVA, p < 0.05, see Tables E.3.3 and E.3.4, Appendix

E). In summary, the fugacities of these chemicals were found to be highest in the sediment and lower in the water and biota. Additionally, fugacities in the higher trophic organisms were lower than those in the water and the prey species (i.e., fsediment > fwater ≅ fprey ≥ fpredator)

(Figures 3.18, 3.20, 3.22, 3.24, and 3.26, and Table E.3.1, Appendix E).

For the isomers with intermediate molecular weights, concentrations in the marine organisms ranged from 11 to 772 ng/g lipid wt. (0.09 to 17 ng/g wet wt.) for di-iso-hexyl phthalate (C6), and from 28 to 2,060 ng/g lipid wt. (0.4 to 45 ng/g wet wt.) for di-iso-heptyl phthalate (C7) (Figures 3.23 and 3.25). For both C6 and C7, fugacities in the sediment (2.0 and 2.8 nPa) and the in the water (freely dissolved fraction: 0.74 and 0.24 nPa) were approximately equal to the highest fugacities in the biota, which ranged from 0.01 to 2.1 nPa for both chemicals (Figures 3.24 and 3.26). ANOVA tests revealed that approximately half of the marine species tested exhibited chemical fugacities that were significantly lower than those in the sediments, while only a few of the species (i.e., Dungeness Crab for C6, and minnows, Pile Perch, and Whitespotted Greenling for C6 and C7) exhibited fugacities that were significantly lower than those in the water (Tables E.3.1 and E.3.2 Appendix E). In terms of the chemical movement through the food chain, the fugacities of di-iso-hexyl phthalate (C6), and di-iso-heptyl phthalate (C7) did not show a statistically significantly

93 increase or decrease with increasing trophic position in the food chain (Table 3.17, Figure

3.18). Overall for these substances, the fugacities appeared slightly higher in the sediments relative to the water and biota, which exhibited comparable fugacities, with the exception of a few higher trophic and pelagic fish species (i.e., fsediment ≥ fwater ≅ fprey ≥ fpredator).

94 10 DiBP

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

1000

100

10 Concentration (ng/g lipid) (ng/g Concentration

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l a s a e e r h F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.17. Concentrations of Di-iso-butyl Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

95 100 DiBP

1 Fugacity (nPa)

0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s b E w s t n M t m e e m r r i p o i g g f l l o t o a h k l l a s e e r h a Fi u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D

100

10 Fugacity (nPa) 1

0.1

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( (

d r . . . n e e s s s h n e s s h h h s e g l r e i l L o a a c c s s E w t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y C e f i a C u S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.18. Fugacities (nPa) of Di-iso-butyl Phthalate in Marine Biota (λ), Sediment (ν), and Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

96 1000 DBP

100

10 Concentration (ng/g wet wt.) 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s s b E w t n M t m e e m r r i p o i g g f l l o t o a h k l l a s e e r h a Fi u r S h l s l n n s a c s n A A i s P P t c C e s i n C y e f i a u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D

100000

10000

1000

100 Concentration (ng/g lipid) (ng/g Concentration 10

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y C e f i a C u S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.19. Concentrations of Di-n-butyl Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

97 DBP 10000

1000

100

10 Fugacity (nPa)

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i p o i g g f l l o t o a h k l l a s a e e Fi r r h l u S n h n s l c s n s a i s A A P P c C e s y e t f i n C u C i f a i S f l . . . . . e g g S S g . O . . r g B M o P G M S P D o o M G Fr S G D D D

10000

1000

100

Fugacity (nPa) 10

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( (

d r . . . n e e s s s h n e s s h h h s e g l r e i l L o a a c c s E w s t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y C e f i a C u S f . . i e f l . . g S . g . . S r g O r . g M o P B G M S P D o o M G F S G D D D Figure 3.20. Fugacities (nPa) of Di-n-butyl Phthalate in Marine Biota (λ), Sediment (ν), and

Total (▬), C18 (°), and Freely Dissolved () Water from False Creek. Harbour.

98 100 BBP

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

10000

1000

100

10 Concentration (ng/g lipid) (ng/g Concentration

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l s e r h a a e F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g M o P B G M S P D o o M G F S G D D D Figure 3.21. Concentrations of Butylbenzyl Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

99 BBP 100

1 Fugacity (nPa) 0.1

0.01

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i p o i g g f l l o t o a h k l l a s e e r h a Fi u r S h l s l n n s a c s n A A i s P P t c C e s i n C y e f i a u C S f . . i f l . . S . e g g S g . O . . r g B M o P G M S P D o o M G Fr S G D D D

100

1 Fugacity (nPa) 0.1

0.01

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( (

d r . . . n e e s s s h n e s s h h h s e g l r e i l L o a a c c s E w s t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.22. Fugacities (nPa) of Butylbenzyl Phthalate in Marine Biota (λ), Sediment (ν), and

Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

100 100 C6

0.1 Concentration (ng/g wet wt.) 0.01

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e r r i i p o i m f l h g g t a l l l o s r o k a a e e F r S s h l u n h n s l c i n s a f s A A P P c C e s y e t i n C u C i f a i S g f l . . . . . e g S S g . O . . r o g B M P G M S P D o D o M G Fr S G D D

10000

1000

100

Concentration (ng/g lipid wt.) (ng/g Concentration 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i i p o i h g g f l l o t o a k l l s e e r h a a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D

Figure 3.23. Concentrations of Di-iso-hexyl Phthalate (C6) in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

101 C6 10

0.1

Fugacity (nPa) 0.01

0.001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s b E w s t n M t m e e m r r i p o i h g g f l l o t o a k l l a s a e e r h Fi u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D

1000

100

0.1 Fugacity (nPa)

0.01

0.001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( ( (

d r . . n e e s s s h n e s s h h h s e g L l r e i l o a a c c s E w s t b n M S t m e e m r r i i p o i h g g f l l o t o a k l l s a e e r h a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o D o M G F S G D D Figure 3.24. Fugacities (nPa) of Di-iso-hexyl Phthalate (C6) in Marine Biota (λ), Sediment

(ν), and Total (▬), C18 (°), and Freely Dissolved () Water from False Creek. Harbour.

102 C7 1000

100

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s b E w s t n M t m e e m r r i p o i h g g f l l o t o a k l l a s e e r h a Fi u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D

100000

10000

1000

100

Concentration (ng/g lipid wt.) (ng/g Concentration 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i i p o i h g g f l l o t o a k l l s e e r h a a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y C e i a C u S g f . . i f l . . S . e g S g . O . . r o g M r P B G M S P D o D o M G F S G D D

Figure 3.25. Concentrations of Di-iso-heptyl Phthalate (C7) in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

103 C7 10

0.1

Fugacity (nPa) 0.01

0.001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

10000

1000

100

Fugacity (nPa) 0.1

0.01

0.001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( (

d r . . n e e s s s h n e s s h h h s e g L l r e i l o a a c c s E w s t b n M S t m e e m r r i i p o i h g g f l l o t o a k l l s e e r h a a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n C y C e i a u S g f . . i f l . . S . e g S g . O . . r o g M r P B G M S P D o D o M G F S G D D Figure 3.26. Fugacities (nPa) of Di-iso-heptyl Phthalate (C7) in Marine Biota (λ), Sediment

(ν), and Total (▬), C18 (°), and Freely Dissolved () Water from False Creek. Harbour.

104 3 4 DiBP DBP

2 3

1 2

0 1 Log Fugacity (nPa) Fugacity Log Log Fugacity (nPa) Fugacity Log

-1 0 012345 012345 Trophic Position Trophic Position

2 BBP

0 Log Fugacity (nPa) Fugacity Log

-1 012345 Trophic Position

1 1 C6 C7

0 0

-1 -1

-2 -2 Log Fugacity (nPa) Fugacity Log (nPa) Fugacity Log

-3 -3 012345 012345 Trophic Position Trophic Position Figure 3.27. Fugacity (nPa) Versus Trophic Position for Di-iso-butyl Phthalate (top left), Di-n-butyl Phthalate (top right), Benzylbutyl Phthalate (middle), Di-iso-hexyl Phthalate (C6) (bottom left), and Di-iso-heptyl Phthalate (C7) ( bottom right).

105 3.4.3.3. High Molecular Weight Phthalates Di-2-ethylhexyl and C8 (di-iso-octyl) phthalate were both present in relatively high concentrations in the marine organisms, ranging between 79 and 16,700 ng/g lipid (1 and

305 ng/g wet weight) for DEHP, and between 17 to 13,900 ng/g lipid wt. (3 to 180 ng/g wet wt.) for C8 (Figures 3.28, and 3.32). Fugacities of both DEHP and C8 in the biota ranged from 0.001 to 1.8 nPa and appeared to decline with increasing trophic position (Figures

3.29, and 3.33). Both di-n-octyl, and di-n-nonyl phthalate were present in the marine organisms at concentrations ranging from 2 to 2,120 ng/g lipid wt. (0.07 to 25 ng/g wet wt.)

(Figures 3.30, and 3.34). The fugacities of these substances in the organisms were relatively low and ranged from 2.8·10-5 to 0.13 nPa (Figures 3.31, 3.35). C9 (di-iso-nonyl) and C10

(di-iso-decyl) phthalate isomers were detected in the organisms at relatively high levels ranging between 260 and 11,000 ng/g lipid wt. (0.8 to 71 ng/g wet wt.) for C9, and between

6 and 13,900 ng/g lipid (0.6 and 72 ng/g wet wt.) for C10 (Figures 3.36, and 3.38).

Fugacities of both substances in the marine biota were low (1·10-3 to 2 nPa for C9 and 1·10-5 to 0.02 nPa for C10), and appeared to decline at higher levels in the food chain (Figures

3.37, and 3.39).

These high molecular weight phthalates exhibited similar environmental distributions and fugacity patterns in the food chain (Figures 3.29, 3.31, 3.33, 3.35, 3.37, and

3.39). For these substances (i.e., DEHP, DnOP, DnNP, C8, C9, and C10 isomers), there is a considerable difference between the fugacities determined from the three water concentrations (i.e., total, C18, and freely dissolved). Specifically, “total” and “freely dissolved” fugacities in the water differ by approximately 4 to 6 orders of magnitude for C8 and C10 phthalates, respectively. As discussed in section 3.2, the freely dissolved

106 concentration best represents the chemical concentration in the water that can be absorbed via the respiratory surface area of the organism. Therefore, the fugacity determined from the freely dissolved chemical concentration is believed to be the most appropriate for inter- media comparison. This is particularly apparent for the high molecular weight phthalates where the freely dissolved fraction appears to be close to an equilibrium with the sediment fugacity, whereas the fugacities based on the “total” and “C18” concentrations are orders of magnitude greater than the fugacities in the sediment and biota compartments. In terms of the environmental distribution of these high molecular weight phthalates, the sediment fugacities were typically up to an order of magnitude greater than the freely dissolved water fugacities, which were approximately equal to the highest fugacities in the biota, usually occurring in the algae and plankton species at the base of the food chain. The fugacities of the high molecular weight phthalates significantly declined with trophic position in the food chain (p < 0.05 for DEHP, DnOP, DnNP, C8, and C9, and p = 0.065 for C10, Table 3.17,

Figure 3.40). Fugacities of these substances in some of the fish and higher trophic species

(e.g., minnows, perch, Dungeness crab, Whitespotted Greenling and Spiny Dogfish), were significantly lower than the freely dissolved water fugacity (ANOVA, p < 0.05, Table E.3.1

Appendix E). Thus, the fugacities in the various compartments appear to decrease from sediment to water to biota (i.e., fsediment ≤ fwater(FD) ≅ fprey < fpredator) (Tables E.3.1, E.3.2, E.3.3 and 3.17).

107 10000 DEHP

1000

100

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

100000

10000

1000

100 Concentration (ng/g lipid) (ng/g Concentration 10

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y C e f i a C u S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.28. Concentrations of Di(2-ethylhexyl) Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

108 DEHP 100

0.1

0.01 Fugacity (nPa)

0.001

0.0001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l a l o t o h k l l a s a e e F r r h l u S n h n s l c s n s a i s A A P P c C e s y e t f i n C u C i f a i S f l . . . . . e g g S S g . O . . r g B M o P G M S P D o o M G Fr S G D D D

10000

1000

100

0.1 Fugacity (nPa) 0.01

0.001

0.0001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( ( (

d r . . . n e e s s s h n e s s h h h s e g l r e i l L o a a c c s E w s t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.29. Fugacities (nPa) of Di(2-ethylhexyl) Phthalate in Marine Biota (λ), Sediment

(ν), and Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

109 DnOP 100

0.1 Concentration (ng/g wet wt.) 0.01

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i p o i g g f l l o t o a h k l l a s e e r h a Fi u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i f l . . S . e g g S g . O . . r g B M o P G M S P D o o M G Fr S G D D D

10000

1000

100

10 Concentration (ng/g lipid) (ng/g Concentration 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.30. Concentrations of Di-n-octyl Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

110 DnOP 1

0.1

0.01 Fugacity (nPa) 0.001

0.0001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

1000

100

0.1 Fugacity (nPa)

0.01

0.001

0.0001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( ( (

d r . . . n e e s s s h n e s s h h h s e g l r e i l L o a a c c s E w s t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.31. Fugacities (nPa) of Di-n-octyl Phthalate in Marine Biota (λ), Sediment (ν), and

Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

111 C8 1000

100

10 Concentration (ng/g wet wt.) 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i p o i h g g f l l o t o a k l l a s a e e Fi r r s h l u S n h n s l c i n s a f s A A P P c C e s y e t i n C u C i f a i S g f l . . . . . e g S S g . O . . r o g B M P G M S P D o o M G Fr S G D D D

100000

10000

1000

100

10 Concentration (ng/g lipid wt.) lipid (ng/g Concentration 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i i p o i h g g f l l o t o a k l l s e e r h a a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D Figure 3.32. Concentrations of Di-iso-octyl Phthalate (C8) in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

112 10 C8

0.1

0.01 Fugacity (nPa) 0.001

0.0001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i i p o i h g g f l a l o t o k l l a s a e e F r r s h l u S n h n s l c i n s a f s A A P P c C e s y e t i n C u C i f a i S g f l . . . . . e g S S g . O . . r o g B M P G M S P D o o M G Fr S G D D D

10000

1000

100

0.1 Fugacity (nPa) 0.01

0.001

0.0001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( ( (

d r . . n e e s s s h n e s s h h h s e g L l r e i l o a a c c s E w s t b n M S t m e e m r r i i p o i h g g f l l o t o a k l l a s a e e r h F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D Figure 3.33. Fugacities (nPa) of Di-iso-octyl Phthalate (C8) in Marine Biota (λ), Sediment

(ν), and Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

113 100 DnNP

0.1 Concentration (ng/g wet wt.)

0.01

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

10000

1000

100

10 Concentration (ng/g lipid) (ng/g Concentration 1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . . n e e s s s s s h h h h n s e g l r e i l L o a a c c s E w s t b n M t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.34. Concentrations of Di-n-nonyl Phthalate in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

114 DnNP 1

0.1

0.01

0.001 Fugacity (nPa)

0.0001

0.00001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

10000

1000

100

0.1

Fugacity (nPa) 0.01

0.001

0.0001

0.00001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( (

d r . . . n e e s s s h n e s s h h h s e g l r e i l L o a a c c s E w s t b n M S t m e e m r r i i p o i g g f l l o t o a h k l l s e e r h a a F u r S h l s l n n s a c s n A A i s P P t c C e s i n y e f i a C u C S f . . i e f l . . g S . g . . S r g O r . g B M o P G M S P D o o M G F S G D D D Figure 3.35. Fugacities (nPa) of Di-n-nonyl Phthalate in Marine Biota (λ), Sediment (ν), and

Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

115 C9 1000

100

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i p o i h g g f l l o t o a k l l a s e e r h a Fi u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i f l . . S . e g S g . O . . r o g B M P G M S P D o o M G Fr S G D D D

100000

10000

1000

100 Concentration (ng/g lipid wt.) (ng/g Concentration 10

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i i p o i h g g f l l o t o a k l l s e e r h a a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o D o M G F S G D D Figure 3.36. Concentrations of Di-iso-nonyl Phthalate (C9) in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

116 C9 10

0.1

Fugacity (nPa) 0.01

0.001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s b E w s t n M t m e e m r r i p o i h g g f l l o t o a k l l a s e e r h a Fi u r S s h l s l n i n s a c n A A f s P P t c C e s i n C y e i a u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D

10000

1000

100

Fugacity (nPa) 0.1

0.01

0.001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( (

d r . . n e e s s s h n e s s h h h s e g L l r e i l o a a c c s E w s t b n M S t m e e m r r i i p o i h g g f l l o t o a k l l s e e r h a a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n C y C e i a u S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D Figure 3.37. Fugacities (nPa) of Di-iso-nonyl Phthalate (C9) in Marine Biota (λ), Sediment

(ν), and Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

117 C10 1000

100

1 Concentration (ng/g wet wt.) 0.1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

100000

10000

1000

100

1 Concentration (ng/g lipid wt.) lipid (ng/g Concentration

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s E w s t b n M t m e e m r r i i p o i h g g f l l o t o a k l l s e r h a a e F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i f l . . S . e g S g . O . . r o g M r P B G M S P D o D o M G F S G D D Figure 3.38. Concentrations of Di-iso-decyl Phthalate (C10) in Marine Biota from False Creek Harbour Expressed in Wet Weight (ng/g wet wt.) (top) and Lipid Weight (ng/g lipid wt.) (bottom).

118 1 C10

0.1

0.01

0.001

0.0001 Fugacity (nPa)

0.00001

0.000001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7

0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ......

1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

r . . n e e s s s s s h h h h n s e g L l r e i l o a a c c s s b E w t n M t m e e m r r i p o i h g g f l l o t o a k l l a s e e r h a Fi u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D

10000 1000 100 10 1 0.1 0.01

Fugacity (nPa) 0.001 0.0001 0.00001 0.000001

r t ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

e n 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 t e 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 a ......

m 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 i ( ( ( ( ( ( ( ( ( ( ( ( ( ( W ( ( ( ( ( (

d r . . n e e s s s h n e s s h h h s e g L l r e i l o a a c c s E w s t b n M S t m e e m r r i i p o i h g g f l l o t o a k l l a s e e r h a F u r S s h l s l n i n s a c n A A f s P P t c C e s i n y e i a C u C S g f . . i e f l . . g S . . . S r o g O r . g B M P G M S P D o o M G F S G D D D

Figure 3.39. Fugacities (nPa) of Di-iso-decyl Phthalate (C10) in Marine Biota (λ), Sediment

(ν), and Total (▬), C18 (°), and Freely Dissolved () Water from False Creek Harbour.

119 2 0 DEHP DnOP 1 -1 0 -2 -1 -3 -2

Log Fugacity (nPa) Fugacity Log -3 (nPa) Fugacity Log -4

-4 -5 012345 012345 Trophic Position Trophic Position

1 0 C8 DnNP -1 0

-2 -1 -3 -2 Log Fugacity (nPa) Log Fugacity

Log Fugacity (nPa) Fugacity Log -4

-3 -5 012345 012345 Trophic Position Trophic Position

1 -1 C9 C10 -2 0

-3 -1 -4

-2 Log Fugacity (nPa) Log Fugacity (nPa) Log Fugacity -5

-3 -6 012345 012345 Trophic Position Trophic Position Figure 3.40. Log Fugacity (nPa) Versus Trophic Position for Di(2-ethylhexyl) Phthalate (top left), Di-n-octyl Phthalate (top right), Di-iso-octyl Phthalate (C8) (middle left) and Di- n-nonyl Phthalate (middle right). and Di-iso-nonyl Phthalate (C9) (bottom left), and Di-isodecyl Phthalate (C10) (bottom right).

120 3.4.4. Summary of Food Chain Bioaccumulation Results Fugacity is plotted as a function of trophic position for all phthalate esters in Figures

3.41 (individual phthalate esters) and 3.42 (isomeric mixtures). Statistical results of the regression between fugacity (f) and trophic position (TP) for all phthalate esters are summarized in Table 3.17. For the low molecular weight phthalates (i.e., DMP and DEP), the fugacity does not increase or decrease in a statistically significant manner with increasing trophic position in the food-chain. For the mid-molecular weight individual phthalates (i.e., DiBP, DBP, and BBP), a declining trend in fugacity, with increasing trophic position, becomes apparent; however, the relationship is not statistically significant (Table

3.17). For C6 and C7 phthalate ester isomeric mixtures, there is no statistically significant increase or decrease in fugacity with increasing trophic position. For the high Kow substances (i.e., DEHP, DnOP, DnNP, C8, C9, and C10), a statistically significant negative correlation between fugacity and trophic position exists.

Table 3.17. Statistical Results of Regression: Fugacity versus Trophic Position (TP) PE Saltwater n “b” “m” p value for R2 KOW y-intercept Slope slope DMP 1.80 18 1.31 0.017 0.860 0.002 DEP 2.77 18 1.45 0.048 0.646 0.014 DiBP 4.58 18 1.04 -0.091 0.322 0.061 DBP 4.58 18 2.41 -0.154 0.199 0.101 BBP 5.03 18 0.45 -0.115 0.353 0.054 DEHP** 8.20 18 0.02 -0.419 0.012** 0.335 DnOP** 8.20 16 -1.13 -0.524 0.006** 0.434 DnNP** 9.11 15 -1.50 -0.490 0.030** 0.315 C6 6.69 16 -0.75 0.014 0.931 0.001 C7 7.44 15 -0.90 -0.038 0.831 0.004 C8** 8.20 18 -0.04 -0.298 0.030** 0.261 C9** 9.11 12 -0.59 -0.335 0.022** 0.422 C10* 10.5 15 -2.08 -0.336 0.065* 0.238 *p < 0.10, **p < 0.05

121 5

3 DMP DEP 2 DiBP DBP DMP Linear 1 DEP Linear DiBP Linear Log Fugacity (nPa) Log Fugacity 0 DBP Linear

-1

-2 012345 Trophic Position

0 BBP DEHP -1 DnOP DnNP BBP Linear -2 DEHP Linear

Log Fugacity (nPa) Log Fugacity DnOP Linear -3 DnNP Linear

-4

-5 012345 Trophic Position

Figure 3.41. Fugacity Versus Trophic Position for Individual Phthalate Esters (DMP, DEP, DiBP, and DBP (top), BBP, DEHP, DnOP, and DnNP (bottom)) in Marine Biota from False Creek Harbour.

122 1

0 C6 C7 C8 -1 C9 C10 -2 C6 Linear C7 Linear C8 Linear -3 Log Fugacity (nPa) Fugacity Log C9 Linear C10 Linear -4

-5 012345 Trophic Position

Figure 3.42. Fugacity Versus Trophic Position for Phthalate Ester Isomeric Mixtures (C6, C7, C8, C9, and C10) in Marine Biota from False Creek Harbour.

3.4.5. Discussion The objective of the field study was to determine the extent of food-chain bioaccumulation of phthalate esters in the marine system and to distinguish between the occurrence of biomagnification, trophic dilution and lipid-water equilibrium partitioning.

Mechanistically, there are several chemical uptake and elimination processes that occur within biological organisms, the relative rates of which determine the resulting levels in the organisms, and the distributional patterns of the chemical in the food chain. For the marine aquatic organisms in the field study, chemical uptake processes include (1) chemical uptake from the water via the gill membrane, and adsorption via the skin, and (2) chemical uptake

123 from diet via the gastrointestinal tract membrane. Chemical elimination processes include

(1) gill elimination, (2) fecal egestion, (3) metabolism, (4) gut hydrolysis and (5) growth dilution (Figure 3.43) (Gobas 1993, Gobas et al. 1999). The results of the field study indicate that the pattern of chemical movement through the food chain is dependent on the chemical’s octanol-seawater partition coefficient. Thus, different patterns of chemical distribution in the food chain were observed for the low, intermediate, and high molecular weight phthalate esters.

Metabolism Gill Elimination Growth Dilution Gill Uptake

Dietary Uptake

Fecal Egestion

Gut Hydrolysis Figure 3.43. Chemical Uptake and Elimination Routes in Fish

For the low Kow phthalates (i.e., DMP and DEP), fugacities in the organisms remain relatively constant throughout the food chain, providing no evidence of either biomagnification or trophic dilution. For these more water-soluble chemicals, chemical uptake from the water, through the gills and/or the skin, is likely the most dominant intake process. For diethyl phthalate, fugacities in the marine biota were similar to, or greater than those in the water, and lower than those in the sediment. Both the comparable fugacities in the water and biota, and the lack of biomagnification indicate that equilibrium partitioning

124 of the chemical between the water and the lipid tissue of the organisms is the dominant process controlling the bioaccumulation of this substance. In the case of dimethyl phthalate, the higher fugacities in the organisms relative to those in the water may reflect exposure of the organisms to a higher chemical fugacity in the sediment matrix, resulting from ventilation of higher fugacity sediment pore water, and/or the ingestion of sediments. The organisms may achieve a steady state fugacity that is in between the higher chemical fugacity in the sediment, and the lower chemical fugacity in the water, and reflects exposure of the organisms to both media. Another possible explanation for the higher fugacities of

DMP in the biota, relative to the water, is that this substance may have an affinity for binding to a non-lipid matrix within the biota (e.g. protein). In summary, lipid-water partitioning mediated by gill uptake and elimination appear to dominate and control the overall mass of the lower molecular weight phthalates in the organisms. Additionally, it appears that exposure of the organisms to significantly higher fugacities in the sediments results in the elevation of the biota fugacities to levels above those in the water.

As Kow increases, fugacities of some of the intermediate molecular weight phthalates

(i.e., DiBP, DBP, BBP) appear to decline slightly with increasing trophic level in the food chain, although this decrease was not statistically significant. However, the fugacities of

DiBP, DBP, and BBP in the dogfish were significantly lower than those in the primary producers and some of the smaller fish (e.g., perch and sculpin). For these phthalates with intermediate molecular weights, the freely dissolved water fugacities were generally comparable to those in the organisms, although the fugacities of DBP in the dogfish liver, and fugacities of C6 and C7 in minnows, crabs, and greenlings, dropped below the levels in the water. Comparable fugacities in the biota and water suggest that equilibrium partitioning

125 between the water and the organisms is occurring, and that the processes of gill uptake and gill elimination drive the resulting chemical body burdens (Figure 3.43). The slight decline in fugacity throughout the food chain that was observed for DiBP, DBP and BBP, suggests that metabolic transformation may occur. However, the general agreement between fugacities in the water and those in the organisms of the food chain, indicate that metabolism is too minor to affect the observed lipid-water partition coefficients. Woffard et al. (1981) and Carr et al. (1997) suggest that biotransformation of di-n-butyl phthalate and butylbenzyl phthalate occurs, although metabolic transformation rates have not been quantified.

Substances with high KOW’s have a high potential to bioaccumulate in marine organisms and biomagnify through the food chain. Significant evidence of food chain bioaccumulation for non-metabolizable substances such as PCB’s in aquatic ecosystems, such as the Great Lakes, has been reported in the literature (e.g., Oliver and Niimi, 1988,

Connolly and Pedersen, 1988, Morrison et al. 1997). However, in the current study, the fugacities of the high KOW phthalates in the marine organisms decreased significantly with increasing trophic position, providing evidence of trophic dilution. Additionally, the water fugacities were generally equal to the levels in the plankton and algae, and greater than those in the higher trophic organisms. This pattern indicates that chemical uptake decreases, and/or elimination rates increase at each step in the food chain. Potential mechanisms and factors that may contribute to the observed pattern of trophic dilution include: (1) a reduced bioavailability of the high KOW substances in the water, and (2) the occurrence of gut hydrolysis and/or (3) metabolism. The reduced bioavailability of these substances in the water is likely to limit chemical uptake through the respiratory surface of the marine organisms and may reduce the overall mass of chemical entering the organisms.

126 Additionally, chemical entering the organism through the dietary pathway may be subject to hydrolysis in the gastrointestinal tract, reducing dietary assimilation and the overall chemical uptake. Dietary assimilation efficiencies for bluegills (Macek et al. 1979), and paneaid shrimp (Hobson et al. 1994) fed a C14 labeled DEHP contaminated diet were estimated by Staples et al. (1997a) to range between 0.25 and 0.30. However, since radiolabeled chemicals were used, they report that the assimilation of parent DEHP may be lower than the estimated value due to metabolism in the gut. Parkerton et al. (2001) determined a dietary assimilation efficiency of 0.20 for di-iso-heptyl phthalate (C7) in rainbow trout based on laboratory dietary uptake experiments. These estimated values are generally lower than dietary assimilation efficiencies reported for PCBs of similar KOW, which range from approximately 0.25 up to >0.60 (Gobas et al. 1988, Gobas et al. 1993,

Morrison et al. 1997). Thus, biotransformation of phthalate esters in the gastrointestinal tract may be the cause of this difference in dietary assimilation efficiencies between the two classes of chemicals. Another possible explanation is that metabolic transformation of these chemicals in the organisms may increase the overall elimination (or transformation) of these substances, and may play a role in driving the fugacities in the organisms to levels below that in their diet and the water. Evidence of metabolic transformation of DEHP has been reported for several aquatic or marine organisms (Metcalf et al. 1973, Stalling et al. 1973, and Wofford et al. 1981, Barron et al. 1995, Sabourault 1998, and Karara and Hayton 1988).

The metabolism of DnOP in aquatic organisms has been described by Sanborn et al., 1975.

However, metabolic rate constants have not been quantified.

Qualifiers: It is important to note that the fugacity versus trophic position correlation is heavily dependent on the two extremes of the food web: the primary producers

127 (e.g., plankton and green algae: trophic position = 1.00) and the top predator (i.e., spiny dogfish: trophic position = 4.07). For all of the phthalates, the fugacities in the algae and plankton tended to be relatively high, while those in the dogfish tended to be relatively low.

However, at both extremes, there is uncertainty or confounding factors that influence the resulting trends.

First, there is uncertainty in the determination of the fugacity capacity for green algae and plankton. Direct measurements of the fugacity capacity of these organisms have not been reported, and there is debate in the literature as to the best method of normalizing concentrations (i.e., whether to base the normalization on organic carbon or lipid content).

Skoglund and Swackhamer (1999) suggest that organic carbon is the best matrix to use for the normalization of PCB accumulation in plankton. Based on a review of available data,

Seth and others (1999) suggested that organic carbon has a sorbing capacity for organic chemicals that ranges between 0.14 and 0.89 that of lipids, and suggest values of 0.35-0.41.

Hiatt (1999) and Tolls and McLachlan (1994), suggest that terrestrial plants behave as though they have 0.1% - 10% octanol equivalence. Cousins and Mackay (2001) recommend using a 1% lipid value for chemical partitioning into terrestrial plants based on a literature assessment and model validations. Alternatively, plankton and algae data may be normalized based strictly on the measured lipid contents, which were 0.1% and 0.2% respectively in our study. Gobas et al. (1991) report that bioconcentration in aquatic macrophytes is effectively a chemical partitioning process between the plant lipids (which were approximately 0.2%) and water. Normalizing the concentrations based solely on the lipid contents of the organisms results in a low fugacity capacity, and therefore a relatively high fugacity. These three methods of calculating the fugacity capacity (i.e., using measured

128 organic carbon contents, a 1% lipid content, or measured lipid contents) yield resulting fugacities that differ by up to two-orders of magnitude. This consequently affects the slope of the regression between fugacity and trophic position. The purpose of lipid normalizing the data or calculating fugacities, is to remove the effect of differences in lipid contents or sorbing matrices between organisms, since these differences greatly affect the overall chemical concentration. Algae and plankton contain low lipid contents (i.e., plankton ≅

0.1%, green algae ≅ 0.2% (wet wt.)), and high organic carbon contents (i.e., plankton ≅ 40% dry wt. (or 0.6% wet wt.), green algae ≅ 34% dry wt. (or 6.1% wet wt.)). Organic carbon serves as the organism’s energy and carbon source, and due to its relatively high content, it is likely to serve as an important site for chemical accumulation. Thus, our normalization

(fugacity capacity calculation) incorporated lipid, organic carbon, and moisture contents of the algae and plankton (Eqn. 2.10), since all are likely to contribute to the overall sorption of the chemicals in these organisms.

The spiny dogfish (Squalas acanthias) was the top predator in the food chain, and they generally exhibited lower fugacities for all of the phthalates, relative to the other species (Tables E.3.3 and E.3.4 in Appendix E). The dogfish are larger and more mobile than the other species in the study and, as a result, inhabit larger spatial ranges. Dogfish tend to move into foraging areas accompanying an incoming tide; and it was during this period in the tidal cycle that the organisms were collected from False Creek. Likely, the dogfish moved into the harbour to forage just prior to collection, and the concentrations in these organisms may be more reflective of their overall exposure to phthalate levels throughout their geographical range (i.e., Georgia Basin), where phthalate levels tend to be lower than in False Creek (Garrett 2002). Additionally, dogfish have a low metabolic rate, and digest

129 their food slowly (Ketchen 1996). Jones and Geen (1977) estimated that 16 days elapse between feedings for dogfish in British Columbia. Given the slow digestion and metabolic rate of the dogfish, as well as the relatively long half-lives of some of these chemicals, it is possible that the dogfish were not exposed to the phthalate levels in False Creek long enough to achieve a steady state with the ambient environment. The concentrations of the higher molecular weight phthalates in the dogfish may therefore be lower than their steady- state levels in False Creek. This factor could be a significant contributor to the lower phthalate ester levels in this species, relative to the levels in other biota.

Additionally, the correlation between fugacity and trophic position is naturally dependent on the determination of trophic position. In this study, trophic position was calculated based on quantitative dietary information from the literature (i.e., dietary proportions of each prey species). This approach has two major advantages: (i) it provides a more complete picture of the dietary preferences of each species, rather than a “snap-shot” representation would have been obtained from a gut-content analysis of the samples, and (ii) it enables us to determine direct trophic linkages, such that food-chain bioaccumulation models for phthalate esters can be constructed. However, because dietary preferences vary with changes in prey abundance, season, and age or life-stage of the predator, there is some natural variability in trophic level that may not be taken into account. Thus, in order to assess some of the potential variability, and as an additional method for the determination of trophic positions, stable nitrogen and carbon isotope ratio analysis will be conducted on the samples (i.e., δN15 and δC13). Stable isotope analysis is becoming increasingly common as a means to assess community structure and ecological function. Nitrogen and carbon isotope ratios in animal tissues are related to those found in their diet, and can be used as tracers to

130 assess trophic position and initial carbon sources. The results of this analysis will be reported in a later publication.

3.5. Biota - Water Distribution Of Phthalate Esters

3.5.1. Overview Bioaccumulation Factors (BAFs), relating the chemical concentrations in the marine biota to those in the water, are reported in Tables F.3.18 to F.3.30 of Appendix F for all phthalate esters. For each chemical, the BAFs are determined on both a wet weight

(Equation 3.9), and lipid weight (Equation 3.10) basis.

BAFwet = Cbiota / Cwater (3.9)

BAFlipid = Clipid / Cwater (3.10)

Where the BAFwet is the wet weight bioaccumulation factor (L/kg wet weight); Cbiota is the wet weight chemical concentration in the organism (ng/kg wet weight); and Cwater is the chemical concentration in the water (ng/L). In the lipid BAF (L/kg lipid) calculation

(Equation 3.10), the lipid normalized chemical concentration in the organism (Clipid, ng/kg lipid) is utilized. Since the concentration data were lognormally distributed (Appendix D), the mean BAFs (ΧBAF) were calculated from the mean logarithmic concentration values in the organism (Χ(C bio)) and water (Χ(C wat)) (Equation 3.11a), and then converted back to the original units. Standard deviations (SDBAF) were determined accordingly on a logarithmic basis (Equation 3.11b).

Log ΧBAF = Log Χ(C bio) - Log Χ(C wat) (3.11a)

Log SDBAF = Log SD(C bio) + Log SD(C wat) (3.11b)

As explained previously, three different water concentrations were measured or estimated in this study: “Total water”, “C18 water”, and “Freely Dissolved water”. As a

131 result, one can express the BAF values for each congener in three ways, depending on the type of water concentration. These three wet weight and lipid weight BAFs are compared to the appropriate Canadian Environmental Protection Act (CEPA, 1999) bioaccumulation criteria (i.e., 5000 L/kg wet wt. or 100,000 L/kg lipid wt.) for each phthalate ester in Figures

3.44 to 3.56. Since, through their respiratory surfaces, organisms are only effectively exposed to freely dissolved chemical in the water phase, the BAF based on this fraction (i.e.,

BAFFD) most accurately represents the actual degree of bioaccumulation of a substance, and is thus, the most appropriate value for comparison with the CEPA BAF criterion.

Additionally, the lipid normalized BAF can be directly compared to the octanol – seawater partition coefficient of a substance to assess whether equilibrium partitioning of the chemical between the water and lipids is occurring. Specifically, the lipid normalized BAF will equal the KOW under equilibrium conditions.

3.5.2. Bioaccumulation Factors (BAFs)

3.5.2.1. Low Molecular Weight Phthalate Esters For the lower molecular weight phthalate esters, i.e., dimethyl phthalate and diethyl phthalate, the majority of the chemical in the water phase is in the freely dissolved form.

Hence, there are no differences in the bioaccumulation factors based on the “total”, “C18”, and “freely dissolved” water concentrations. For DMP, there was significant variability in the BAFs between the different species. The mean wet weight BAFs ranged between 53 (23

- 107) and 790 (380 - 3,100) L/kg wet weight, with most falling below 180 L/kg, while the lipid-based values ranged between 1,090 (386 - 3,050) and 61,500 (14,000 - 269,000) L/kg lipid (Table F.3.18, Figure 3.44). For DEP, the mean wet weight BAFs ranged between 9 (3

- 27) and 169 (41 - 693) L/kg wet wt., while the lipid-based BAFs varied between 254 (54 -

132 1,200) and 8,620 (1,550 - 48,000) L/kg lipid wt. (Table F.3.19, Figure 3.45). Since the observed water concentrations (i.e., total, C18, and freely dissolved) for both DMP and DEP were relatively consistent, the intra-species variability in the BAFs can be mainly attributed to variability in the biota concentrations. Both chemicals exhibited BAFs that were higher than expected based on equilibrium partitioning of the substances between the organisms and the water. The lipid based BAFs of DMP were approximately 100 - 300 fold greater than the chemical’s Kow of 62, while those for DEP were approximately 2 - 10 times greater than expected from DEP’s KOW of 587 (Figures 3.44, and 3.45). For both DMP and DEP, all of the mean BAFs were lower than the CEPA bioaccumulation criteria, both on a wet weight and lipid weight basis (i.e., 5000 L/kg wet wt., and 100,000 L/kg lipid wt., respectively).

133 DMP BAFs - Wet Weight 1 E+4

1 E+3 TOT C18 FD CEPA 1 E+2 BAF (L/kg wet wt.) wet (L/kg BAF

1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 4 0 0 2 4 5 7 8 0 0 . . . . .4 . . .5 . . . . .4 .5 ...... 0 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 4 4 ( ( ( ( ( ( ( ( ( ( 3 ( ( ( ( 4 ( ( ( ( ( ( n s s r . . e e s ls s h h h h n s le g L o r c c s s e i n M E t a a w m e e m r r i i t p b o i h g g o t f o l a l k l l a s a e e F r r S n s h h n n l s s l c u fi s s A A y P P ta c e i i a n C u C e S C g f f l . . i . . g S . e . M O . r S . r o g g P B G M S P o M G F S D G D o D D

DMP BAFs - Lipid Normalized 1 E+6

1 E+5 TOT 1 E+4 C18 FD CEPA 1 E+3

BAF (L/kg lipid) (L/kg BAF Kow

1 E+2

1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 4 4 0 0 2 4 7 8 0 0 ...... 5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( ( 2 ( ( ( ( 3 3 ( ( 4 ( ( ( ( ( ( ( ( ( r . . n e e s s ls s s h h h h n s e g L o r c c s s e i l n E t a a w m e e m r r i i t p b o i h M g g t f o l a l k l l o a s a e e F r r S n s h h n n l s s l c u fi s s A A y P P ta c e i i a n C u C e S C g f f l . . i . . g S . e . M O . r S . r o g g P B G M S P o M G F S D G D o D D Figure 3.44. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for dimethyl phthalate in False Creek marine biota. The

BAFs are calculated from “total” (▬), “C18” (σ), and “freely dissolved” (○) water concentrations. The CEPA bioaccumulation criterion ( —°― ), and octanol-seawater partition coefficient (⎯) are presented. Error bars represent one standard deviation.

134 DEP BAFs - Wet Weight 1 E+4

1 E+3 TOT C18 1 E+2 FD CEPA BAF(L/kg wet wt.) 1 E+1

1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( r . n e e s ls s h h h h n s le g L o r c c s s e i n M. E t a a w ms e e ms r r i i t p b o i h g g t f l a l k l l o a s a e e F r o r S n s h h n n l s s l c u fi s s A A y P P ta c e i i a n C C e S C g f f l . . . . g S . e . O . r S . r o g g P B G Mi Mu S P o M G F S D G D o D D

DEP BAFs - Lipid Normalized 1 E+6

1 E+5 TOT C18 1 E+4 FD CEPA 1 E+3 Kow BAF (L/kg lipid)

1 E+2

1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( r . n e e s ls s s h h h h n s le g L o r c c s s e i n M. E t a a w ms e e m r r i i t p b o i h g g t f l a l k l l o a s a e e F r o r S n s h h n n l s s l c u fi s s A A y P P ta c e i i a n C C e S C g f f l . . . . g S . e . O . r S . r o g g P B G Mi Mu S P o M G F S D G D o D D Figure 3.45. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for diethyl phthalate in False Creek marine biota. The BAFs are calculated from “total” (▬), “C18” (σ), and “freely dissolved” (○) water concentrations. The CEPA bioaccumulation criterion ( —°― ), and octanol-seawater partition coefficient (⎯) are presented. Error bars represent one standard deviation.

135 3.5.2.2. Intermediate Molecular Weight Phthalate Esters For the mid molecular weight phthalate esters (i.e., di-iso-butyl, di-n-butyl, and butylbenzyl phthalate), the fraction of chemical in the water that was estimated to be freely dissolved was comparable to the C18-bound fraction (which includes both freely dissolved and dissolved organic carbon bound chemical), and ranged from approximately 0.50 for

BBP to 0.75 for DiBP, and DBP. Thus, the observed BAFs differed for the three water concentrations. Specifically, the BAFs based on the “C18” and “freely dissolved” water concentrations were approximately 40 to 50% greater than those based on the “total” water concentration (Figures 3.46, 3.47, and 3.48).

For DiBP and DBP, the mean wet weight BAFs ranged from approximately 29 to

1,100 L/kg, and for BBP they ranged from 187 up to 8,700 (“total”), 17,000 (“C18”), and

43,000 (“freely dissolved”) L/kg wet wt. The lipid normalized BAFs ranged from 1,330 to

107,000 L/kg lipid for DiBP, from 807 to 64,500 L/kg lipid for DBP (excluding the BAFFD for plankton, which was 254,000 L/kg lipid), and from 4,370 up to 1.99 million L/kg lipid for BBP (Tables F.3.20, F.3.21, and F.3.22, Figures 3.46, 3.47, and 3.48).

For both DiBP and DBP, the highest lipid BAFs in the marine species were approximately equal the chemicals’ octanol-seawater partition coefficients (i.e., 37,900).

The majority of BAFs for other species were within an order of magnitude below the KOW, indicating lower than expected concentrations in the organisms, relative to the water. For

BBP, the lipid based BAFs were comparable to the chemical’s KOW (i.e., 106,740), and fell within an order of magnitude above and below the KOW.

The mean wet weight and lipid weight BAFs of DiBP and DBP in the marine organisms fell below the CEPA bioaccumulation criteria, with the exception of plankton in

136 DBP on a lipid weight basis (which was 106,000 and 157,000 L/kg lipid for “total” and

“C18” water concentrations, respectively). However, the upper standard deviations of the lipid BAFs of DiBP and DBP in plankton, brown algae, green algae, geoduck clams, striped seaperch, surf scoters, staghorn sculpin, english sole, and whitespotted greenling exceeded the criteria, indicating that a certain proportion of the individuals in these distributions (i.e., at least 16%) exhibited BAFs that exceeded the guideline (Figures 3.46, and 3.47).

For BBP, there were several species with BAFs that exceeded the CEPA bioaccumulation criteria. On a wet weight basis, the mean BAFs for brown and green algae, spiny dogfish (liver and embryo), dungeness crabs, and surf scoters exceeded the criterion, based on one or more of the water concentrations. As well, the upper standard deviations of the BAFs for all the species except whitespotted greenling exceeded the guideline, again demonstrating that a certain fraction of the individuals in these populations had BAFs greater than 5,000 L/kg wet wt (Figure 3.48). On a lipid basis, BAFs for BBP in plankton, green algae, geoduck clams, striped seaperch, pile perch, pacific staghorn sculpin, and surf scoter exceeded the CEPA criterion based on all three water concentrations (i.e., “total”,

“C18” and “freely dissolved”). The mean BAFs for the all of the other marine species, except forage fish, starfish, and dogfish (liver, muscle, embryo), were greater than 100,000

L/kg lipid, based on at least the “freely dissolved” water concentration (Figure 3.48).

137 DiBP BAFs - Wet Weight 1 E+4

1 E+3 TOT C18 FD CEPA 1 E+2 BAFs (L/kg wet wt.)

1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( r . . n e e s ls s s h h n s le g L o r ch c s sh e i n M E a a ws m e e m r r i i t p b i h g f a lg o a e F r o r So nl s nkt l st la c fi sh sh Al A y P Pe ta e i i a nn C uss C e S C g f f l . i . g Scul . e . O . r S . r o g g P B. G M M S P. o M G F S D G D o D D

DiBP BAFs - Lipid Normalized 1 E+6

1 E+5 TOT C18 FD 1 E+4 CEPA

BAF (L/kg lipid) Kow 1 E+3

1 E+2

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( r . . n e e s ls s s h n s le L o r ch ch s sh e i ng M E a a ws m e m r r i i t p b o i h g g f a a F r o r S nl s nkt l st la c ul fi sh sh Al Al y Pe Pe ta c e i i a nno C usse C e S C g f f l . i g S . e . O . r S . r o g g P B. G M M S. P. o M G F S D G D o D D Figure 3.46. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-iso-butyl phthalate in False Creek Marine Biota.

The BAFs are calculated from “total” (▬), “C18” (σ), and “freely dissolved” (○) water concentrations. The CEPA bioaccumulation criterion ( —°― ), and octanol-seawater partition coefficient (⎯) are presented. Error bars represent one standard deviation.

138 DBP BAFs - Wet Weight 1 E+4

1 E+3 TOT C18 1 E+2 FD CEPA

BAFs (L/kg wet wt.) 1 E+1

1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 ( ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( n s h r . e e s ls r s in s le g L o a a ws ch ch s sh te b n M E. m e e m r r i fi p li h g lg o a s F r a s l st la r So n i sh nkt Al A y Pe Pe a e f ish i a nn C us C e Sco C g f f . i g Scul . e . O . r St r o g g Pl B. G M M S. P. o M G F S. D G D o D D

DBP BAFs - Lipid Normalized 1 E+7

1 E+6

TOT 1 E+5 C18 FD 1 E+4 CEPA BAF (L/kg lipid) Kow

1 E+3

1 E+2

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( n s h r . . e e s ls r s in s le g L o a a ws ch ch s sh te b n M E m e m r r i fi p i h g g a se F r a s h l st la ul r So nl i s nkt Al Al y Pe Pe a c e f ish i a nno C us C e Sco C g f f . i g S . e . O . r St r o g g Pl B. G M M S. P. o M G F S. D G D o D D Figure 3.47. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-n-butyl phthalate in False Creek marine biota. The

139 BBP BAFs - Wet Weight 1 E+6

1 E+5

TOT 1 E+4 C18 FD 1 E+3 CEPA BAF (L/kg wet wt.) wet (L/kg BAF 1 E+2

1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 ( ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( n s h r . e e s ls r s in s le g L o a a ws ch ch s sh te b n M E. m e e m r r i fi p i h g lg o a a F r a s h n l st l r So nl i s nkt Al A y Pe Pe a e f ish i a n C uss C e Sco C g f f . i . g Scul . e . O . r St r o g g Pl B. G M M S. P o M G F S. D G D o D D

BBP BAFs - Lipid Normalized 1 E+7

1 E+6 TOT 1 E+5 C18 FD 1 E+4 CEPA Kow BAF (L/kg lipid) 1 E+3

1 E+2

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( . n e e s s s s h h r n s L . l r c ch s sh e i le E o a a ws r r i i t b ing h M m te m f p g g a a F r a s l s l ul r So nl i sh nkt Al Al y Pe Pe a c e f ish i a nno C usse C e Sco C g f f l . i g S . e . O . r St r o g g P B. G M M S. P. o M G F S. D G D o D D Figure 3.48. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for butylbenzyl phthalate in False Creek Marine Biota.

140 3.5.2.3. Intermediate Molecular Weight Phthalate Ester Isomeric Mixtures For di-iso-hexyl (C6) and di-iso-heptyl (C7) phthalate, the BAFs based on the “total” and “freely dissolved” water concentrations varied by approximately 2 orders of magnitude for C6, and 3 orders of magnitude for C7. Additionally, there was approximately 2 orders of magnitude difference in the BAFs that were calculated from the same water concentration, due to interspecies differences in phthalate ester concentrations.

Wet weight BAFs based on “total” and “C18” water concentrations ranged from 9 to

1,270 L/kg for C6, and from 12 to 7,130 L/kg for C7. Those based on the “freely dissolved” water fraction ranged from 1,560 to 301,000 and from 12,600 to 3.28 million L/kg wet wt. for C6 and C7, respectively (Tables F.3.26, and F.3.27, and Figures 3.49, and 3.50).

The lipid weight BAFs based on the “total” and “C18” water concentrations ranged from 1,080 L/kg up to 161,000 L/kg for C6, and from 1,350 to 287,000 L/kg for C7. The

“freely dissolved” lipid BAFs ranged from 194,000 to 14.0 million for C6, and from 1.38 million to 132 million L/kg for C7. Figures 3.49 and 3.50 reveal that the “freely dissolved” lipid based BAFs are, on the whole, slightly lower than expected based on equilibrium partitioning of the chemical between the organisms and water. Specifically, they range from approximately ½ an order of magnitude above the chemicals’ octanol - seawater partition coefficients to 1 ½ orders of magnitude below KOW, where KOW is ca. 4.88 million (C6) and

27.8 million (C7).

With the exception of the lipid based BAFs in geoduck clams, and surf scoter birds, all of the “total” and “C18” BAFs for C6 were below the CEPA bioaccumulation criteria both on a wet weight and lipid weight basis. Similarly for C7, only the lipid based BAFs for

141 plankton, surf scoters, and staghorn sculpin, and the wet weight BAF for dogfish (liver) exceeded the CEPA bioaccumulation criteria, using the “total” and “C18” water concentrations. However, based on the “freely dissolved” water concentrations, the majority of species exhibit BAFs that exceed the CEPA criteria. The only species with BAFs that did not exceed CEPA criteria were striped seaperch and pile perch for C6 phthalate, determined on a wet weight basis (Figures 3.49, and 3.50).

142 C6 BAFs - Wet Weight 1 E+7

1 E+6

1 E+5 TOT 1 E+4 C18 FD 1 E+3 CEPA

1 E+2 BAFs (L/kg wet wt.)

1 E+1

1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( . n e e s s s s s h h h r n s L . l r ch c s s e i le E to a a w r r i i t b ing M m te m f a l k g a se a F r o r sh h no l s l ulp So n i sh n Alg Al y Pe Pe a e f is i a n C us C e Sc C g f f . i g Sc . e . O . r St r o g g Pl B. G M M S. P. o M G F S. D G D o D D

C6 BAFs - Lipid Normalized 1 E+8

1 E+7

1 E+6 TOT C18 1 E+5 FD CEPA 1 E+4 Kow BAF (L/kg lipid) 1 E+3

1 E+2

1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( . n e e s s s s s h h r n s e L l r ch ch s s e i l E. to a a w r r i i t b ing M m te m f lp a l g g a se a F r o r sh h no l s l u So n i sh nk Al Al y Pe Pe a e f is i a n C us C e Sc C g f f . i g Sc . e . O . r St r o g g Pl B. G M M S. P. o M G F S. D G D o D D Figure 3.49. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-iso-hexyl phthalate in False Creek marine biota.

143 C7 BAFs - Wet Weight 1 E+8

1 E+7

1 E+6 TOT 1 E+5 C18 1 E+4 FD CEPA 1 E+3

BAFs (L/kgwet wt.) 1 E+2

1 E+1

1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 .7 . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( 4 ( ( ( ( ( ( ( ( r . . n e e s ls s s h h n s le g L o r c ch s sh e i n M E a a ws m e e m r r i i t p b i h g f l a lg o a F r r So nl s nkt l st la fi sh sh Al A y Pe Pe ta e i i a nn C uss C e Sco C g f f . i g Scu . e . O . r S r o g g Pl B. G M M S. P. o M G F S. D G D o D D

C7 BAFs - Lipid Normalized 1 E+10 1 E+9 1 E+8 TOT 1 E+7 C18 1 E+6 FD 1 E+5 CEPA Kow BAF (L/kg lipid) 1 E+4 1 E+3 1 E+2 1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 .7 . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( 4 ( ( ( ( ( ( ( ( n s h r s . . e e s ls r s in le L o a a ws ch ch s sh te b ng M E m e m r r i fi p i h g a a e F r a s no l st l ul r So nl i sh nkt Al Alg y Pe P a c e f ish i a n C usse C e Sco C g f f . i . g S . e . O . r St r o g g Pl B. G M M S. P o M G F S. D G D o D D Figure 3.50. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-iso-heptyl phthalate in False Creek marine biota.

144 3.5.2.4. High Molecular Weight Phthalate Esters Due to the high octanol-water partition coefficients of these substances (i.e., di-2- ethylhexyl, di-n-octyl, and di-n-nonyl phthalate), the majority of the chemical in the water phase are associated with particulate matter and dissolved organic carbon, which greatly reduces their bioavailability for uptake via the respiratory surface area. While the observed fractions on the C18 extraction disks (which include freely dissolved and dissolved organic carbon-bound chemical) ranged from 40% for DnNP to 45% for DEHP, the model estimated freely dissolved fractions were only 0.017% for DEHP and DnOP, and 0.002% for DnNP, suggesting that a substantial amount of these chemicals was bound to small diameter particulate matter. As a result, there are large differences between the BAFs based on the

“total”, “C18” and “freely dissolved” water concentrations. Specifically, the BAFs for

DEHP and DnOP varied by 3 orders of magnitude, while those for DnNP varied by 3.5 orders of magnitude.

In addition to the variability in BAFs due to the different water concentrations, there was also substantial interspecies variability in the BAFs. On a lipid weight basis, this resulted in approximately 3 orders of magnitude variability in BAFs that were based on the same water concentration. For both DEHP and DnOP, the wet weight BAFs based on the

“total” and “C18” water concentrations ranged between 5 and 2,560 L/kg wet weight, while those based on the “freely dissolved” fraction ranged from 26,900 to 6.41 million L/kg wet weight. For DNP, the “total” and “C18” BAFs ranged from 1 to 762 L/kg wet weight, and the

“freely dissolved” BAFs ranged from 43,800 to 14.5 million L/kg wet weight (Tables

F.3.23, F.3.24, F.3.25 and Figures 3.51, 3.52, and 3.53).

145 Mean lipid-based BAFs for DEHP were between 202 to 135,000 L/kg (“total” and

“C18”), and 1.17 million to 353 million L/kg based on the “freely dissolved” water concentrations. For DnOP, the lipid based BAFs ranged from 154 to 368,000, L/kg lipid, based on the “total”, “C18”, water concentrations and from 894,000 and 912 million based on the “freely dissolved” water concentration. For DNP, lipid-based BAFs ranged from 21 to

67,700 L/kg lipid for “total” and “C18” water concentrations, and from 993,000 to 1.29 billion L/kg lipid for the “freely dissolved” water concentration (Tables F.3.23, F.3.24, and

F.3.25 and Figures 3.51, 3.52, and 3.53).

For these high molecular weight phthalate esters, the “freely dissolved” lipid-based

BAFs were generally lower than expected based on equilibrium partitioning, indicating lower than expected concentrations in the biota, relative to those in the water. For DEHP and DnOP, the “freely dissolved” lipid-based BAFs range from approximately the chemicals’ octanol-seawater partition coefficients (i.e., ~158 million) for plankton and algae, to 2 orders of magnitude below that, for the higher trophic species. The BAFs based on

“total” and “C-18” water concentrations were 3 to 6 orders of magnitude below the octanol - seawater partition coefficients’ of DEHP and DnOP (Figures 3.51, and 3.52). The pattern was similar for DnNP, where the “freely dissolved” lipid based BAFs range from the

BAF=KOW equilibrium line (i.e., 1.28 billion) to 3 orders of magnitude below that, and the

“total” and “C18” BAFs are 4 to 7 orders of magnitude lower than expected based on equilibrium partitioning (Figure 3.53).

With the exception of the lipid-based BAFs for DEHP in green algae and DEHP and

DnOP in plankton, all of the lipid and wet weight BAFs based on the “total” and “C18” water concentrations fell below the CEPA bioaccumulation criteria for all three chemicals.

146 However, all of the BAFs of these substances based on the “freely dissolved” water concentration exceeded the guidelines, both on a wet weight and lipid weight basis (Figures

3.51, 3.52, and 3.53).

147 DEHP BAFs - Wet Weight 1 E+8

1 E+7

1 E+6

1 E+5 TOT C18 1 E+4 FD 1 E+3 CEPA

BAFs (L/kgwet wt.) 1 E+2

1 E+1

1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 .7 . . . .0 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( . n e e s s s s h h h r n s g L . l r e i le o a a ws c ch is is t b n M E m e e m r r f lp o i h kt lg lg o a a F r o a s l st l r S nl i sh n A A y Pe Pe a e f ish i a nn C uss C e Sc C g f f . . i g Scu . e . O . r St r o g g Pl B G M M S. P. o M G F S. D G D o D D

DEHP BAFs - Lipid Normalized 1 E+10 1 E+9 1 E+8 1 E+7 TOT C18 1 E+6 FD 1 E+5 CEPA

BAF (L/kg lipid) 1 E+4 Kow 1 E+3 1 E+2 1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( r . . n e e s ls s s h h n s le L o r c ch s sh e i b ng M E t a a ws m e m r r i i t p i h g f a a a F r r So nl s nk l st l ul fi sh sh Al Alg y Pe Pe ta c e i i a nno C usse C e Sco C g f f . i g S . e . O . r S . r o g g Pl B. G M M S. P. o M G F S D G D o D D Figure 3.51. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-2-ethylhexyl phthalate in False Creek marine biota. The BAFs are calculated from “total” (▬), “C18” (σ), and “freely dissolved” (○) water concentrations. The CEPA bioaccumulation criterion ( —°― ), and octanol-seawater partition coefficient (⎯) are presented. Error bars represent one standard deviation.

148 DnOP BAFs - Wet Weight 1 E+8

1 E+7

1 E+6 TOT 1 E+5 C18 1 E+4 FD CEPA 1 E+3

BAFs (L/kg wet wt.) 1 E+2

1 E+1

1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( r . . n e e s ls s s h h h n s le g L o r ch c s s e i n M E a a ws e m r r i i t p b i h m e f a g lg o a e F r o r s h n l st la So nl i sh nkt Al A ss y P Pe a e f is i a n C u C e Sc C g f f . i . g Scul . e . O . r St r o g g Pl B. G M M S P. o M G F S. D G D o D D

DnOP BAFs - Lipid Normalized 1 E+10 1 E+9 1 E+8 TOT 1 E+7 C18 1 E+6 FD 1 E+5 CEPA

BAF (L/kg lipid) 1 E+4 Kow 1 E+3 1 E+2 1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( n s h r s . . e e s ls r s in le L o a a ws ch ch s sh te b ng M E m e m r r i fi p i h kt lg o a F r a s l st la ul r So nl i sh n A Alg y Pe Pe a c e f ish i a nn C usse C e Sco C g f f . . i g S . e . O . r St r o g g Pl B G M M S. P. o M G F S. D G D o D D Figure 3.52. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-n-octyl phthalate in False Creek marine biota. The

149 DnNP BAFs - Wet Weight 1 E+9 1 E+8 1 E+7

1 E+6 TOT 1 E+5 C18 1 E+4 FD 1 E+3 CEPA 1 E+2 BAFs (L/kgwet wt.) 1 E+1 1 E+0 1 E-1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( . n e e s s s s s h r n s e L . l r ch ch sh s e i l E o a a w r r i i t b o ing h M m te m f lp kt lg g a a e e F r o a s h h l s l c r S nl i s n A Al sse y P P a e f is i a nno C u C e S C g f f l . . i . . g Scu . e . O . r St . r o g g P B G M M S P o M G F S D G D o D D

DNP BAFs - Lipid Normalized 1 E+11 1 E+10 1 E+9 1 E+8 TOT 1 E+7 C18 FD 1 E+6 CEPA 1 E+5 Kow BAF (L/kg lipid) (L/kg BAF 1 E+4 1 E+3 1 E+2 1 E+1

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 .7 . . . .0 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( . n e e s s s r n s e L ls r ch ch sh sh e i l E. to a a ws r r i i t b ing M m te m f lp a l lg g a se a e e F r o r sh h no l s l c So n i sh nk A Al s y P P a e f is i n C u C e S C g f f . i . . g Scu . e . O . r St r o g g Pla B. G M M S P o M G F S. D G D o D D Figure 3.53. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-n-nonyl phthalate in False Creek marine biota.

150 3.5.2.5. High Molecular Weight Phthalate Ester Isomeric Mixtures For di-iso-octyl (C8), di-iso-nonyl (C9), and di-iso-decyl (C10) phthalate, the BAF results are similar to those for the high molecular weight individual phthalates. Specifically, due to their high octanol-seawater partition coefficients (i.e. log KOW’s of 8.20 for C8, 9.11 for C9, and 10.6 for C10), these substances are mainly associated with large and small diameter particulate matter in the water phase. The observed fractions on the C18 extraction disks (which include freely dissolved and small diameter particulate bound chemical) ranged from 47% for C8 and C9 to 33% for C10, while the model estimated freely dissolved fractions were only 0.017% for C8, 0.002% for C9, and 7.7 · 10-5 % for C10. As a result, there is a large difference between the “total” and “freely dissolved” water concentrations for these substances. Consequently, the corresponding “total” and “freely dissolved” BAFs differed by approximately 3.8, 4.7, and 6.1 orders of magnitude for C8, C9, and C10, respectively. In addition to variability due to the water concentration, differences in concentrations between the marine species resulted in approximately 2.0 (C8), 1.1 (C9), and

3.5 (C10) orders of magnitude variability in the BAFs that were based on the same water concentration.

For C8 to C10 inclusive, the wet weight BAFs based on the “total” and “C18” water concentrations ranged between 9 and 2,740 L/kg wet weight. “Freely dissolved” wet weight

BAFs ranged from 66,700 to 4.13 million L/kg for C8; 353,000 to 36.5 million L/kg for C9; and 13.9 million to 1.20 billion L/kg for C10 (Tables F.3.28, F.3.29, and F.3.30).

The “total” and “C18” lipid-based BAFs for C8, C9, and C10 varied from 59 to

536,000 L/kg. The “freely dissolved” lipid based BAFs were between 344,000 and 605

151 million L/kg for C8; 135 million and 5.71 billion L/kg for C9; and 97.1 million and 236 billion L/kg for C10 (Tables F.3.28, F.3.29, and F.3.30).

For these high molecular weight isomeric mixtures, the “freely dissolved” lipid- based BAFs were slightly lower than expected from equilibrium partitioning; generally falling one order of magnitude below the chemicals’ octanol - seawater partition coefficients. Figures 3.54, 3.55, and 3.56 also indicate an apparent decline in the BAFs with increasing trophic position in the food chain.

A comparison of the wet weight bioaccumulation factors to the CEPA guideline of

5000 L/kg wet wt. reveals that, for C8, C9, and C10, all of the “total” and “C18” based BAFs are lower than the criteria, while all of the BAFs based on the “freely dissolved” water concentration exceed the criteria (Figures 3.54, 3.55, and 3.56). This is generally true for the lipid based BAFs, where all of the “freely dissolved” BAFs exceeded the 100,000 L/kg lipid wt. CEPA guideline. However, a few of the “total” and/or “C18” lipid based BAFs also exceeded the criteria (i.e., C8 in plankton, C9 in plankton, green algae, blue mussels and geoduck clams, and C10 in plankton, green algae, and striped seaperch) (Figures 3.54, 3.55, and 3.56).

152 C8 BAFs - Wet Weight 1 E+8

1 E+7

1 E+6 TOT 1 E+5 C18 1 E+4 FD 1 E+3 CEPA

BAFs (L/kg wetwt.) 1 E+2

1 E+1

1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( r . n e e s ls s s h n s le L o r ch ch sh s e i ng M E. a a ws m e m r r i i t b o i h g g t f a l kt l l a e e F r r S n s h h n no la sse s l fi s s A A y P P ta e i i a n C u C e Sco C g f f . i g Sculp . e . O . r S r o g g Pl B. G M M S. P. o M G F S. D G D o D D

C8 BAFs - Lipid Normalized 1 E+10 1 E+9 1 E+8 1 E+7 TOT 1 E+6 C18 1 E+5 FD CEPA 1 E+4

BAF (L/kg lipid) Kow 1 E+3 1 E+2 1 E+1 1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 .7 . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( 4 ( ( ( ( ( ( ( ( n s h r s . . e e s ls r s in le g L o a a ws ch ch s sh te b n M E m e m r r i fi p i h g lg a e F r a s h l st la ul r So nl i s nkt Al A y P Pe a c e f ish i a nno C usse C e Sco C g f f . i . g S . e . O . r St r o g g Pl B. G M M S P. o M G F S. D G D o D D Figure 3.54. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-iso-octyl (C8) phthalate in False Creek marine biota. The BAFs are calculated from “total” (▬), “C18” (σ), and “freely dissolved” (○) water concentrations. The CEPA bioaccumulation criterion ( —°― ), and octanol-seawater partition coefficient (⎯) are presented. Error bars represent one standard deviation.

153 C9 BAFs - Wet Weight 1 E+9 1 E+8 1 E+7 1 E+6 TOT 1 E+5 C18 1 E+4 FD 1 E+3 CEPA

BAFs (L/kg wet wt.) 1 E+2 1 E+1 1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( 3 3 ( ( ( 4 ( ( ( ( ( ( ( ( ( r . n e e s ls s s h h n s le g L o r c ch s sh e i n M E. a a ws e r r i i t p b i h m e m f a g lg o a a F r o r s n l st l So nl i sh nkt Al A y Pe Pe a e f ish i a n C uss C e Sc C g f f . i g Scul . e . O . r St r o g g Pl B. G M M S. P. o M G F S. D G D o D D

C9 BAFs - Lipid Normalized 1 E+11

1 E+10

1 E+09 TOT 1 E+08 C18 1 E+07 FD CEPA 1 E+06

BAF (L/kg lipid) (L/kg BAF Kow 1 E+05

1 E+04

1 E+03

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 .7 . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( 4 ( ( ( ( ( ( ( ( n s s h r s . . e e s ls r s in le L o a a w ch ch s sh te b ng M E t m e m r r i fi p i h g o a se a F r a s l st l ul r So nl i sh nk Al Alg y Pe Pe a c e f ish i a nn C us C e Sco C g f f . i g S . e . O . r St r o g g Pl B. G M M S. P. o M G F S. D G D o D D Figure 3.55. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-iso-nonyl (C9) phthalate in False Creek marine biota. The BAFs are calculated from “total” (▬), “C18” (σ), and “freely dissolved” (○) water concentrations. The CEPA bioaccumulation criterion ( —°― ), and octanol-seawater partition coefficient (⎯) are presented. Error bars represent one standard deviation.

154 C10 BAFs - Wet Weight 1 E+10 1 E+9 1 E+8 1 E+7 TOT 1 E+6 C18 1 E+5 FD 1 E+4 CEPA 1 E+3 BAFs (L/kg wet wt.) wet (L/kg BAFs 1 E+2 1 E+1 1 E+0

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 7 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 . . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( ( 4 ( ( ( ( ( ( ( n s r s . . e e s ls r s in le g L o a a ws ch ch sh sh te b n M E m e e m r r i fi o i g lg o a s e F r a sh l st la r S nl i sh nkt Al A y Pe P a e f ish i nn C us C e Sco C g f f . i . g Sculp . e . O . r St r o g g Pla B. G M M S. P o M G F S. D G D o D D

C10 BAFs - Lipid Normalized 1 E+12 1 E+11 1 E+10 1 E+09 TOT 1 E+08 C18 1 E+07 FD 1 E+06 CEPA Kow BAF (L/kg lipid) 1 E+05 1 E+04 1 E+03 1 E+02 1 E+01

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 7 0 0 0 3 4 0 0 2 4 8 0 0 . . . . .4 .4 . .5 . . . . .4 .5 .5 .7 . . . .0 1 1 1 2 2 3 3 3 3 3 3 3 4 4 ( ( ( ( 2 2 ( 2 ( ( ( ( 3 3 ( ( ( 4 ( ( ( ( ( ( ( ( n s h r s . . e e s ls r s in le L o a a ws ch ch s sh te b ng M E m e m r r i fi p o i h g g a se e F r a s l st la ul r S nl i sh nkt Al Al y Pe P a c e f ish i a nno C us C e Sco C g f f . i . g S . e . O . r St r o g g Pl B. G M M S. P o M G F S. D G D o D D Figure 3.56. Bioaccumulation factors expressed on a wet weight (L/kg wet wt.) (top), and lipid weight (L/kg lipid wt.) (bottom) basis for di-iso-decyl (C10) phthalate in False Creek marine biota. The BAFs are calculated from “total” (▬), “C18” (σ), and “freely dissolved” (○) water concentrations. The CEPA bioaccumulation criterion ( —°― ), and octanol-seawater partition coefficient (⎯) are presented. Error bars represent one standard deviation.

155 3.5.3. Chemical Distribution in the Food Chain The lipid based BAFs determined from the “total” water concentration are plotted as a function of trophic position for all phthalate esters in Figures 3.57 (individual phthalate esters), and 3.58 (isomeric mixtures). Results of the linear regression between the lipid – based BAFs and trophic position for each phthalate ester are presented in Table 3.31, where

“p” values indicate whether the slope of the regression is statistically significantly different from zero.

Figures 3.57 and 3.58 and Table 3.31 reveal that the lipid-based BAFs exhibit the same patterns as those in the fugacity plots. Specifically, the BAFs do not increase or decrease in a statistically significant manner throughout the food chain for the low and mid molecular weight phthalates (i.e., DMP, DEP, DiBP, DBP, BBP, C6, and C7). However, for phthalates with carbon chains greater than or equal to eight (i.e., DEHP, DnOP, DnNP, C8,

C9, and C10), there was a statistically significant decline in the lipid normalized BAF with increasing trophic position.

Table 3.31. Statistical Results of Regression: Log BAF (L/kg lipid wt.) versus Trophic Position PE Kow n “b” “m” p value for R2 y-intercept Slope slope DMP 1.80 18 4.30 -0.12 0.26 0.077 DEP 2.77 18 3.14 0.04 0.74 0.007 DiBP 4.58 18 4.23 -0.09 0.32 0.062 DBP 4.58 18 4.25 -0.15 0.22 0.093 BBP 5.03 18 5.10 -0.11 0.38 0.049 DEHP* 8.20 18 4.93 -0.46 2.27E-03* 0.451 DnOP* 8.20 16 5.02 -0.56 3.72E-03* 0.463 DnNP* 9.11 14 4.27 -0.45 4.15E-02* 0.303 C6 6.69 17 3.87 -0.01 0.96 1.60E-04 C7 7.44 15 4.17 -0.04 0.82 0.004 C8* 8.20 18 4.71 -0.30 3.08E-02* 0.260 C9* 9.11 13 5.25 -0.34 1.69E-02* 0.418 C10* 10.5 16 4.86 -0.35 3.98E-02* 0.268 * p < 0.05

156 6

DMP 4 DEP DiBP DBP 3 Linear (DMP) Linear (DEP)

Log BAF (L/kg lipid) (L/kg Log BAF Linear (DiBP) 2 Linear (DBP)

1 012345 Trophic Position

BBP DEHP 4 DnOP DnNP Linear (BBP) 3 Linear (DEHP) Linear (DnOP) Log BAF (L/kg lipid) 2 Linear (DnNP)

1 012345 Trophic Position Figure 3.57. Lipid based Bioaccumulation Factors (L/kg lipid wt.) plotted as logarithms versus trophic position for individual phthalate esters (DMP, DEP, DiBP, and DBP (top), BBP, DEHP, DnOP, and DnNP (bottom)) in marine biota from False Creek Harbour.

157 6

5 C6 C7 4 C8 C9 C10 3 Linear (C6) Linear (C7)

Log BAF (L/kg lipid) (L/kg Log BAF Linear (C8) 2 Linear (C9) Linear (C10)

1 012345 Trophic Position Figure 3.58. Lipid Based Bioaccumulation Factors (L/kg lipid wt.) plotted as logarithms Versus Trophic Position for Phthalate Ester Isomeric Mixtures (C6, C7, C8, C9, and C10) in Marine Biota from False Creek Harbour.

3.5.4. Relationship between the Lipid BAFs, based on the “Total” water concentration, and the Octanol – Seawater Partition Coefficient The lipid-based BAFs, based on the “Total” water concentration, are plotted as a function of KOW in Figures 3.59a and 3.59b for all of the marine species included in the study. Figure 3.59 (“total water”) reveals that there is substantial variability in the BAFs between the various marine species. This variability may in part be due to the observed trophic dilution in the food chain, where the fugacities, or lipid normalized chemical concentrations, of the high KOW phthalates were highest for the plankton and algae species, and lowest in the large predatory fish species (e.g., spiny dogfish).

158 Figure 3.59 also reveals that the BAFs of the low molecular weight phthalate esters

(DMP, and DEP) are greater than expected from equilibrium partitioning of the chemical between the organisms and the water (i.e., the BAFLipid = KOW line). This result may be due to the sediment - water disequilibrium that was present in the system, where the organisms are being exposed to high chemical fugacities in the sediments, and relatively low fugacities in the water. Thus, the organisms achieve steady state concentrations in between those in the two surrounding media. The lipid normalized BAFs of the mid molecular weight phthalates (i.e., dibutyl and butylbenzyl) are approximately equal to the values expected from equilibrium partitioning of the chemical between the organisms and water, while those for the higher molecular weight phthalates fall below the equilibrium line.

With the exception of DMP, the BAFs tend to increase with increasing KOW up to

BBP, and then either decline, or remain relatively constant for the higher KOW substances

(i.e., exhibiting either parabolic or logarithmic curve patterns). The magnitude of this relationship varies for the different organisms. For example, plankton exhibits the highest

BAFs, which increase with increasing KOW initially, up to a value of 200,000 L/kg lipid or for BBP; the levels then remain around 100,000 L/kg lipid for the higher KOW phthalates.

Similarly, for green algae, the BAF increases up to a maximum value of approximately

100,000 L/kg lipid for BBP; the BAFs then remain relatively constant (above 10,000 L/kg) for the higher molecular weight phthalates. For geoduck clams, a similar relationship is observed, but the BAFs are approximately 10 fold lower than those in green algae. The maximum BAF value reaches approximately 100,000 L/kg lipid for BBP; levels then slightly decline in the higher KOW substances (i.e., ca. 30,000 - 40,000 L/kg lipid). The lipid- based BAFs for the dungeness crabs increase with increasing KOW to approximately 30,000

159 L/kg lipid for BBP; BAFs then significantly drop off to approximately 1,000 L/kg lipid for the high molecular weight phthalates. For the fish species, the initial increase in the BAF values with increasing Kow is followed by a subsequent decline for the higher molecular weight phthalates. In the fish, the lipid BAFs are highest for the smaller forage fish species

(e.g., striped seaperch maximum BAF was 230,000 L/kg lipid), followed by the larger fish species (e.g., whitespotted greenling maximum BAF was 40,000 L/kg Lipid), and lowest for the top-predator (i.e., spiny dogfish maximum BAF was 15,000 L/kg lipid). In the surf scoter marine bird species, the lipid BAFs increase up to a value of 400,000 L/kg lipid for

BBP. The BAFs then drop off for the higher molecular weight phthalates, particularly for the C8, and C9 substances.

Figure 3.59b illustrates that the lipid normalized BAFs determined from the “total” water concentration, generally, do not exceed the CEPA bioaccumulation criteria, although the BAFs of several of the high KOW phthalates in plankton exceed the criteria. The other exception is butylbenzyl phthalate, where the BAFs for approximately one-third of the species exceed the criteria.

160 1 E+11

1 E+10

1 E+09 Plankton 1 E+08 Algae Benthos 1 E+07 Bird Small Fish 1 E+06 Large Fish Kow 1 E+05 CEPA BAF (L/kg lipid)

1 E+04

1 E+03

1 E+02

1 E+01 1E+01 1E+03 1E+05 1E+07 1E+09 1E+11 Kow Figure 3.59a. Lipid Normalized Bioaccumulation Factors, Based on “Total” Water Concentrations, of Phthalate Esters in Marine Biota from False Creek Harbour Versus the Octanol - Seawater Partition Coefficient. The CEPA Criteria (⎯) and BAFLipid = KOW line (▬) are presented.

1 E+06

1 E+05

Plankton 1 E+04 Algae Benthos Bird Small Fish 1 E+03 BAF (L/kg lipid) Large Fish CEPA

1 E+02

1 E+01 1E+01 1E+03 1E+05 1E+07 1E+09 1E+11 Kow

Figure 3.59b. Lipid Normalized Bioaccumulation Factors, Based on “Total” Water Concentrations, of Phthalate Esters in Marine Biota from False Creek Harbour Versus the Octanol - Seawater Partition Coefficient. The CEPA Criteria (⎯) is presented.

3.5.5. Relationship between the Lipid BAFs, based on the “Freely Dissolved” water concentration, and the Octanol – Seawater Partition Coefficient Figure 3.60 illustrates the lipid BAFs, determined from the freely dissolved water concentration, as a function of the octanol-seawater partition coefficient, and indicates that the lipid BAFs generally follow the “BAFLipid = KOW” line. Comparing Figures 3.59 and

3.60 reveals that the BAFs of DMP and DEP remain unchanged by the water concentration, since most of the chemical is in the freely dissolved form. The BAFs for the mid molecular weight phthalates increase slightly (i.e., by a factor of two), since approximately 50% of the chemical is in the freely dissolved form. The water concentration used in the BAF calculation has the greatest effect on the BAF values of the high molecular weight phthalates, indicated by the contrast between the “total water” BAFs and the “freely dissolved water” BAFs, since only a minute fraction of the chemical is estimated to be in the freely dissolved phase (i.e., ≤ 0.017%).

Similar to Figure 3.59 (total water concentrations), the lipid normalized BAF values for DMP and DEP were generally greater than their octanol - seawater partition coefficients, those for DiBP, DBP, BBP, C6, and C7 were approximately equal to KOW, while those for the C8 to C10 phthalates were generally lower than expected from equilibrium partitioning.

However, in contrast to the Figure 3.59 (BAFs determined from “total” water concentrations), the maximum “freely dissolved” BAFs of the high molecular weight phthalates approximately equal or exceed equilibrium levels. Again, for these high KOW substances, the highest BAFs occurred in plankton and algae, and the lowest occurred in the higher trophic organisms, and these differences are likely due to the occurrence of trophic dilution in food chain for these substances. The observation that the “freely dissolved” lipid

163 BAFs of these high molecular weight phthalates generally did not exceed the equilibrium partitioning line (i.e., BAFLipid = KOW) except in plankton and algae, gives evidence that these chemicals are not biomagnifying in the food chain. Therefore, although the lipid normalized BAFs of some of the intermediate and, in particular, the high molecular weight phthalates exceed the CEPA bioaccumulation criteria (Figure 3.60), this is really only due to the low bioavailability of the substances, and not due to biomagnification in the food chain.

164 1E+12

1E+11

1E+10

1E+09 Plankton Algae 1E+08 Benthos 1E+07 Bird Small Fish 1E+06 Large Fish

BAF (L/kg lipid) (L/kg BAF 1E+05 Kow CEPA 1E+04

1E+03

1E+02

1E+01 1E+01 1E+03 1E+05 1E+07 1E+09 1E+11 Kow

Figure 3.60. Lipid normalized Bioaccumulation Factors, based on “Freely Dissolved” water concentrations, of phthalate esters in marine biota from False Creek Harbour versus the octanol - seawater partition coefficient. The CEPA criteria (⎯) and BAFLipid = KOW line (▬) are presented.

3.6. Biota - Sediment Distribution Of Phthalate Esters

3.6.1. Overview Biota - sediment accumulation factors are reported for all thirteen phthalate esters in

Tables F.3.32 to F.3.34 of Appendix F. The BSAF (kg OC/kg lipid) is the ratio between the chemical concentration in an organism to that in the sediments (Equation 3.12).

BSAF = Clipid / Csediment (3.12)

Where “Clipid” (μg PE/kg lipid) is the lipid normalized phthalate ester concentration in the organism, and “Csediment” (μg PE/kg OC) is the organic carbon normalized concentration in the sediments. Similar to the BAFs, the BSAFs were calculated from the mean logarithmic concentration values in the organism and sediments, and then converted back to the original units (see Equation 3.11). For species which exhibited spatial differences phthalate ester concentrations (e.g., plankton, green algae, geoduck clams, and pacific oysters), station – specific BSAFs were derived and are presented.

3.6.2. Biota - Sediment Accumulation Factors (BSAFs) The BSAFs are presented in Figures 3.61 to 3.67 for all phthalate esters. A BSAF value of unity suggests that equilibrium partitioning of the chemical between the sediments and the organism is occurring, assuming that organic carbon and lipid have equal sorbing capacities for the substance. The BSAFs of all of the phthalate esters were generally less than unity, which indicates that the chemical concentrations in the organic carbon compartment of the sediments were greater than those in the lipid tissue of the organisms.

For DMP, the BSAFs were relatively low and ranged from 0.003 to 0.1 kg OC/kg lipid

(Figure 3.61, Table F.3.32). The BSAFs for DEP, DiBP, DBP, BBP, C6, and C7 were

166 similar and ranged from 0.01 to over 1 kg OC/ kg lipid (Figures 3.61, 3.62, 3.63, and 3.65, and Tables F.3.32, F.3.33, and F.3.34). The absolute values of the BSAFs for the higher molecular weight phthalates (i.e., DEHP, DnOP, DnNP, C8, C9, and C10), were relatively low and ranged from 0.0008 to just less than 1 kg OC/ kg lipid (Figures 3.63, 3.64, 3.66, and

3.67, and Tables F.3.33, and F.3.34).

In terms of patterns of the BSAFs throughout the food chain, the BSAF values appeared relatively constant throughout the food chain for the low and mid molecular weight phthalates (i.e., DMP, DEP, DiBP, DBP, BBP, C6, and C7). However, a declining pattern in the BSAFs with increasing trophic level is apparent for the higher molecular weight phthalates (i.e., DEHP, DnOP, DnNP, C8, C9, and C10), which is consistent with the trends in fugacity throughout the food chain.

167 DMP BSAFs 1

0.1

0.01

0.001 BSAF (kg OC/kg lipid) OC/kg (kg BSAF

0.0001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3) 5 5 5) 7 9) 1 5 4 1 7 7 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 07 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

e e s s s s s h h h r n e g L . . l e l on a a w e ch c s is t pi bs n M E t g g m er m r r i f l a o li h k l l o a s t a e e F r o r s h n l s l c u S n i h n A A P P a c C e f s is n C us y C e t S g i f la . . i . . . e f . O . g S . S r o g g P B G M M S P r D o M G F S G D o D D

10 DEP BSAFs

0.1

0.01 BSAF(kg OC/kg lipid)

0.001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0) 3) 0 8 8 5 5) 7 9 1) 5 4 1 7 7 7 0 00 0 3 4 4 4 53 0 0 25 4 4 5 5 7 8 0 0 0 ...... 1 1. 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

n e e s s h r n . . s l s s h h h i s le g L o a a r c c s s e n E t w m e e m r i i t p o i h M g g o t f o l ab k l l a s a e er F r r S s h h n l s s l c u nl i n A A P P a c C e f s is a n C u y C e t S i f l . . i . . g S . e . O . r S . r og gf g P B G M M S P D o M G F S G D o D D

Figure 3.61. Biota - Sediment Accumulation Factors (kg OC / kg lipid) of dimethyl phthalate (top), and diethyl phthalate (bottom) in marine biota from False Creek Harbour. Error bars represent one standard deviation.

168 DIBP BSAFs 10

0.1 BSAF (kg OC/kg lipid) OC/kg (kg BSAF

0.01

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0) 0 3 0 8 8 3 5 5 5 7 9 1 5 4 1 7 7 0 0 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 07 ...... 1 1 1 2 2 2 2 2. 3 3 3 3 3 3 3 3 3. 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

e s r . . n e s s l s s h h h h in s le g L o a a c c s s e b n E t w m e er m r r i i t p o i h M g g o t f o l a l k l l a s a e e F r u r S s h h n l s s l c n i n A A P P a c C e f s is n C u y C e t S g i f la . . i . . . e . O . g S . S r o gf g P B G M M S P r D o M G F S G D o D D

DBP BSAFs 100

0.1

BSAF (kg OC/kg lipid) OC/kg (kg BSAF 0.01

0.001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 0 3 0 8 8 3) 5 5 5 7 9) 5 4 1 7 7 7 0 0 0 3 4 4 4 5 0 0 2 4 4 51 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

e s r n . . n e s s l s s h h h h i s le g L o a a r c c s s e b n E t w m e e m r r i i t p o i h M g g o t f o l a l k l l a s a e e F r u r S s h h n l s s l a c n i s n A A P P t c C e f s i a n C u y C e S g i f l . . i . . S . e f . O . g S . r o g g P B G M M S P r D o M G F S G D o D D Figure 3.62. Biota - Sediment Accumulation Factors (kg OC / kg lipid) of di-iso-butyl phthalate (top), and di-n-butyl phthalate (bottom) in marine biota from False Creek Harbour. Error bars represent one standard deviation.

169 BBP BSAFs 10

0.1

0.01 BSAF(kg OC/kg lipid)

0.001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 3 0 8 8 3) 5 5 5) 7 9) 1) 5 4 1) 7 7 7 0 00 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3. 3 3 3 3. 3 4 4. 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

e e s s h r n . . s l s s h h i s le g L on a a r c ch s s e b n E t w m e e m r r i i t p o i h M g g o t f o l a l k l l a s a e e F r r S s h h l s s l c u n i n A A P P a c C e f s is a nn C u y C e t S g i f l . . i . . . e f . O . g S . S r o g P B G M M S P r D og M G F S G D o D D

DEHP BSAFs 10

0.1

0.01

0.001 BSAF (kgOC/kg lipid) 0.0001

0.00001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 3 0 8 8 3 5 5 5 7 1) 5 4 1 7 7 7 0 00 0 3 4 4 4 5 0 0 2 4 49 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4. 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

n e e s s h h r n . . s l s s h h s le g L o a a c c s s e n E w m e er m r i i t pi o i h M g g o t f o l ab l kt l l a s a er e F r u r S s h h l s l a c n i s n A A P P t c C e f s i a nn C us y C S g fi f l . . i . . ge S . e . O . r S . r o g g P B G M M S P D o M G F S G D o D D Figure 3.63. Biota - Sediment Accumulation Factors (kg OC / kg lipid) of butylbenzyl phthalate (top), and di(2-ethylhexyl) phthalate (bottom) in marine biota from False Creek Harbour. Error bars represent one standard deviation.

170 DnOP BSAFs 10

0.1

0.01

BSAF (kg OC/kg lipid) OC/kg (kg BSAF 0.001

0.0001

) ) 7) 00) 00) 00) 33 48) 05) 05) 51) 55) . 40 48) 53) 25) 47) 49) 74) 81) 07) 07) 0 1. 1 1. 2. 2. 2. 2. 2. 3. 3. 3. 3. 3. 3. 3. 3. 3. 4. 4. 4. ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

s s s s s n s e L . . er E on ch ch sh ish pi ng M gae gae er i f l ab ol i l l am t am er er F r sh l l cot u S i A A P P ar c C enl sh ish nnow C ussel ys C t S i lankt . . i . . . e . O . ge S . S r ogf P B G M M S P r D ogf M G F S G D ogf D D

DNP BSAFs 100

0.1

0.01 BSAF (kgOC/kg lipid)

0.001

0.0001

) ) 3) 5) 9) 1) 00 00) 00) 33) 40 48) 48) 5 05) 05) 2 47) 4 51) 55) 74) 8 07) 07) 07) . . 1. 1. 1. 2. 2. 2. 2. 2. 3. 3. 3. 3 3. 3. 3. 3 3. 4. 4. 4. ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

s s s s s n e L . . er E on ae ch ch sh ish t pi ng M g gae er i f abs ol i l l am am er er F r sh l l co S i A A P P ar cul C enl sh ish nnow C ussel yst C t S i lankt . . i . . . e . O . ge S . S r ogf gf P B G M M S P r D ogf M G F S G D o D D Figure 3.64. Biota - Sediment Accumulation Factors (kg OC / kg lipid) of di-n-octyl phthalate (top), and di-n-nonyl phthalate (bottom) in marine biota from False Creek Harbour. Error bars represent one standard deviation.

171 C6 BSAFs 100

0.1

BSAF (kg OC/kg lipid) OC/kg (kg BSAF 0.01

0.001

) 7) 00) 00) 00) 33) 40 48) 48) 53) 05) 05) 25) 47) 49) 51) 55) 74) 81) 07) 07) 0 1. 1. 1. 2. 2. 2. 2. 2. 3. 3. 3. 3. 3. 3. 3. 3. 3. 4. 4. 4. ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

e s s s s s r n e L . . e l E on ch ch sh ish pi ng M ga gae er i f l abs o i l l am t am er er F r sh now l s l cot S i A A P P ar cu C enl sh ish n C ussel y C e t S i lankt . . i . . . e . O . g S . S r ogf P B G M M S P r D ogf M G F S G D ogf D D

C7 BSAFs 100

0.1

BSAF (kg OC/kg lipid) OC/kg (kg BSAF 0.01

0.001

) 3) 5) 9) 1) 00) 00) 00) 33) 48) 48) 05 05) 47) 51) 55) 07) 40) 5 2 . 4 74) 8 07) 07) 1. 1. 1. 2. 2. 2. 2. 2. 3. 3. 3. 3 3. 3. 3. 3. 3. 4. 4. 4. ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

s s s s s n e L . . er i E on ch ch sh ish t p ng M gae gae er i f abs ol i l l am am er er F r sh l st l co S i A A P P ar cul C sh ish nnow C ussel y C t S i lankt . . i . . . eenl . O . ge S . S r ogf gf P B G M M S P r D o M G F S G D ogf D D Figure 3.65. Biota - Sediment Accumulation Factors (kg OC / kg lipid) of di-iso-hexyl (C6) phthalate (top), and di-iso-heptyl (C7) phthalate (bottom) in Marine Biota from False Creek Harbour. Error bars represent one standard deviation.

172 C8 BSAFs 10

0.1

0.01

0.001 BSAF (kgOC/kg lipid) 0.0001

0.00001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 3 0 8 8 3) 5 5 5) 7 9) 1 5 4 1 7 7 0 00 0 3 4 4 4 5 0 0 2 4 4 5 5 7 8 0 0 07 ...... 1 1 1 2 2 2 2 2 3 3 3 3. 3 3 3 3 3 4 4 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

n e e s s h r n . . s l s s h h i le g L o a a r c ch s s e bs n E t w m e e m r r i i t p o i h M g g o t f o l a l k l l a s a e e F r u r S s h h n l s s l a c n i s n A A P P t c C e s i a n C u y C e S gf fi f l . . i . . g S . e . O . r S . r o g g P B G M M S P D o M G F S G D o D D

C9 BSAFs 10

0.1

0.01 BSAF (kg OC/kg lipid) OC/kg (kg BSAF

0.001

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 0 0 3 0 8 8 3 5 5 5 7 1 5 4 1 7 7 7 0 00 0 3 4 4 4 5 0 0 2 4 49 5 5 7 8 0 0 0 ...... 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4. 4 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (

n e e s s s s s h h h h r n s e g L . . l e l o a a w e c c s is t pi n M E g g m er m i f l ab o li h kt l l o a s t a er er F r o r s h l s l c u S n i h n A A P P a c C e s is a nn C us y C t S gf i f l . . i . . ge S . e . O . r S . r o gf g P B G M M S P D o M G F S G D o D D Figure 3.66. Biota - Sediment Accumulation Factors (kg OC / kg lipid) of di-iso-octyl (C8) phthalate (top), and di-iso-nonyl (C9) phthalate (bottom) in marine biota from False Creek Harbour. Error bars represent one standard deviation.

173 C10 BSAFs 10

0.1

0.01

BSAF (kg OC/kg lipid) OC/kg (kg BSAF 0.001

0.0001

) ) ) ) ) ) ) ) ) ) ) ) 0) 5 7) 9 5 4) 1 7) 7 7) 00 0 00 33 40) 48) 48) 53 05 05 2 4 4 51 5 7 8 0 0 0 ...... 1 1 1 2. 2. 3. 3. 3 3. 3 4 4. ( ( ( ( 2. ( 2. 2. ( ( 3 ( 3 ( ( 3 3 4 ( ( ( ( ( ( ( ( ( ( . n e s s s s s h n e L . l r er i l E o ae a w e ch ch sh is p bs ng M t g g m e r r i f a o i h l l a t am e e F ot r s h l s l c ul S i A A P P ar c C enl f sh is nno C uss y C e t S g i f lank . . i . . S . e . O . g S . r o gf g P B G M M S P r D o M G F S G D o D D Figure 3.67. Biota - Sediment Accumulation Factors (kg OC / kg lipid) of di-iso-decyl (C10) phthalate in marine biota from False Creek Harbour. Error bars represent one standard deviation.

3.6.3. Relationship Between the BSAF in Benthic Species and the Octanol-Seawater Partition Coefficient Biota - sediment accumulation factors in the benthic marine species from False

Creek Harbour are plotted as a function of the octanol - seawater partition coefficient for all phthalates esters in Figure 3.68. The figure demonstrates that, even for these benthic species, there is significant variability in the BSAFs between the various species. In general, the BSAFs are highest for the burrowing geoduck clams, followed by the other bivalves

(i.e., blue mussels and pacific oysters); values are lowest for the dungeness crab and common seastar, an epibenthic species. Differences in the BSAFs between species may, to some degree, reflect differences in habitat usage between the species. As presented in

174 Section 3.3, we observed a sediment-water disequilibrium in the system, particularly for the low KOW substances, where chemical fugacities in the sediments were greater than those in the water. Thus, benthic organisms, such as the geoduck clam, that are closely tied to the sediments and it’s associated pore water, are likely being exposed to higher chemical levels in the sediments (relative to those in the water), resulting in greater internal body residues.

For the higher molecular weight phthalate esters, there were greater differences in the

BSAFs between species. These differences may be partly due to the observed trophic dilution, where the fugacities, or lipid-normalized concentrations, decrease at higher levels in the food chain (i.e., dungeness crab, and seastar). Higher order species such as the crabs, may have a more developed enzymatic system relative to the clams, and thus may be better able to metabolize these chemicals. It is also possible that the diet is a relatively more important exposure route in the crab and seastar than in the filter feeding bivalves.

Therefore, if these chemicals are being metabolized in the gastrointestinal tract of the organisms prior to assimilation, then the crab / seastar would uptake lower levels of the chemical.

Additionally, the BSAFs tend to exhibit a parabolic trend with KOW. In general, the

BSAFs are lowest for DMP, they increase for the mid molecular weight phthalates, and then tend to drop off for the high molecular weight phthalates. The BSAFs in the geoduck clams are greatest for C6 phthalate and then appear to decrease slightly for the larger molecular weight substances (i.e., BSAFs are lowest for the C8 and C10 phthalates, although those for

C9 approach unity). This pattern is the same for the BSAFs in mussels and oysters, although the absolute values are about 3 times lower (or 0.5 orders of magnitude lower).

175 Again, the same pattern is observed in the crabs and starfish; however, the C8 and C10

BSAFs drop off more significantly in these organisms.

With the exception of C6 phthalate in geoduck clams and blue mussels, all BSAFs are less than unity. Thus, while the BSAFs approach this line of equilibrium, they tend not to exceed it, indicating that biomagnification is not occurring.

176 0.5

-0.5 G. Clams Mussels Oysters -1.5 Starfish D. Crabs BSAF=1

-2.5 Log BSAF (kg OC / kg lipid) / OC (kg Log BSAF

-3.5 1357911 Log Kow

Figure 3.68 Biota - Sediment Accumulation Factors (kg OC / kg lipid) on a Logarithmic Scale versus Log Octanol - Seawater Partition Coefficients for Phthalate Esters in Benthic Marine Biota from False Creek Harbour.

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APPENDIX A

BACKGROUND INFORMATION ON PHTHALATE ESTERS

195

APPENDIX B

TROPHODYNAMIC INTERACTIONS AND LIFE HISTORY

INFORMATION ON SELECTED RESIDENT MARINE SPECIES IN

SOUTHWESTERN BRITISH COLUMBIA

210

APPENDIX C

DIETARY MATRIX FOR CALCULATION OF TROPHIC POSITIONS

267

APPENDIX D

QUALITY ASSURANCE AND CONTROL OF DATA (QA/QC)

TABLES AND FIGURES FROM SECTION 2.4

270

APPENDIX E

STATISTICAL ANALYSES ON PHTHALATE ESTER

CONCENTRATION DATA

I) NORMALITY TESTS ON DISTRIBUTIONS OF PHTHALATE ESTER CONCENTRATION DATA

II) STATISTICAL TESTS ON THE SPATIAL DISTRIBUTION OF PHTHALATE ESTERS IN FALSE CREEK HARBOUR

III) STATISTICAL TESTS ON THE DISTRIBUTION OF PHTHALATE ESTERS IN THE ENVIRONMENTAL MEDIA AND SPECIES OF FALSE CREEK HARBOUR

287

APPENDIX F

DATA TABLES FROM SECTION 3 (RESULTS & DISCUSSION)

I) MEAN PHTHALATE ESTER CONCENTRATIONS AND FUGACITIES IN SEDIMENT, SEAWATER AND BIOTA FROM FALSE CREEK HARBOUR

II) COMPARISON OF REPORTED PHTHALATE ESTER CONCENTRATIONS IN VARIOUS LOCATIONS THROUGHOUT THE WORLD TO OBSERVED CONCENTRATIONS IN FALSE CREEK HARBOUR

III) MEAN BIOACCUMULATION FACTORS

IV) MEAN BIOTA-SEDIMENT ACCUMULATION FACTORS

310

APPENDIX G

ORIGINAL RAW DATA OF PHTHALATE ESTER

CONCENTRATIONS IN SEDIMENT, SEAWATER AND MARINE

BIOTA SAMPLES FROM FALSE CREEK HARBOUR

349