UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effects of pulp mill effluent on marine mussels in an on-site, flow-through bioassay Kinnee, Karen Judith 2005

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2005-0506.pdf [ 6.56MB ]
Metadata
JSON: 831-1.0092115.json
JSON-LD: 831-1.0092115-ld.json
RDF/XML (Pretty): 831-1.0092115-rdf.xml
RDF/JSON: 831-1.0092115-rdf.json
Turtle: 831-1.0092115-turtle.txt
N-Triples: 831-1.0092115-rdf-ntriples.txt
Original Record: 831-1.0092115-source.json
Full Text
831-1.0092115-fulltext.txt
Citation
831-1.0092115.ris

Full Text

EFFECTS OF PULP MILL EFFLUENT ON MARINE MUSSELS IN AN ON-SITE, FLOW-THROUGH BIO ASSAY by K A R E N JUDITH KINNEE B.Sc. (Hons.), Simon Fraser University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Resource Management and Environmental Studies THE UNIVERSITY OF BRITISH COLUMBIA May 2005 © Karen Judith Kinnee 2005 ABSTRACT The potential effect of pulp mill effluent on the survival, growth, and condition index of marine mussels (Mytilus edulis) was investigated. Mussels were exposed to five environmentally relevant concentrations (0.23, 0.46, 1.01, 2.07, and 4.88% v/v) of pulp mill effluent diluted with ambient seawater, and a seawater control for 89 d. This study was conducted on-site at the Norske Canada pulp and paper mill in Campbell River, BC, to satisfy provincial biological monitoring requirements. Mussels were sorted into 1-mm size classes and distributed into individual cages, made of oyster netting. Whole wet weight and length were measured at experimental initiation and termination. At test initiation, 400 mussels were sacrificed; tissue and shell weights were measured to determine baseline conditions. Dissolved oxygen, temperature, pH, salinity, and effluent flow rates were monitored during the experiment. At test termination, tissue and shell weights were measured; and condition index, defined as the ratio of wet tissue weight to shell weight, was calculated. In addition, mussel tissue was analysed for resin acids, lipids, and moisture. Statistically significant reductions in survival were observed in the 0.46 to 4.88% v/v concentrations, as compared to the control, and an LC20 of 3.80% v/v, with 95% confidence intervals of 2.09 to >4.88% v/v, was calculated. No significant reductions in growth, based on changes in length and whole wet weight, and condition index were observed. Condition indices and tissue lipid concentrations of the mussels declined significantly over the exposure, as compared to the T = 0 group. In addition, the mussels exposed to 1.01 to 4.88% v/v effluent had significantly decreased lipid concentrations as compared to the control. Only dehydroabietic acid was detected in the mussel tissues and concentrations of <0.2 to 0.15 |ig/g (wet weight basis) ii were measured. This pulp mill effluent discharge may have an adverse effects on the long-term survival of mussels, if they are continually exposed to 0.5 % v/v effluent. It is not expected that reduced lipid content in native mussels in Discovery Passage would be observed, as effluent concentrations exceeding 1% unlikely to occur outside of a 250 m radius of the effluent discharge. iii TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures viii List of Abbreviations and Acronyms x Acknowledgements xii Dedication xiii Chapter 1 General Introduction 1 1.1 Study Background 1 1.2 Pulp Mills and Their Effluent 2 1.2.1 Distribution and Types of Pulp Mills in Canada 2 1.2.2 Chemical Components of Pulp Mill Effluent 3 1.2.3 Toxic Effects of Pulp Mill Effluent 9 1.3 Mill History 13 1.3.1 Operations and Production 13 1.3.2 Pulping Processes 14 1.3.3 Bleaching Processes 16 1.3.4 Effluent Treatment System 17 1.3.5 Effluent Discharge 18 1.3.6 Effluent Dispersion 18 1.3.7 Effluent Characteristics 19 1.4 Mussels 20 1.4.1 Use of Mussels to Monitor Effects of Environmental Contaminants 20 1.4.2 Mussels and Pulp Mill Effluent 24 1.4.3 Life History 32 1.5 Study Objective for Mussel Study 38 1.5.1 Study Objective 38 1.5.2 Rationale for Study Design 38 1.5.3 Null Hypotheses 40 Chapter 2 Materials and Methods 41 2.1 Study Design 41 2.2 Experimental Set-Up 42 2.3 Seawater 44 2.4 Effluent 45 2.5 Test Vessels 45 2.6 Bag Construction 45 2.7 Mussel Source 48 iv 2.8 Mussel Sorting and Distribution 49 2.9 Flow Rates 50 2.10 Study Initiation 50 2.11 Daily Measurements and Maintenance 51 2.12 Baseline Condition of Mussels 52 2.13 Study Termination, Observations, and Measurements 52 2.14 Lipid and Moisture Analyses 54 2.15 Resin Acid Tissue Concentrations 54 2.16 Calculations and Statistical Analyses 55 Chapter 3 Results 58 3.1 Effluent Characteristics 58 3.2 Seawater and Effluent Flow Rates 58 3.3 Water Quality Measurements 59 3.3.1 Dissolved Oxygen 59 3.3.2 Temperature 60 3.3.3 pH 61 3.3.4 Salinity 61 3.4 Mussel Data 62 3.4.1 Initial Lengths and Weights 62 3.4.2 Survival / Mortality : : : 63 3.4.3 Growth 65 3.4.4 Condition Index 68 3.5 Lipids and Moisture 70 3.5.1 Lipids 70 3.5.2 Moisture 72 3.6 Resin Acid Tissue Concentrations 73 3.7 Quality Assurance/Quality Control 74 3.7.1 Mussel Measurements 74 3.7.2 Instrument Calibration 75 Chapter 4 Discussion 76 4.1 Water Quality 76 4.1.1 Dissolved Oxygen 76 4.1.2 Temperature 77 4.1.3 pH 77 4.1.4 Salinity 77 4.1.5 Trends in Water Quality Measurements 78 4.2 Survival / Mortality 78 4.3 Growth '. 80 4.4 Condition Index and Lipid Concentrations 81 4.5 Resin Acid Tissue Concentrations 83 4.6 Improvements to Study Design 84 4.7 Conclusions 85 Literature Cited 87 Appendices 100 Appendix A - Summary of Mussel Survival and Mortality 101 Appendix B - Statistical Calculations 104 v LIST OF TABLES Table 1-1 Fatty Acids Present in Pulp and Paper Mill Effluent (McLeay and Associates Ltd. 1987) 4 Table 1-2 Resin Acids Present in Pulp and Paper Mill Effluent (McLeay and Associates Ltd. 1987) 5 Table 1-3 Concentrations of Pulp Mill Effluent Constituents in Effluent, Receiving Water, and Sediment 6 Table 1-4 Bioconcentration Factors for Mussels of Pulp and Paper Mill Effluent Constituents 12 Table 1-5 Description of the Bleaching Stages for Semi-Bleached Pulp and Fully-Bleached Pulp Produced at Norske Canada, Elk Falls Division, Campbell River, BC (J. Kauffman, NorskeCanada, pers. comm.) 17 Table 1-6 Summary of Norske Canada, Elk Falls Division Final Effluent Measurements - June 9 to September 7, 1999:. 19 Table 1-7 Summary of Norske Canada, Elk Falls Division EEM Toxicity Testing -Cycle II (Hatfield Consultants Ltd. 2000) 20 Table 1-8 Effects of Pulp and Paper Mill Effluent and/or its Components on Marine Mussels 25 Table 1-9 Effects of Pulp and Paper Mill Effluent and/or its Components on Freshwater Mussels 26 Table 3-1 Total Flow Rates (L/hour) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 59 Table 3-2 Mean Effluent Concentrations (% v/v) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 59 Table 3-3 Dissolved Oxygen (DO) Concentrations (mg/L) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (n = 56) 60 Table 3-4 Dissolved Oxygen (DO) Concentrations (% Saturation) during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (n = 56) 60 Table 3-5 Test Temperatures (°C) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (n = 56) 61 vi Table 3-6 pH Measurements during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (n = 55) 61 Table 3-7 Salinity Measurements (%o) during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (n = 56) 62 Table 3-8 Mussel Lengths at Test Initiation of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 62 Table 3-9 Mussel Weights at Test Initiation of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 63 Table 3-10 Summary of Survival and Mortality of Mussels at Test Termination of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 64 Table 3-11 89-d LC20 and LC25 Values, 95% Confidence Intervals and NOEC and LOEC for the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 64 Table 3-12 Summary of Growth in Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 66 Table 3-13 Summary of Condition Index of Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 69 Table 3-14 Mean Lipid and Moisture Concentrations in Mussel Tissues including Baseline Mussels (T = 0), from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 71 Table 3-15 Resin Acid Concentrations in Mussel Tissues (ug/g wet weight basis), from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 74 Table 3-16 Re-Measured Mussel Data (% Differences) for Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 75 vii LIST OF FIGURES Figure 1-1 Location of Norske Canada, Elk Falls Division Pulp and Paper Mill, Campbell River, British Columbia, Canada (http://srmwww.gov.bc.ca/ bmgs/2mil/bcmap.gif) 14 Figure 2-1 Schematic of Seawater and Effluent Flow Routes and Arrangement of Treatments, Used in the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 43 Figure 2-2 Experimental Set-Up of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 44 Figure 2-3 Experimental Set-Up Showing Seawater Headtank and Hoses Attached from Seawater Manifold to the Mixing Buckets for 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 46 Figure 2-4 Barrel and Peristaltic Pumps Used to Hold and Pump Effluent for the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 47 Figure 2-5 Test Exposure Barrels, Mixing Buckets, and Cross-Bars Used for Mussel Bag Attachment in the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 48 Figure 2-6 Mussels Being Placed into Oyster Netting Bags for the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 51 Figure 3-1 Mortality of Mussels Exposed from the 89 d, June to September 1999 of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC (mean ± SEM; see Table 3-10 for n) (Hatched bars indicate a statistically significant difference as compared to the 0% v/v (Z = 3.10 (0.46% v/v) to 7.43 (4.88% v/v; p < 0.05)) 65 Figure 3-2 Growth, Based on Change in Length, in Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (mean ± SEM; see Table 3-12 for n) Hatched bars indicate a statistically significant difference as compared to 0% v/v (F5, | 8 6 9 = 7.49, p < 0.0001; Bonferroni's testp< 0.05) 67 viii Figure 3-3 Growth, Based on Change in Whole Weight, in Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (mean ± SEM, see Table 3-12 for n) Hatched bars indicate a statistically significant difference as compared to 0% v/v (F5, 1869 = 8.42 p < 0.0001; Bonferroni's;? < 0.05) 68 Figure 3-4 Mussel Condition Index of Mussels Exposed from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (mean ± SEM; see Table 3-13 for n) Hatched bars indicate a statistically significant difference as, compared to 0% v/v (Bonferroni's test, p < 0.05) Asterisks indicate a statistically significant difference as compared to the T = 0 (Bonferroni's test, p < 0.05) 70 Figure 3-5 Mean Lipid Concentrations in Mussel Tissues, including Baseline Mussels (T = 0) from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC,June to September 1999 (% Wet Weight) (mean ± SEM, n = 4) Hatched bars indicate a statistically significant difference from 0% v/v (F$, is = 26.89; LSD test p < 0.05) Asterisks indicate a statistically significant difference from T = 0 (F 6 , 2 i = 146.71; LSD test p < 0.05) 72 Figure 3-6 Mean Moisture Concentrations in Mussel Tissues, including Baseline Mussels (T = 0), from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 (mean ± SEM, n = 4) Asterisks indicate a statistically significant difference from T = 0 (F62i -28.69; LSD test p < 0.05) 73 ix LIST OF ABBREVIATIONS AND A C R O N Y M S A E C Adenylate Energy Charge A N O V A Analysis of Variance AOX Adsorbable Organic Halides BCF Bioconcentration Factor BOD Biochemical Oxygen Demand C4-PCPH Polychlororetenes CEPA Canadian Environmental Protection Act CG-3 4,5,6-Trichloroguaiacol COD Chemical Oxygen Demand DO Dissolved Oxygen EC25 25% Effective Concentration EC50 Median effective concentration E E M Environmental Effects Monitoring EOX Extractable Organic Halogens EROD 7-Ethoxyresorufin-O-Deethylase HMW High Molecular Weight KSL Kolmogorov-Smirnoff-Lillifors IC25 25% Inhibition Concentration LC20/25 20% / 25% Lethal Concentration LC50 Median Lethal Concentration L M W Low Molecular Weight LOEC Lowest Observable Effect Concentration LSD Least Significant Difference MFO Mixed Function Oxygenase uS Microsiemens NADPH Nicotinamide Adenine Dinucleotide Phosphate NOEC No Observable Effect Concentration PAH Polycyclic Aromatic Hydrocarbons PBB Polybrominated Biphenyls x PCB Polychlorinated Biphenyls PCP Pentachlorophenol PCT Polychlorinated terphenyls PCYMD Polychlorocymenenes PVC Polyvinyl Chloride RA Resin Acids SD Standard Deviation SEM Standard Error of the Mean SPF Spruce, Pine, and Interior Douglas Fir t 1 / 2 Half-Life T = 0 Mussels that were sacrificed at beginning of study TBT Tributyl Tin TCDD 2,3,7,8-Tetrachlorodibenzo-p-Dioxin TCDF 2,3,7,8-Tetrachlorodibenzofuran TMP Thermomechanical Pulp TSS Total Suspended Solids UDP-GT Uridine diphosphate glucuronosyltransferase UNOX Union Carbide Oxygen; a process for pure oxygen bio-oxidation of industrial waste water WLAP Ministry of Water, Land and Air Protection % v/v Percent volume per volume xi A C K N O W L E D G E M E N T S 1 would like to thank my committee members, Drs. Ken Hall, Les Lavkulich, and Sheldon Duff, for all of their support and assistance with my research and writing my thesis. I would also like to recognise the support of the staff at Vizon SciTec Inc. (formerly BC Research Inc.), especially, Dr. Carol Ann McDevitt, Lorrie Hunt, and Susan Ewing, who were instrumental in their help with the experimental design and set-up, and for measuring the mussels used in this study. I would also like to thank Dr. Tony Kozak for his advice on the statistical analysis of my data. My sincere gratitude goes to Martin Davies and Dr. Wayne Dwernychuk of Hatfield Consultants, Ltd., for providing valuable comments on my thesis. I am indebted to Chuck Easton, Sarah Peplow, and the Steam Recovery Plant Employees at Elk Falls, who were invaluable in the maintenance of the experiment. I would also like to thank the many people that were involved in take-down of the experiment: Ruth Fraser and the students of Lord Byng Secondary School; Rosalind Kellett and the students of University Hill Secondary School; Chris Lowe, Hatfield Consultants, Ltd.; and Laura Kitchen, Heather Ewing, Trevor Davies, and Shelly Lybeck. The loan of equipment from the UBC Soil Science Laboratory, Dr. Francis Law, Simon Fraser University, and Silvagen Inc. was most appreciated. Finally, I would like to thank my parents, Denver and Susan Kinnee, my sister, Debra Dennehy, and my fiance, Curtis Eickhoff, for their many years of love and support during my university career. xii DEDICATION This thesis is dedicated to Curtis Eickhoff, whose love, support, and unending encouragement, made the completion of this thesis possible. x i i i CHAPTER 1 G E N E R A L INTRODUCTION 1.1 Study Background Pulp mill effluent is a mixture of dissolved organic and inorganic compounds in solution and suspension, including solids, that enters the aquatic environment through the discharge of effluent from pulp and paper mills. Therefore, organisms in aquatic environments near pulp mill effluent discharges are exposed to effluent components, which have the potential to affect their health. Effects can be related to the chemical constituents of the effluent, from the physical effects of the solids and/or from the oxygen demand of the effluent. The effects of pulp mill effluent in the receiving environment can be monitored using a variety of techniques and studies, such as benthic invertebrate and fish population surveys; toxicity testing of effluent; and determining tissue concentrations of pulp mill effluent components in exposed organisms. The discharge of pulp mill effluent, in Canada, is regulated by the Pulp and Paper Regulations (1992) under the Fisheries Act (Environment Canada 2003). These regulations limit the total suspended solids and biochemical oxygen demand concentrations of the effluent, require that the 96-hour LC50 using rainbow trout is >100% v/v effluent and an Environmental Effects Monitoring (EEM) program be conducted (Environment Canada 2003). This program evaluates the long-term effects of pulp and paper mill effluent discharges on the aquatic environments. In addition, a second purpose of this program was to determine whether the improvements in effluent quality through process changes (e.g. reduction in the use of elemental chlorine for bleaching) and the introduction of secondary effluent treatment resulted in an improvement in the receiving environments of pulp and paper mills (Environment Canada 2003). 1 Pulp mills are also regulated under the Canadian Environmental Protection Act (CEPA) that requires dioxins and furans concentrations be undetectable in effluents from mills who use chlorine or chlorine dioxide to bleach pulp; controls of quality of defoamers used in bleaching processes; and prohibits the use of polychlorinated phenol treated wood chips (Environment Canada 2003). Mussels are common inhabitants of coastal areas, which may be near pulp mill effluent discharges, and have been used extensively to monitor pollution. Mussels are also an important fisheries resources and food source for humans. In addition, mussels were used in this study as they were identified as a sentinel species in the Environment Canada's E E M Program Pre-Design Reference Document (Hatfield Consultants Ltd. 1994). The objective of this study was to determine the effects of pulp mill effluent on the survival, growth, and condition index of the marine, blue mussel, Mytilus edulis. This study was conducted on-site at Norske Canada (formerly Fletcher Challenge Canada Ltd.), Elk Falls Division in Campbell River, BC, to satisfy provincial biological monitoring requirements. An in-situ test or a wild mussel survey were not feasible at the study site given logistic and safety issues associated with the rapid currents in the receiving environment near the mill. 1.2 Pulp Mills and Their Effluent 1.2.1 Distribution and Types of Pulp Mills in Canada There are over 120 pulp and paper mills in Canada (Environment Canada website), with twenty-three mills in British Columbia (BC WLAP website). Of the twenty-three mills in BC, there are nine mills which discharge effluent to the marine environment (BC WLAP website). Pulp and paper mills typically manufacture products from wood, recycled paper, and non-wood fibres (Environment Canada 2003). There are three major processes for producing pulp: chemical 2 (e.g. kraft, sulphite), mechanical, and semi-chemical (Environment Canada 2003). Products manufactured from kraft pulp are writing paper, paper bags, and cardboard (Environment Canada 2003). Mechanical pulp is used to make newsprint and catalogue paper, while semi-chemical pulp is used to produce corrugated cardboard (Environment Canada 2003). Pulp mill effluent is typically treated using a minimum of secondary treatment (Environment Canada 2003). Typically, secondary treatment involves the use of aerated stabilisation basins or activated sludge (Environment Canada 2003). However, there are a few mills with tertiary effluent treatment (Environment Canada 2003). 1.2.2 Chemical Components of Pulp Mi l l Effluent Major constituents of pulp and paper effluent include organic acids, sulphur-containing compounds, chlorinated phenolics, chlorinated neutral compounds, and chlorinated organic acids (McLeay and Associates Ltd. 1987, Owens 1991, LaFleur 1996). Toxicity of pulp and paper mill effluents is due to resin and fatty acids, chlorinated phenols, lignin degradation products, and many neutral compounds (Leach and Thakore 1977, Walden and Howard 1977, McLeay and Associates Ltd. 1987). Volatile compounds, such as sulphides, mercaptans, chlorine and sulphur dioxide can also contribute to the toxicity of pulp mill effluent (Leach and Thakore 1977). Physical factors (e.g. pH, colour, and suspended solids) and biochemical oxygen demand (BOD) may also be implicated in pulp mill effluent toxicity (Leach and Thakore 1977, Owens 1991). The distribution of toxic compounds present in pulp mill effluent is dependent upon the tree species which are used, and the pulping, washing, and bleaching process (Leach and Thakore 1977, Carlberg and Stuthridge 1996). For example, pine species have higher resin acid concentrations than do fir species; and chlorinated organic compounds only occur in effluents where chlorine is used in the bleaching process (Leach and Thakore 1977). 3 In mills that use elemental chlorine and/or chlorine dioxide for pulp bleaching, common toxic chemicals in the effluent include chlorinated organics, such as resin acids (e.g. mono- and dichloro-dehydroabietic acid), guaiacols (e.g. trichloro- and tetrachloro-guaiacol) and fatty acids (e.g. 9,10-dichlorostearic acid) (Leach and Thakore 1977, LaFleur 1996). In addition, during the bleaching process, chlorinated organic compounds, such as chlorinated phenolics, adsorbable organic halides (AOX), dioxins, and furans are produced (Muir and Servos 1996). The source, toxicity, environmental concentrations, fate, and degradation of the five major toxic classes of compounds found in pulp mill effluent, are discussed in the following sections. 1.2.2.1 Resin and Fatty Acids Resin acids are carboxylic acids and have lipophilic properties (Morales et al. 1992). Resin and fatty acids are found in parenchyma cells of coniferous trees (Mahood and Rogers 1975), originate from the pulping of coniferous trees and are often recovered in crude tall oil (Holmbom 1977, Morales et al. 1992). Tall oil, also known as liquid rosin, is a mixture of rosins, fatty acids, sterols, high-molecular alcohols, and other alkyl chain materials and is used in soaps, lubricants, surfactants, and paper sizings (Smook 1992, Chemical Land 21 website). The most common fatty and resin acids found in pulp and paper effluent are listed in Table 1-1 and Table 1-2. The five most common and abundant resin acids are pimaric, palustric, isopimaric, abietic, and dehydroabietic acids (Leach and Thakore 1976). Table 1-1 Fatty Acids Present in Pulp and Paper M i l l Effluent (McLeay and Associates L td . 1987) Dichlorostearic Acid Linoleic Acid Oleic Acid Epoxystearic Acid Linolenic Acid 4 Table 1-2 Resin Acids Present in Pulp and Paper Mi l l Effluent (McLeay and Associates L td . 1987) Abietic Isopimaric Levopimaric Neoabietic Palustric Chlorodehydroabietic Dehydroabietic Pimaric Sandaracopimaric Dichlorohydroabietic Resin acids can contribute to the overall effluent toxicity (Owens 1991) and prior to secondary treatment of pulp mill effluent, 60 to 90% of the toxicity in the effluent may be due to resin acids (Leach and Thakore 1976). Dehydroabietic acid is the most abundant resin acid and the least toxic (Leach and Thakore 1976, Leppanen et al. 1998). Isopimaric acid is the most toxic non-chlorinated resin acid of the five most common resin acids. (Leach and Thakore 1976, 1977). Environmental concentrations of resin acids can be below detection limits due to secondary biological treatment of the effluent (Leppanen et al. 1998, Table 1-3). Chlorinated resin acids are more persistent and more toxic than unchlorinated versions (McLeay and Associates Ltd. 1987, Li et al. 1996) and are formed during pulp bleaching processes (Li et al. 1996). Resin acids can undergo microbiological degradation, which results in detoxification (Servizi et al. 1986). During microbiological degradation, the resin acids can be hydroxylated, reduced or undergo decarboxylation to form fichtelite, dehydroabetin, and retene (LaFleur 1996). Levopimaric/palustric and dehydroabietic sodium salts were the most readily biodegradable and pimaric and isopimaric sodium salts were the least biodegradable, when exposed to either river water or activated sludge (Hemingway and Greaves 1973). 12,14-dichlorodehydroabietic acid is the most resistant to microbial degradation (Servizi et al. 1986). Hydroxylation also reduces the toxicity of chlorinated resin acids; however the hydroxylated metabolites are more toxic than their non-chlorinated counterparts (Servizi et al. 1986). Secondary biological treatment is effective in reducing non-chlorinated resin acid concentrations and they can be reduced by 47 to 100% (LaFleur 1996). Fatty acid concentrations in effluents are often reduced by 80 to 100% after secondary treatment (LaFleur 1996). However, 5 chlorinated resin and fatty acids can be more difficult to treat (McLeay and Associates Ltd. 1987) and are reduced by 38 to 100% in biological treatment systems (LaFleur 1996). Fatty acids are less toxic to fish than resin acids and are degraded during biological treatment (McLeay and Associates Ltd. 1987). Concentrations of resin and fatty acids have been detected in waters and sediments near pulp and paper mills (McLeay and Associates Ltd. 1987, Lee and Peart 1990 & 1992, Morales et al. 1992, Tavendale et al. 1995, Leppanen and Oikari 1999). A major removal mechanism of resin acids from the water column is binding to suspended and colloidal matter, which may result in a reduction in their microbial degradation (Carlberg and Stuthridge 1996). Resin acid products of microbial degradation are often more hydrophobic than their parent compounds and can accumulate in sediments and fish (Carlberg and Stuthridge 1996). Retene, a resin acid degradation product, has also been detected in receiving waters and sediments near pulp mills and is bioavailable to juvenile rainbow trout (Oikari et al. 2002). Fichtelite, a decarboxylated resin acid, has been detected in pulp mill effluents and in biota near pulp mills (Burggraaf et al. 1996). Table 1-3 Concentrations of Pulp Mi l l Effluent Constituents in Effluent, Receiving Water, and Sediment Chemical Class Effluent Concentration Receiving Water Concentration Sediment Concentration (Hg/g) AOX (mg/L) 0.30-33.1 a A f ' h 0.052-4 mg/L f g 0.4-5270 d , s BOD (mg/L) 4 _ Q _ 7 8 a , b , e , h - -Chlorinated Phenols (ug/L) <0.1 - 125.3 b c J l < 0.001 -0.28 1 0.001 - 1.9 free" 0.2 - 149.0 bound d COD (mg/L) 170-460 a ' e ' h 29.8-47.0 y -Extractable Organic Halogens (EOX) (mg/L) 0.87-0.48 mg/L f < 0.5 -0.05 c ' f < 0.5 -61 c f Resin and Fatty Acids (ug/L) 0.1 -2400 a ' M 0.01 -0.049 c 1 - 1600d TCDD / TCDF (pg/L) < 1.0-31 c h < 1.0-5.0 c N D C " Vertae/a/. 1996; b Halle/ al. 1996; c Swanson et al. 1996; d Carlberg & Stuthridge et al. 1996; e Eklunde/o/. 1996; 'craige/a/. 1990; 8 Hayere/a/. 1996; h Kovacsero/. 1995; ' Robinson et al. 1994; ND = non-detectable. 6 1.2.2.2 Chlorophenols Chlorophenols present in pulp mill effluent, include, as a group, compounds such as chlorophenols, chlorocatechols, and chloroguaiacols. These compounds are formed during lignin degradation and bleaching, where phenol-containing residues produced during the pulping process are chlorinated (McLeay and Associates Ltd. 1987). These compounds can be metabolised to anisoles and veratroles, which have increased toxic and bioaccumulative potential and are more persistent than their parent compounds (Carlberg and Stuthridge 1996). Reductions in chlorophenol concentrations may occur if chlorine dioxide rather than elemental chlorine is used for bleaching (McLeay and Associates Ltd. 1987, Herve et al. 1996). Chlorophenols are more resistant to biological treatment than resin and fatty acids and may be present in untreated and treated effluents (McLeay and Associates Ltd. 1987, Herve et al. 1996, Table 1-4). Chlorophenols have been detected in receiving waters close to and far away from discharges of treated bleached kraft mill effluent (Carlberg and Stuthridge 1996, Herve et al. 1996, Table 1-4). Mussels have been used to monitor environmental concentrations of chlorophenols due to their bioaccumulative nature (Herve et al. 1988, 1996). For example, tetrachloroguaiacol and pentachlorophenol have bioconcentration factors of 74 and 150, respectively (Makela et al. 1991). Chlorophenols have also been detected in sediments near pulp and paper mills (Tavendale etal. 1995, Table 1-4). 1.2.2.3 Neutral Compounds Neutral compounds, such as, chloroform, juvabione, and dichloromethyl methyl sulphone, account for only a small portion of effluent toxicity and there is limited data on their environmental fate and effects (McLeay and Associates Ltd. 1987). These compounds range in toxicity and persistence. For example, chloroform has low toxicity, while juvabione, a neutral balsam fir extractive, undergoes biological degradation, but has high toxicity to fish (96-h LC50 7 is 1.5 ug/L) (McLeay and Associates Ltd. 1987, Kovacs et al. 1997) and can cause induction of MFO activity (Kovacs et al. 1997). Dichloromethyl methyl sulphone is not readily biodegradable and it has low toxicity (96-h LC50 > 10,000 ug/L) (McLeay and Associates Ltd. 1987). 1.2.2.4 Dioxins and Furans Dioxins and furans are formed during the bleaching process when high concentrations of molecular chlorine are present along with dioxin precursors (Owens 1991, Yunker and Cretney 1996). In addition, polychlorinated phenols can be converted to dioxins and furans during pulp digestion (Yunker and Cretney 1996). Dioxin and furans are very toxic and persistent. Effluent concentrations of dioxins and furans have decreased with the reduction of elemental chlorine in the bleaching process (Gifford 1996). In most effluents, dioxin and furan levels are usually at concentrations less than analytical detection limits, which is the requirement under the Canadian Environmental Protection Act (Swanson et al. 1996). 1.2.2.5 Tannins and Lignins Tannins are phenolic acids that are found in the bark of many tree species and protect trees them against microbial infections (Sjostrom 1981). Lignin, a three dimensional polymer, consisting of phenylpropane units, aids in holding together the cellulose fibres in trees (Sjostrom 1981, Smook 1992). High concentrations of lignin are found within the middle lamella of tree cells (Sjostrom 1981, Smook 1992). Lignin derived macromolecules are present in pulp mill effluents and have been shown to prevent fertilisation in echinoderms (Pillai et al. 1997). In addition, tannins and lignins have been shown to cause acute toxicity in rainbow trout (Bailey et al. 1999). 8 1.2.3 Toxic Effects of Pulp Mi l l Effluent The toxicity of pulp mill effluent has been evaluated in many laboratory and field studies, as summarised below. The effects on pulp mill effluent on survival, growth, and reproduction, along with biochemical and physiological effects are discussed. In addition, studies on the bioaccumulation of chemicals present in pulp mill effluent are presented. 1.2.3.1 Survival, Growth and Reproduction Effects Secondary treated pulp mill effluent is typically non-toxic to Daphnia magna and rainbow trout (Kinnee, pers.obs, Priha 1996). Pulp mill effluent is considered to be non-toxic if the 48-h Daphnia and 96-h rainbow trout median lethal concentrations (LC50) are greater than 100% v/v effluent (Government of Canada 1992). Typically, no mortality is observed in acute toxicity tests using these two species (Kinnee, pers.obs.). Sublethal effects of secondary treated pulp mill effluent to freshwater and marine larval fish, invertebrates and algae have been monitored in laboratory bioassays over the last 11 years in Canada through the E E M program (Scroggins et al. 2002) and by various researchers (e.g. Eklund et al. 1996, Hall et al. 1996, Kovacs etal. 1997). Typically, pulp mill effluent has little to no effect on the survival and growth of larval fish, such as fathead minnows and topsmelt, in 7 d tests, as most of the LC50s and EC50s were >100% v/v effluent for fathead minnows and >60% v/v for topsmelt (Scroggins et al. 2002, Hall et al. 1996). For the rainbow trout embryo viability test, 46% of the effluents tested had EC25 of >100% v/v (Scroggins et al. 2002). However, 25% reductions in egg viability were observed at concentrations of 5 to 13% v/v in 18% of the effluents tested (Scroggins et al. 2002). Fathead minnow life cycle tests were conducted using < 24-hour-old fathead minnows that were exposed to pulp mill effluent concentrations of 1.25 to 20% v/v until 3 months after reproduction (Kovacs et al. 1997). Prior to upgrades in process and effluent treatment in one of the mills, reproductive 9 effects, such as reduced egg production and increased number of male fish, were observed in the effluent of one out of two mills (Kovacs et al. 1997). Some of the process changes at this mill were using elemental chlorine free bleaching, increased condensate treatment, and increased efficiency of the steam stripper (Kovacs et al. 1997). Improvements to the effluent treatment system, included increased retention time, lagoon dredging, and elimination of nutrient addition (Kovacs et al. 1997). However, no effects on survival, growth, viable egg production, gonad size and histology, sex ratio, and time to spawn were observed after the mill process and treatment upgrades. Reductions in Ceriodaphnia reproduction were mainly observed at effluent concentrations of 22 to >60% v/v (Scroggins et al. 2002). No effects were observed in 27% of the effluents tested (i.e. IC25 >100% v/v), while 22% of the effluents showed reproduction effects at concentrations of >0.6 to >13% v/v (Scroggins et al. 2002). Similar results in Ceriodaphnia reproduction were also observed by Kovacs et al. (1997), as one mill showed no effects in reproduction to 100% v/v effluent in three tests and an IC25 of 65% v/v, while a second mill had a mean IC25 of 41% v/v effluent. Effects, such a 25% reduction in echinoderm fertilisation, have been observed to occur at concentrations of < 5% v/v effluent (Hall et al. 1996, Scroggins et al. 2002). Most of the mills in the Cycle II of the EEM program reported IC25 between 4.67 to >60% v/v effluent (Scroggins et al. 2002). No effect on Nitocra spinipes (an estuarine invertebrate) reproduction was observed after a 7-d exposure (Eklund et al. 1996). Reductions in cystocarp (a vesicle that contains the reproductive spores) production in a red macroalgae (Ceramium strictum) have been observed and EC50 values after a 2-d exposure to secondary treated pulp mill effluent from a elemental chlorine free process were 26.2% v/v, with 95% confidence intervals of 23.0 - 29.2% v/v (Eklund et al. 1996). Effects (e.g. a 25% reduction in the number of cystocarps, as compared to the control) in the reproduction of Champia parvula, a marine red macroalgae, have been detected at concentrations as low as 0.36% v/v; however, 10 most of the mills tested in the EEM program show effects between 7.78 to 60% v/v effluent (Scroggins et al. 2002). Sandstrom (1996) reviewed Canadian and Swedish studies of pulp mill effluent exposed fish. The effects on fish growth ranged from significant decreases, to no effect, to significant increases. Effects of pulp mill effluent on the condition factor of fish, a weight to length relation, ranged from significant increases to no effects. In addition, exposed fish were observed to reach sexually maturity at a later age. Increases in condition factors in the exposed fish may have been due to altered metabolism (Borton et al. 1996). Changes in bleaching sequence, such as reduction in elemental chlorine use, the increased use of chlorine dioxide, and the addition of secondary biological effluent treatment at pulp and papers mills has resulted in large reductions in effluent toxicity. These changes were made in response to new Pulp and Paper Effluent Regulations that were introduced in 1992. 1.2.3.2 Biochemical/Physiological Effects and Bioaccumulation Exposure of fish to pulp mill effluent can lead to induction of the mixed function oxygenase (MFO) enzymes, such as 7-ethoxyresorufin-O-deethylase (EROD), and Phase II biotransoformation enzymes, such in UDP-glucuronyltranferase (UDP-GT) (Oikari and Kunnamo-Ojala 1987, Hewitt et al. 1996, Hodson 1996, Lehtinen 1996, Priha 1996, Sandstrom 1996, Kovacs et al. 1997). Many compounds in pulp mill effluent, such as chlorophenols, resin and fatty acids and chloroguaiacols, can be detoxified in fish by conjugation to glucuronides and sulphate esters, with subsequent excretion (Oikari et al. 1984a, Muir and Servos 1996). Retene has been shown to induce EROD at a concentration of 1000 ug/g (dry weight) in juvenile rainbow trout (Oikari et al. 2002). Simulated kraft pulp mill effluent caused significant effects in rainbow trout blood, such as decreases in hematocrit and plasma proteins; and increases in leucocrit and plasma ammonia and bilirubin concentrations (Oikari and Nakari 1982, Oikari et al. 11 1984b). Reductions in UDP-GT, liver glycogen and, glucose concentrations have also been observed in rainbow trout (Oikari and Nakari 1982, Oikari et al. 1984b). Resin acids, chlorinated phenolics, extractable organic halogens (EOX), dioxins and furans can bioaccumulate in organisms, such as fish and mussels (Miettineri et al. 1982, Oikari et al. 1982, Suntio et al. 1988, Pellinen et al. 1993, Muir and Servos 1996, Rantio et al. 1996, Tavendale etal. 1996, Leppanen et al. 1998) (Table 1-4). Table 1-4 Bioconcentration Factors for Mussels of Pulp and Paper Mi l l Effluent Constituents Species/Size Chemical Length of Exposure / Units Bioconcentration Factor Mytilus edulis 1 Pentachlorophenol Not listed / 304-370 Size not listed a No units listed Anodonta anatina 1 Trichlorophenol 18 d / 30-125 6 - 10 g soft tissueb Tetrachlorophenol No units listed 76-156 Pentach 1 oropheno 1 81-461 Trichloroguaiacol 103 -237 Tetrachloroguaiacol 45-154 Total chlorophenolics 67-172 Hydriella menziesi / Pimaric Acid 28 d / 2500 ± 200 63.1 ± 2 . 3 mm c Isopimaric Acid L/kg lipid 3200 ± 6 0 0 Dehydroabietic Acid weight 2200 ± 3 0 0 Abietic Acid 6 0 0 0 ± 1000 14-Chloroabietic Acid 2000 ± 500 Fichtelite 89,000 ± 7000 Anodonta cygnea / EOX 91 dl Gills: 1455- 1917 85.4 ± 4.9 mm d No units listed Gonads: 1182 -1292 Digestive Glands: 2375 - 2864 Anodonta piscinalis Polychlorocymenes 4 weeks/ 6000- 166,000 1 Size not listed e Polychlorocymenenes No units listed 1800-49,000 Alkyl(4Q- 1,410,000 polychlorophenathrenes 42,000 - 267,000 Alkyl(5C)polychlorobibenzyIs aGeyere(a/. 1982; b Makela et al. 1991; c Burggraaf etal. 1996; d Hayer and Pihan 1998; e Rantio etal. 1996. 12 1.3 Mill History 1.3.1 Operations and Production The Elk Falls mill, located in Campbell River, BC, opened in 1952 and produced groundwood pulp and paper (Hatfield Consultants Ltd. 1994) (Figure 1-1). At present, the Elk Falls Division produces newsprint, kraft pulp, and linerboard (Hatfield Consultants Ltd. 1994). In 1999, 737,000 t of pulp and paper were produced. Coastal Douglas fir; yellow cedar; hemlock/balsam fir (Hembal); and spruce, pine, and interior Douglas fir (SPF or Whitewood) wood chips are used to produce pulp. Sawdust, containing a variety of tree species, is also used. 13 Figure 1-1 Location of Norske Canada, E lk Falls Division Pulp and Paper M i l l , Campbell River, British Columbia, Canada (http://srmwww.gov.bc.ca/bmgs/ 2mil/bcmap.gif) 1.3.2 Pulping Processes Three separate pulping processes occur at the Elk Falls mill and these produce kraft pulp, thermo-mechanical pulp (TMP), and linerboard. The purpose of chemical pulping is to separate the cellulose (pulp) from the lignin and wood extractives (LaFleur 1996). These processes are described in the sections below. 14 1.3.2.1 The Kraft Process The kraft pulping process is a type of chemical pulping, in which sodium hydroxide (NaOH), sodium sulphide (Na2S), and high temperatures are used to digest the wood chips (Smook 1992, Hatfield Consultants Ltd. 1994). The NaOH and Na2S are chemically recovered after the" digestion process is complete (Smook 1992, Hatfield Consultants Ltd. 1994). The brownstock pulp is washed and stored prior to bleaching. After the chips have been digested and washed, the residual material, known as black liquor, is burned and evaporated to create green liquor, which contains sodium carbonate (Na 2C0 3) and Na2S (Smook 1992). Black liquor also contains chemical residues, dissolved lignin and other wood constituents (McLeay and Associates Ltd. 1987). The black liquor is concentrated and subsequently incinerated to form a liquid smelt, containing inorganic compounds (Smook 1992). The organic portion of the black liquor is combusted to produce steam and electricity. The smelt is dissolved in water to produce green liquor (Na2S and Na 2 C0 3 ) (Smook 1992, Hatfield Consultants Ltd. 1994). The green liquor is combined with reburned lime (CaO) in the recaustising process to regenerate white liquor (NaOH and Na2S04) (Smook 1992). Sulphur-containing gases, turpentine, and tall oil by-products are produced using digestion and recovery (McLeay and Associates Ltd. 1987). 1.3.2.2 The Newsprint Process Thermomechanical pulp is produced from wood chips, using mechanical refiners and screeners (Hatfield Consultants Ltd. 1994). TMP comprises of approximately 25% of the total production of pulp and paper at Elk Falls. Newsprint is made from TMP, de-inked, recycled pulp and up to 10% kraft pulp (Hatfield Consultants Ltd. 1994). Dye, clay and retention aids, along with other chemicals, such as slimicides, roll and felt cleaners, and wire life extenders, may also be added (Hatfield Consultants Ltd. 1994). 15 1.3.2.3 The Linerboard Process Linerboard is made from pulp from a variety of sources: unbleached, semi-, and fully-bleached kraft pulp, TMP, and waste kraft paper (Hatfield Consultants Ltd. 1994). Dye and a variety of additives, such as clay and alum are added prior to producing the final product (Hatfield Consultants Ltd. 1994). 1.3.3 Bleaching Processes 1.3.3.1 The Kraft Process For the kraft process at Elk Falls, there are two types of bleaching plants (Hatfield Consultants Ltd. 1994). Semi-bleached pulp is bleaching using a three stage process, while fully-bleached pulp is whitened using a five stage process (Hatfield Consultants Ltd. 1994). A description of the bleaching stages is listed in Table 1-5. The semi-bleached pulp is first bleached with a chlorine dioxide and the second stage involves alkali extraction using hydrogen peroxide and oxygen (J. Kauffman, NorskeCanada, pers. comm.). Chlorine dioxide is used in the third stage to bleach the pulp. For the fully-bleached pulp, chlorine dioxide is used in the first bleaching step. This stage is followed by alkali extraction using hydrogen peroxide and oxygen (J. Kauffman, NorskeCanada, pers. comm.). The first two bleaching steps are repeated and the final step in the bleaching process uses chlorine dioxide. 16 Table 1-5 Description of the Bleaching Stages for Semi-Bleached Pulp and Fully-Bleached Pulp Produced at Norske Canada, E lk Falls Division, Campbel l River, B C (J. Kauffman, NorskeCanada, pers. comm.) Product Bleaching Sequence Explanation Semi-Bleached Pulp DE o p D D = Chlorine Dioxide (C102) E 0= Alkaline Extraction with 0 2 P = Hydrogen Peroxide Fully-Bleached Pulp D E o p D E o p D D = Chlorine Dioxide (C102) E 0= Alkaline Extraction with 0 2 P = Hydrogen Peroxide 1.3.3.2 The T M P Bleaching Process Newsprint and speciality grades are brightened using sodium hydrosulphite and hydrogen peroxide (Hatfield Consultants Ltd. 1994). 1.3.4 Effluent Treatment System Treatment is used to reduce the toxicity, BOD, and total suspended solid (TSS) of the effluent. Primary treatment consists of clarifiers that allow the suspended solids to settle. Secondary treatment involves biological oxidation to remove BOD and remove potentially acutely toxic compounds from the effluent (Leach and Thakore 1977, Smook 1992, Hatfield Consultants Ltd. 1994), such as resin and fatty acids and chlorinated phenolics (LaFleur 1996). At the Elk Falls mills, there is one treatment train for the kraft effluent and one for the TMP/newsprint effluent. The kraft effluent treatment consists of a primary clarifier, an equalisation basin, UNOX secondary treatment, and a secondary clarifier. The TMP/newsprint effluent is treated similarly, however, there is no equalisation basin for this effluent. UNOX activated sludge secondary treatment systems were installed at the Elk Falls mill in 1991 (Hatfield Consultants Ltd. 1994). The UNOX treatment system uses aerobic micro-organisms and the addition of nitrogen, phosphorus and pure oxygen, to digest the organic matter present in the 17 effluent (Hatfield Consultants Ltd. 1994). After treatment, the effluent is transferred to secondary clarifiers for 5 to 9 hours prior to discharge (Hatfield Consultants Ltd. 1994). 1.3.5 Effluent Discharge The effluents from the kraft mill and the TMP/newsprint operations are combined into one effluent stream after secondary treatment (Hatfield Consultants Ltd. 1994). This combined effluent is discharged into Discovery Passage using a 500 m long submarine diffuser, at water depths of approximately 15 to 40 m. (Hatfield Consultants Ltd. 1994). The effluent discharge rate is approximately 230,000 m3/d. (Hatfield Consultants Ltd. 1994). 1.3.6 Effluent Dispersion There is rapid mixing of the effluent in Discovery Passage due to current and tidal influences (Hatfield Consultants Ltd. 1994). Two dye dispersion injections were conducted to determine the dispersion of the effluent in Discovery Passage. One study was conducted during a small ebb tide, while a second study was conducted two hours after a small flood tide (Hodgins and Knoll 1991). It was found that the effluent was dispersed from Seymour Narrows (north) to Cape Mudge (south) (Hodgins and Knoll 1991). Effluent concentrations of 1% or greater were only found within 100 m of the diffuser and for less than one hour after the dye injection (Hodgins and Knoll 1991). In addition, effluent concentrations considerably below 1% were measured within 2 km of the outfall (Dwernychuk 1990). Therefore, a conservative effluent field of a 250 m radius around the diffuser was designated, in which concentrations of 1% or greater may occur (e.g. within 100 m of diffuser) and a large margin of safety to account for variable effluent flows and oceanographic conditions (Hatfield Consultants Ltd. 1994). 18 1.3.7 Effluent Characteristics Conductivity, pH, temperature, BOD, TSS, and adsorbable organic halides (AOX) of the effluent are monitored at various frequencies (Table 1-6) (Hatfield Consultants Ltd. 1994). Effluent toxicity is measured by using weekly 48-h Daphnia magna and monthly 96-h rainbow trout toxicity testing. No acute toxicity was observed to either of these species during the testing period (unpublished data, BC Research 1999). Typically, pulp mill effluent from BC mills is not acutely toxic to these two species (unpublished data, BC Research 1996 - 2003). Table 1-6 Summary of Norske Canada, E lk Falls Division Final Effluent Measurements - June 9 to September 7,1999 Parameter Frequency of Measurement Units Average SD Min imum Maximum Flow Daily m3/Day 221626 8110 193940 238340 TSS Daily mg/L 30 12 11 80 TSS Daily t/Day 6.8 2.7 2.2 19.1 pH Daily - 6.7 0.2 6.2 7.1 Conductivity Daily LiS/cm 1386 219 1055 2119 Temperature Daily °C 38.8 1.1 36.0 41.0 BOD See Footnote mg/L 16 6 7 30 BOD See Footnote t/Day 3.53 1.35 . 1.52 7.15 AOX Weekly mg/L 2.0 0.5 0.8 2.5 BOD on Final Effluent every other day and on separate streams into treatment plant once/week. The effluent is also monitored for sublethal effects, two times per year, as part of Environment Canada's E E M Program. Three tropic levels of marine organisms are tested and include a fish (Atherinops affinis, topsmelt), an invertebrate (Strongylocentrotus purpuratus, sea urchin; or Dendraster excentricus, sand dollar) and a macroalgae (Champia parvula). The mean . sublethal toxicity of the effluent during 1998 and 1999 ranged from 26.2% v/v effluent for the echinoderm test to >67.3% v/v in the topsmelt test (Table 1-7) (Hatfield Consultants Ltd. 2000). These concentrations are approximately one order of magnitude higher than maximum predicted effluent concentration in the receiving environment. Therefore, it is not expected that the effluent 19 has negative survival, growth and reproduction effects on these species in the receiving environment (Hatfield Consultants Ltd. 2000). Typically, pulp mill effluent has little to no effect on the survival and growth of topsmelt, in 7 d tests (Scroggins et al. 2002). Most of the mills in the Cycle II of the E E M program reported IC25 values between 4.67 to >60% v/v effluent for the 20-min echinoderm fertilisation test and 7.78 to 60% v/v for the 2-d Champia parvula reproduction (Scroggins et al. 2002). Therefore, the sublethal toxicity of the Elk Falls mill effluent is similar to other pulp and paper mills in Canada. Table 1-7 Summary of Norske Canada, E lk Falls Division E E M Toxicity Testing -Cycle II (Hatfield Consultants L td . 2000) Test Organism A. affinis Survival LC50 A. affinis Growth IC25 D. excentricus Fertilisation IC25 C. parvula Reproduction IC25 Summer 1998 Winter 1999 >67a >67 >67 >67 Summer 1999 Geometric Mean >68 >67.3 >68 >67.3 37.04 18.65 25.89 26.2 (35.43 - 38.60)b (16.98-20.62) (23.04-28.23) 12.36 32.38 18.49 19.5 (11.17-13.72) (29.22-35.38) (16.02-20.40) a All units are % v/v effluent; 95% confidence intervals in parentheses. 1.4 Mussels 1.4.1 Use of Mussels to Monitor Effects of Environmental Contaminants Mussels are useful biomonitors because they are sedentary, widely distributed, relatively resistant to pollution, and a potential source of exposure of chemicals to humans through consumption (Cossa et al. 1980, Cossa 1989). Mussels are also euryhaline (i.e. can tolerate a wide range of salinity), can be transplanted, and are easily collected for studies (Davies & Pirie 1978, Cossa et al. 1980, Cossa 1989). Mussels also have sufficient tissue masses for chemical analyses and they bioconcentrate the pollutant of interest at detectable levels (Cossa 1989). In addition, 20 bivalves, such as mussels, clams, and oysters, have been shown to be just as sensitive to a variety of toxicants as some of the most commonly used testing organisms such as Daphnia, fathead minnows, and amphipods (Herve et al, 1988, 1996). Bivalves have been used to monitor exposure to environmental contaminants by collecting native organisms and analysing their tissues for contaminants of concern. Native freshwater and marine bivalves have been used to measure uptake and exposure to petroleum hydrocarbons (Broman and Ganning 1985, Goldberg et al. 1978), pesticides (Metcalfe and Charlton 1990, Hickey et al. 1997), polychlorinated biphenyls (PCB) (Metcalfe and Charlton 1990, Pugsley et al. 1985, Hickey et al. 1997), metals (Davies and Pirie, 1978, Goldberg et al. 1978, Cossa et al. 1980, Gordon et al. 1980, Salanki et al. 2003), organic contaminants (Kauss and Hamdy 1985, Pugsley et al. 1985, Muncaster et al. 1989, Metcalfe and Charlton 1990, Muncaster et al. 1990, Gustavson and Jonsson 1999), industrial effluents (Broman and Ganning 1985), radionuclides (Goldberg etal. 1978), and sewage (Moles and Hale 2003). Native mussels exposed to untreated and treated sewage effluent had decreased aerial exposure survival rates, byssal thread production and greater presence of parasites as compared to mussels collected from unimpacted reference sites (Moles and Hale 2003). Transplanted mussels have also been used to monitor pollution from a variety of point and non-point sources. These studies involve the placement of mussels at various distances from the source of the pollution. The mussels are sampled at various time(s) of exposure and are evaluated for effects and/or their tissues are analysed. Bivalves have also been transplanted into areas where there are pollution gradients from metals (Foster and Bates 1978, De Kock 1983, Mikac et al. 1996, Veldhuizen-Tsoerkan et al. 1991, Salazar and Salazar 1995, Salazar et al. 1995, Grout and Levings 2001), PCB (De Kock 1983, McDowell Capuzzo et al. 1989, Prest et al. 1995, Veldhuizen-Tsoerkan et al. 1991, Peven et al. 1996), organochlorines (Herve et al. 1988, 21 Kauss and Hamdy 1985, Prest et al. 1995, Herve et al. 1995, 1996, Peven et al. 1996, Herve et al. 2001, 2002, St-Jean et al. 2003), power plant discharges (Foe and Knight 1987), chlorinated hydrocarbons (Rantio et al. 1996), tributyl tin (TBT) (Salazar and Salazar 1991, 1995), and hydrocarbons (Peven et al. 1996, Zhou et al. 1996). Herve et al. (1988, 1996, 2001, 2002) determined that mussels were useful for monitoring low concentrations of environmental contaminants and for showing improvements in water quality after pulp mill process changes by analysing pulp and paper mill effluent constituents in mussel tissues (Herve et al. 1988). Increased clam mortality and tissue losses, reduction of condition index (dry tissue weight/shell volume), decreases in oxygen consumption and clearance rate, and increased ammonia production were observed in freshwater clams transplanted near a power plant discharge (Foe and Knight 1987). Mussels were transplanted to several locations with PCB and metals contamination and to a reference site for 2.5 to 5 months (Veldhuizen-Tsoerkan et al. 1991). Mussels exposed from polluted sites had decreased condition index (soft tissue dry weight/soft tissue dry weight + shell weight), anoxic tolerance and survival times, as compared to the mussels from the reference site. In addition, adenylate energy charge (AEC), a measure of cellular energy status, was measured in the mussels from all sites after 2.5 and 5 month exposure periods. There were no differences in A E C in the mussels from the polluted sites, as compared to the reference site mussels. However, the mussels from the polluted sites, when exposed to anoxic conditions, had decreased A E C , as compared to the mussels from the reference site. The synthesis of heat shock proteins, after a heat shock treatment, was also measured. Altered synthesis of heat shock proteins in mussels transplanted to contaminated environments, as compared to the mussels from the reference site were observed (Veldhuizen-Tsoerkan et al. 1991). 22 TBT was shown to reduce growth in mussels (Salazar and Salazar 1991). No effects on mortality, average wet weight, or lipid concentrations in organochlorine-exposed clams were observed (Kauss and Hamdy 1985). Mussels (Quadrula quadruld) were transplanted 0.1 km from the discharge of a copper electroplating plant and after 14 d, the mussels had accumulated a lethal concentration of copper (20.1 ug copper/ g tissue (wet weight)) (Foster and Bates 1978). In addition, 60 and 39% mortality was observed within 5 km and 21 km, respectively, of the discharge location, after a 45-d exposure. MytUus edulis were transplanted to an acid mine drainage gradient and mussel dry weight was reduced in mussels exposed to >20 ug/g copper while survival and condition index (wet tissue weight/shell weight) were reduced in mussels with >40 ixg copper/g dry weight (Grout and Levings 2001). Mussels (juveniles and adults, MytUus trossulus) were transplanted at 12 locations at a Superfund site, where the main contaminants of concern were tributyl tin, copper, lead, and zinc (Salazar et al. 1995). No significant differences in juvenile and adult mussel survival between transplant locations were observed; however, adult survival was significantly lower than juvenile mussel survival. Whole animal wet weight and lengths, and growth rates, based on weight and length, were significantly decreased in mussels after an 82-d exposure at the Superfund site, as opposed to the mussels from the reference site. The uptake, transformation and excretion of mercury by transplanted MytUus galloprovincialis was monitored over a 7 month period (Mikac et al. 1996). A reduction in condition index (tissue wet weight/shell weight) over the study period was observed. The reduction in condition index was a result of the mussels spawning during the study period. Condition index is known to decrease upon spawning (Bayne et al. 1985, Gosling 2003). Total and methylmercury tissue concentrations were well correlated with shell, wet and dry tissue weights of the mussels, that is, as tissue and shell weights increased, mercury concentrations decreased. 23 Mussels have also been used in laboratory experiments to determine the effects of various toxicants such as chlorinated phenolics (Makela et al. 1991), pulp mill effluent constituents (Fahraeus-Van Ree and Payne 1999, McKinney and Wade 1996), PCB (Pruell et al. 1986, Gilek et al. 1996), methyl mercury (Kernaghan et al. 1999a), metals (Latouche and Mix 1982, Kraak et al. 1994, Grout and Levings 2001), pesticides (McLeese et al. 1980, Roberts 1975), polycyclic aromatic hydrocarbons (PAH) (Pruell et al. 1986), contaminated river water (Stuijfzand et al. 1998), and chlorinated nuclear power station cooling water (Lawrence and Nicholson 1998). 1.4.2 M u s s e l s a n d P u l p M i l l E f f l u e n t Several studies have examined the effects of pulp mill effluent on freshwater and marine mussels, using either transplanted mussels (i.e. those placed into the receiving environment near pulp mill outfalls) (Wu and Levings 1980, Burggraaf et al. 1996,Hayer et al. 1996, Hayer.and Pihan 1996, 1998, Salazar 2000, Herve et al. 2001, 2002) or by performing experiments on-site at pulp and paper mills (Kernaghan et al. 1999b) (Table 1-8 and Table 1-9). In addition, mussels have been used in monitoring programs to quantify concentrations of pulp mill effluent in the receiving environment (Herve et al. 1988, Pellinen et al. 1993, Herve et al. 1996, Rantio et al. 1996) (Table 1-8 and Table 1-9). The effects of pulp mill effluent on mussels using laboratory bioassays have also been investigated (Kallqvist et al. 1989, Makela and Oikari 1990, Makela et al. 1991, McKinney and Wade 1996) (Table 1-8 and Table 1-9). Also, one study investigated the cellular effects of a resin acid mixture on marine mussels (Fahraeus-Van Ree and Payne 1999) (Table 1-9). 24 Table 1-8 Effects of Pulp and Paper Mi l l Effluent and/or its Components on Marine Mussels Species / Size Exposure Time / Type Effluent Type Effects Mytilus edulis 1 Maximum 4 months / Untreated bleached Decreased survival and growth adults a field transplanted kraft mill effluent Mytilus edulis 1 5-d, static renewal / Bleached sulphite 5-d EC50 29 mL/L (filtration adultsb lab mill effluent rate) Macoma balthica / Field collected Bleached kraft mill No increase in shell corrosion adults0 effluent Mytilus edulis 1 15-d, static renewal / 22 mg rosin/L Digestive cell degeneration; adultsd lab increased digestive cell lipid storage; increased enzyme activity in digestive glands Mytilus edulis 1 68 d / field Secondary treated Decreased growth, condition adults e transplanted mussels sulphite pulp and paper mill effluent index, and lipids Mytilus edulis / 90 d / field Secondary treated Increased growth; decreased adults f transplanted mussels bleached kraft pulp mill effluent phagocytic activity and lysosome retention; increased mortality in bacterial clearance a Wu and Levings 1980; b Kallqvist et al. 1989;0 Landner et al. 1994; d Fahraeus-Van Ree and Payne 1999; e Salazar 2000;f St. Jean etal. 2003. Wu and Levings (1980) conducted an experiment that investigated the mortality and growth of transplanted mussels near the outfall of a coastal bleach kraft pulp mill in BC over a 4-month period. The mussels that were transplanted near the pulp mill suffered greater mortality than those at the control location did. These mussels also were significantly smaller and had less tissue (based on dry weight) than the mussels placed at the control site. The decreased survival and growth of mussels near the pulp mill may be due to reduced food availability, low dissolved oxygen, low salinity, high turbidity, and/or resin acid exposure. The authors concluded that mussels may be useful in monitoring pollution from pulp mills. 25 Table 1-9 Effects of Pulp and Paper Mi l l Effluent and/or its Components on Freshwater Mussels Species / Size Exposure Time / Type Effluent Type Effects A. piscinalis I adults a A. anatina I adultsb A. anatina I adultsc A. antina I adults c A. anatina I adults c A. imbellicis I juveniles d A. anatina I adultse A. anatina I adultse A. anatina I adultse A. cygnea I adults f A. piscinalis I adults B H. menziesi / adults h A. cygnea I adults 1 E. buckleyi/adults' A. cygnea, U. pictorum, D. polymorpha 1 4 week / field transplanted (1984 - 2000) 24-h static / laboratory 8-d static-renewal / lab 8- d static / lab 4-d flow-through; 72-h depuration / lab 9- d; static renewal / laboratory 6-d; static / lab 7-d; flow-through, 5-d depuration / lab 5 months / field transplanted 28-d / field transplanted 4 weeks / field transplanted Max. 56-d exposure; max. 21-d depuration / field transplanted 91-d / field transplanted 56-d / on-site flow-through system 28-d / field transplanted Secondary treated bleached kraft pulp mill effluent (6 mills) 7 or 14 ttg/L PCP and 23 or 48 ug/L CG-3 Artificial effluent Artificial effluent Artificial effluent Effluent from 6 pulp and paper mills Secondary treated bleached pulp effluent; fractionated into LMW (< 1000) and HMW (> 10,000) Same effluent as above Same effluent from above, without fractionation Chlorine bleached pulp and paper mill effluent Secondary treated softwood bleached pulp mill effluent Kraft pulp and paper mill effluent Chlorine bleached pulp and paper mill effluent 5 concentrations of papermill effluent Chlorine bleached pulp and paper mill effluent Decreases in chlorophenol/guaiacols; constant concentrations of chlorophenol/guaiacol/veratroles BCF in soft tissue for PCP were 145 - 342 and 34 to 125 for CG-3 Chlorophenol uptake rates 33 - 254 ng/g hr"'; temperature independent bioaccumulation at steady state Total chlorophenol BCF highest at 8°C (105) and lowest at 18°C (67) Chlorophenol BCF 49 to 150; t,/2 of < 24 h 9-d LC50 2.2 to >100% v/v effluent No EOX accumulation; increased TCDD accumulation for LMW vs. HMW EOX accumulation for LMW fraction 2 to 3 fold increase in lipid EOX Tissue EOX concentrations higher downstream; steady state not reached BCF of organochlorines 1800 (PCYMD) to 1.41 x 106 (C4-PCPH) RA detected in tissues after 7 d; BCF for fichtelite 10X higher than for resin acids; resin acid t|/2 < 3 d; fichtelite t\a ~ 12 d [EOX] concentrations: digestive glands > gills > gonads Decrease in sex steroid concentration and gravid females in 40 & 80% v/v Decreased dry tissue weight (A. cygnea); decreased lipids (£>. polymorpha); EOX accumulation in tissues; steady state not reached a Herve etal. 1988, 1996, 2001, 2002;b Makela and Oikari 1990;c Makela etal. 1991; d McKinney and Wade 1996;e Pellinen etal. 1993;1 Hayer and Pihan 1996; s Rantio etal. 1996;h Burggraaf et al. 1996;'Hayer and Pihan 1998;' Kernaghan etal. 1999b;k Hayer etal. 1996 Freshwater mussels (Hydridella menziesi) were transplanted near an outfall of a pulp and paper mill in New Zealand for exposures of 0 to 56 d (Burggfaaf et al. 1996). Mean total resin acid concentrations in the water ranged from 39 to 46 (ig/L during the exposure period. No effect on survival, filtration rate, condition or lipid concentration was observed in the control mussels as compared to those exposed to effluent for 7 to 28 d. Mussels were removed at 7-d intervals for the first 28 d and analysed for resin acids and fichtelite. In addition, after 56 d of exposure, the mussels were placed into clean water for depuration and removed for analysis at 0, 2, 5, 10, and 21 d. The mussels reached steady state resin acid concentrations after 7 d of exposure. Most of the resin acids detected were parent compounds, which indicates that mussels have limited abilities to metabolise these compounds. Mussel tissue concentrations of total resin acids were approximately 4 to 9 u.g/g dry weight. Bioconcentration factors were the lowest for dichlorodehydroabietic acid (2000 L/kg lipid weight) and was the highest for abietic acid (6000 L/kg lipid weight). The bioconcentration factor (BCF) for fichtelite was 89,000 L/kg. During the depuration period, >90% of the resin acids and 70% of the fichtelite were excreted over a 21-d period. The half-lives were 3 and 12 d for the resin acids and fichtelite, respectively (Burggraaf et al. 1996). Burggraaf et al. (1996) reported that there was significantly lower BCF for mussels exposed to resin acid concentrations of 120 to 160 u.g/L, as compared to mussels exposed to concentrations of 4 to 22 |J.g/L. There were no differences between condition index (dry tissue weight/shell weight) and filtration rate in clean water in any of mussels. The authors suggested that mussels may stop filtering water when exposed to high concentrations of resin acids and therefore, their exposure time is reduced. Three species of bivalves (Anodonta cygnea, Unio pictorum, and Dreissena polymorphd) were transplanted for 28 d to one site upstream and two sites downstream of a chlorine bleaching 27 pulp and paper mill (Hayer et al. 1996). No significant mortality was observed. Significant decreases in the dry weight of A. cygnea was observed at all three sites, while significant decreases in the lipid concentration in D. polymorpha at the site closest to the mill. Over the 28-d exposure period, significant accumulation of EOX at both downstream sites were observed in Anodonta cygnea and Unio pictorum, while the EOX increase in D. polymorpha was only observed at the near mill site. Steady state concentrations were not reached at the end of the experiment. Hayer and Pihan (1996) transplanted freshwater mussels {Anodonta cygnea L.) to three sites, one upstream and two downstream, close to a pulp and paper mill that uses chlorine and chlorine dioxide in its bleaching sequence. The mussels were collected in 3 to 4 d intervals during the 28-d exposure period and their tissues were analysed for EOX. There were no significant differences in mortality or lipid content in the upstream and downstream mussels. EOX concentrations in the downstream mussels were significantly higher than the upstream mussels! EOX was found to accumulate at the highest concentration in the digestive glands, followed by the gills and the gonads. Steady state accumulation of EOX was not observed during the 28-d exposure period. A second mussel study was conducted subsequently at the same mill, with a 91 -d exposure period (Hayer and Pihan 1998). The mussels were collected 5 times during the exposure period and their tissues were analysed for EOX. There were no significant differences in mortality or condition index (wet tissue weight/length3) between the upstream and downstream mussels. Significant decreases in the dry to wet weight ratio of gonad tissue and in the lipid content of the gills and gonads were observed at the station nearest to the pulp mill. The digestive glands accumulated the most EOX, followed by the gills and gonads. 28 Transplanted marine mussels were used to determine the effects on mussel survival, growth, and lipid content at a BC sulphite pulp mill (Salazar 2000). Mussels were deployed at three stations 0.3, 3, and 10 km from mill diffuser and at 2, 4, and 6 depths at each station. There were paired stations at each of the three deployment locations. The mussel survival at all stations was approximately 95%. Mussel growth, condition index (dry tissue/shell weight), and lipid content were observed to increase after a 68-d exposure, along the gradient of decreasing effluent. It was concluded that spent sulphite liquor and dissolved oxygen concentrations were the primary causative factors in the gradient effects. Spent sulphite liquor concentrations at the mussel deployment sites differed between stations and depth of sampling and ranged from 26.1 to 61.9 mg/L at 2 m to 14.2 to 80.4 mg/L at 6 m. Mussel tissues were analysed for five plant sterols at the end of the exposure period. There were increased concentrations of campesterol in the mussels closest to the effluent source. There were no trends with the other four plant sterols. However, the author concluded that a causal relationship could not be established between campesterol tissue concentrations and effects on mussel growth (Salazar 2000). Immune system effects on Mytilus edulis, transplanted at 3 sites near a pulp and paper mill in Pictou Harbour, NS, were studied by St-Jean et al. (2003). Haematocyte function, lysosome retention, phagocytic activity, and bacterial challenge were tested after a 90-d exposure. Phagocytic activity and lysosome retention were significantly lower in mussels closer to the pulp mill effluent discharge. There was a significant difference in mortality during the bacterial challenge test for the mussels exposed to the pulp mill effluent. No difference was observed in hematocyte function. The authors suggested that the use of immune system response variables may be useful in determining the cellular effects of anthropogenic discharges. Freshwater mussels were exposed for 56 d to four concentrations (10, 20, 40, 80% v/v) of pulp and papermill effluent using an outdoor, flow-through, experiment (Kernaghan et al. 1999b). Some mussels were exposed within the water column, while others were exposed near sediments. 29 Body condition index (total wet weight/shell length); mantle glycogen and sex steroid concentrations; and histopathological condition were measured. At the end of the exposure, mussel survival was high. There were no differences in the measured endpoints between the mussels exposed to the sediment and those exposed to the water column. Decreased sex steroid concentrations were observed in mussels exposed to the two highest effluent concentrations. In addition, fewer female mussels exposed to these effluent concentrations were found to be gravid, whereas 75% of the females exposed to 0, 10 and 20% effluent were gravid. The authors concluded that pulp mill effluent could result in significant endocrine and reproductive effects in mussels. Herve et al. (1988, 1996, 2001, 2002) and Pellinen et al. (1993) used incubated mussels as a tool to monitor chlorinated organic chemicals originating from kraft pulp mills in the receiving environment, instead of monitoring water concentrations. Herve et al. (1998) incubated mussels for 4-week periods each year and observed decreases of chlorophenol concentrations over a period of 4 years. Herve et al. (1996, 2001, 2002) found that the concentrations of these compounds in the mussel tissue showed decreases over a ten-year period. It was concluded that these decreases were directly related to changes in the pulping and bleaching processes and the introduction of secondary treatment at pulp and paper mills in Finland. The EOX concentrations in mussels transplanted near a pulp mill site were two to three times higher than those upstream from the pulp mill, with higher concentrations in the digestive gland lipid as compared to the soft tissue lipid (Pellinen et al. 1993). EOX is the total concentration of extractable organically bound halogen present in tissue. The EOX concentrations in the mussels ranged from approximately 79 \iglg dry weight upstream from the pulp mill to 190 ug/g dry weight three kilometres downstream from the discharge point. Pellinen et al. (1993) concluded that EOX in pulp mill effluent was a much less sensitive parameter for 30 bioaccumulation of pulp mill effluent components than individual low molecular mass compounds, such as chlorophenols. Freshwater mussels were exposed in-situ to pulp mill effluent and their tissues were analysed for several aromatic chlorohydrocarbons, such as polychlorocymenes, polychlorocymenenes, alkylpolychloro-napthalenes, -bibenzyls and -phenanthrenes, to determine tissue concentrations (Rantio et al. 1996). Water concentrations were 0.075 (polychlorobibenzyls) to 52.6 ug/L. Bioconcentration factors of 1800 for polychlorocymenenes (PCYMD) to 1,410,000 for polychlororetenes (C4-PCPH) were calculated. Survival, growth, and condition index of the mussels were not measured. The bioconcentration factors of pentachlorophenol (PCP) and 4,5,6-trichloroguaiacol (CG-3) among the organs of Anodonta anatina L., a freshwater mussel, were determined. The highest bioconcentration factors for PCP (447 -637) and CG-3 (275- 324) were observed in the digestive gland, followed by the kidneys, which had bioconcentration factors of 300 - 326 and 189 - 211, for PCP and CG-3, respectively. The hemolymph had the lowest bioaccumulation factors (PCP: 2.6 - 21; CG-3: 3.5 - 15). Most of the PCP and CG-3 was found in the digestive gland, followed by the kidneys and the foot/mantle. Limited metabolism of PCP and CG-3 was observed. Anodonta anatina, were exposed to an artificial pulp mill effluent to determine bioconcentration factors and depuration rates of chlorophenols, chloroguaiacols, and resin and fatty acids (Makela et al. 1991). Total chlorophenol exposure concentrations ranged from 6 to 56 |ag/L. Bioconcentration factors ranged from 30 for 2,4,6-trichlorophenol to 150 for pentachlorophenol. The shortest elimination half-life (ti/2) of 1.5 hours was observed for 2,4,6-trichlorophenol with the longest ti/2 of 23 hours for 2,3,4,6-tetrachloroguaiacol. 31 Juvenile freshwater mussels (Anodonta imbellcillus) were exposed to concentrations of pulp mill effluents (12.5 to 100% v/v) sampled from 6 different mills in a static renewal test system for 9 d in the laboratory (McKinney and Wade 1996). Mussel survival was reduced in four of the six effluent types tested and the 9-d LC50 values for these four effluents ranged from 2.2 to >92.5% v/v. Mussels that were exposed to 22 mg/L rosin, a resin acid mixture, were found to have degenerated digestive cells and other cellular effects (Fahraeus-Van Ree and Payne 1999). Rosin also increased the enzymatic activity (e.g. NADPH-Diaphorase and glucose-6-phosphate dehydrogenase) of cells present in the digestive glands of the mussels. These enzymes are associated with the MFO enzyme system, which assists in the detoxification of xenobiotics. In addition, the mussels exposed to the rosin had significantly increased concentrations of lipids as compared to the controls. The authors concluded that rosin may be toxic to mussels; monitoring of bivalves around pulp mill effluent discharge sites should be conducted and mussels would be useful biomonitors for pulp and paper mills. The filtration rates of MytUus edulis exposed to five concentrations of bleached sulphite pulp mill effluent were measured and 5-d EC50 was estimated to be 29 mL/L (Kallqvist et al. 1999). Macoma balthica were sampled from the Baltic Sea, in vicinity of a bleached kraft pulp mill outfall (0 to 20 km) and the degree of shell corrosion was evaluated (Landner et al. 1994). No differences in the degree of corrosion of the mussels exposed to the effluent were observed. 1.4.3 Life History 1.4.3.1 Habitat and Geographical Distribution M. edulis, commonly known as the blue mussel, is a semi-sessile, epibenthic bivalve (Newell 1989). They are found attached to a variety of substrates, ranging from large rocks and 32 gravel to mud and sand substrates. M. edulis attaches itself to substratum by byssal threads that are produced by a gland located in their foot (Newell 1989). They typically inhabit the littoral and shallow sublittoral or intertidal zones of the ocean, but can also be found in some estuaries (Newell 1989, Seed and Suchanek 1992). Mussels require adequate water movement to ensure a steady supply of oxygen and food particles (Newell 1989). M. edulis is euryhaline and can tolerate a salinity range from 5 to 34 parts per thousand ( % o ) (Newell 1989, Seed and Suchanek 1992). They are ectothermic and can survive in temperatures below 0°C and up to >25°C (Newell 1989, Seed and Suchanek 1992). M. edulis can regulate the osmotic pressure of their intracellular fluid to that of the surrounding environment, by altering the concentrations of free amino acids (Newell 1989). M. edulis is found along the coasts of the Arctic and North Atlantic Oceans, as far south as North Carolina and North Africa (Newell 1989, Seed and Richardson 1990, Koehn 1991). It is not native to the Pacific Coast of British Columbia (Seed and Richardson 1990). 1.4.3.2 Respiration and Feeding Adult M. edulis respire and feed by bringing in water through an inhalent siphon (Newell 1989). The water is pumped over the gills, also known as ctenida, which are comprised of 4 demibranchs and smaller ciliated filaments (Newell 1989, Morton 1992). The oxygen is transferred from the water into the hemolymph, which is circulated by the heart (Newell 1989). Respiration rates increase with increasing temperature, food concentration, body size in the presence of food, and decreasing oxygen concentrations in the water (Gosling 2003). Increased concentrations of pollutants, such as metals, can result in reductions in respiration rates (Gosling 2003). During times of air exposure, mussels close their valves to prevent desiccation and use anaerobic metabolism to maintain cellular functions (Bayne et al. 1985, Newell 1989). 33 Mussels are suspension filter feeders. They retain particles, such as phytoplankton, bacteria, and organic matter, from water (Newell 1989, Seed and Suchanek 1992). The particles are collected along mucous covered oral food grooves, to labial palps, where the particles are sorted based on their organic composition. Typically particles that are < 5 um are retained (Bayne et al. 1985, Newell 1989). Rejected particles are combined with mucous and excreted through the inhalent siphon (Newell 1989). This waste is also known as pseudofaeces (Newell 1989). Ingestion rates of organic material increase up to a steady rate, as the concentration of organic material in the water increases (Bayne et al. 1989). In the stomach, food particles are digested with enzymes and mechanical manipulation. The food particles enter digestive cells by endocytosis, where they undergo intracellular digestion. Upon digestion, the nutrients are transferred into the haemolymph (Newell 1989). Any undigested matter is excreted through the exhalent siphon (Newell 1989). Nitrogenous wastes are excreted as ammonia (80 to 90%), amino-nitrogen (5 to 10%), and urea (5%) through the kidney, pericardial glands, and the gills (Bayne et al. 1985, Gosling 2003). 1.4.3.3 Growth and Condition Growth of the mussels is dependent on environmental factors, such as age and reproductive stage, temperature, salinity, light, food availability and quality, dissolved oxygen concentrations, and duration of air exposure (Bayne et al. 1985, Newell 1989, Seed and Richardson 1990, Seed and Suchanek 1992). Growth rate decreases with increasing mussel length and age (Almada-Villela et al. 1982, Bayne et al. 1985). Typically, younger mussels allocate more energy to somatic growth, while older mussels put more energy into reproductive output (Gosling 2003). During gametogenesis and spawning, energy is allocated to these two activities rather than somatic growth. Typically, at higher water temperatures, growth will increase (Newell 1989). Growth can also be affected by extreme salinity (< 20%o and > 35%o) and temperatures (< 34 5°C and > 20°C) (Bayne et al. 1985, Seed and Suchanek 1992). Exposure to periods of light > 7 hours can result in decreases in mussel growth (Seed and Suchanek 1992). However, mussels that are grown in the dark, often have thinner shells (Seed and Suchanek 1992). Food availability and quality are important for growth, as low levels or poor quality of the food will not allow the mussel to obtain sufficient energy that can be allocated to somatic growth. The ratio of total seston to particulate matter appears to be important in the efficiency of extracting energy from ingested particles (Seed and Suchanek 1992). Mussels that are exposed to air have reduced growth rates, as compared to mussels that are fully submerged (Seed and Suchanek 1992). This is most likely due to reduced feeding opportunities during air exposure (Seed and Suchanek 1992). Mussel nutrient reserves, such as glycogen and lipids, change seasonally, in response to reproduction and food availability (Gabbott 1976, Bayne et al. 1985). Glycogen reserves are utilised during gametogenesis, while lipid reserves often increase during gametogenesis (Gabbott 1976). Glycogen reserves are replenished after spawning, especially when food availability is high (Gabbott 1976). Condition index is the ratio of tissue weight to shell weight, length, or volume and is indicative of the nutrient state of bivalves. It is influenced by water temperature, season, food availability, and reproductive stage (Gabbott 1976, Bayne et al. 1985). High water temperatures and low food availability can lead to decreases in condition index (Gabbott 1976, Bayne et al. 1985). Condition index increases during energy storage and gametogenesis and decreases upon spawning (Bayne et al. 1985, Gosling 2003). Typically, condition index is low in the early months of the year, increases during the spring, and then decreases in the fall (Bayne et al. 1985). Condition index decreases when the metabolic rate exceeds that of the ingested food, and to compensate for this, carbohydrate reserves (e.g. glycogen) are metabolised, followed by lipid and protein reserves (Gabbott 1976). 35 1.4.3.4 Reproduction and Larval Development M. edulis is diecious and usually becomes sexually mature within one year (Newell 1989). The occurrence and success of reproduction depends on the health of the adult, food availability, temperature, salinity, air exposure, nutrient reserves, hormonal cycle, and genotype (Newell 1989, Gosling 2003). The reproductive cycle consists of three parts: gametogenesis, spawning, and gonad reconstruction (Gosling 2003). M. edulis typically undergoes gametogenesis during the winter and summer with spawning in the spring and fall (Gosling 2003). Gonad reconstruction typically occurs during fall and winter (Gosling 2003). Spawning is triggered by changes in water temperatures and physical disturbances, such as storms (Gosling 2003). During spawning, adult mussels release their gametes into the water column, where fertilisation occurs. Within 24 h of fertilisation, the larvae become ciliated and motile and rely on nutrients from their yolk (Newell 1989, Morton 1992). This stage, known as the trochophore, subsequently develops into a veliger (Newell 1989, Morton 1992). The veliger stage has four distinct developmental forms: prodissoconch I and II; eyed larvae, and pedveliger (Newell 1989). The prodissoconch I stage involves the secretion of a straight-hinged shell from shell gland (Newell 1989). As the prodissoconch II stage, the larval mussel develops a mantle, which produces an umbo-shaped shell (Newell 1989). In the third veliger stage, the larvae develop pigmented eyespots. In the last veliger stage, the larvae develop a foot and are known as a pediveliger (Newell 1989, Morton 1992). During the veliger stage, the digestive system develops which enables the veliger to feed (Newell 1989) on plankton, dissolved organic material, detritus, and bacteria (Gosling 2003). In addition, the veliger moves using cilia at its anterior end (Newell 1989). At the end of the veliger stage, the larvae attach to filamentous substrates, using byssal threads. In the next stage of development, the larvae is known as a plantigrade and this stage can 36 last from approximately 20 d to several months, depending on environmental conditions (Newell 1989, Morton 1992). When the plantigrade is approximately 1.5 mm long, it releases itself from the substrate and is carried by currents to the ocean bottom where it attaches itself to a hard substrate using byssal threads (Newell 1989). Once the plantigrade is attached to a secure substratum, it undergoes metamorphosis, a process by which the body organs are reorganised and the adult dissoconch shell is secreted (Gosling 2003). 1.4.3.5 Detoxification Mechanisms Mussels have enzymes systems designed to aid in the detoxification and excretion of xenobiotic chemicals. Phase I and II enzymes are mainly responsible for detoxifying organic chemicals, while metallothioneins are responsible for binding metals. Most of the detoxification enzymes present in mussels are found in their digestive organs (Lee 1981, Livingstone and Pipe 1992, Gosling 2003). Metallothionein is present in the gills and digestive glands and aids in the binding and excretion of heavy metals (Gosling 2003). The function of Phase I enzymes is to add polar functional groups, such as hydroxyl, carboxyl, and amine, to xenobiotics (Livingstone and Pipe 1992). Phase I enzymes include cytochrome P-450 monooxygenase and flavoprotein monooxygenase, also known as the MFO system (Lee 1981, Livingstone and Pipe 1992, Gosling 2003). MFO activity is observed in the digestive glands, gills, and hemolymph (Bayne et al. 1985, Livingstone and Pipe 1992). The MFOs in marine mussels are present at lower concentrations than those present in fish (Bayne et al. 1985). The Phase II enzymes, such as glutathione S transferase and uridine diphosphate glucoronyltranferase, react with xenobiotics by the addition of a polar molecule, which makes the chemical more water soluble and hence more easily excreted (Livingstone and Pipe 1992). B-glucoronidase and arylsulphatase are present in invertebrates and these enzymes aid in the conjugation of xenobiotics (Bayne et al. 1985). These reactions are often faster than those 37 utilising MFO (Bayne et al. 1985). A variety of xenobiotics such as PAH, PCB, dioxins, furans, metals, pesticides, polybrominated biphenyls (PBB), polychlorinated terphenyls (PCT), and bleached kraft mill effluent have been shown to induce MFOs in fish (Whyte et al. 2000). However, more research is needed to determine whether pollutants, such as pulp mill effluent, can cause induction of this enzyme system in invertebrates (Bayne et al. 1985). 1.5 Study Objective for Mussel Study 1.5.1 Study Objective The objective of this study was to determine the effects of pulp mill effluent on the survival, growth, and condition index of the mussel, Mytilus edulis. This study was conducted on-site at the Norske Canada (formerly Fletcher Challenge Canada Ltd.), Elk Falls Division pulp and paper mill in Campbell River, BC, to satisfy provincial biological monitoring requirements. Mussels were used in this study as they were identified as a sentinel species in the E E M Pre-Design Reference Document (Hatfield Consultants Ltd. 1994). 1.5.2 Rationale for Study Design Neither an in-situ (i.e. transplanted) mussel study nor a wild mussel survey (i.e. collecting mussels from the mill receiving environment) were not feasible at the study site given logistic and safety issues in the receiving environment near the mill. Therefore, this study was conducted on-site (i.e. on land, adjacent to the pulp mill) as opposed to a traditional field or laboratory study for several reasons. In Discovery Passage, which is adjacent to the mill, strong currents (up to 14 knots) and high boat traffic preclude the placement of fixed gear over long periods of time. If a mussel study was conducted in-situ, these currents would pose a danger to personnel deploying and retrieving equipment and could cause mussel cages to be dislodged and removed from their 38 original locations. In addition, effluent is rapidly dispersed and variably mixed in the receiving environment, and it would have been very difficult to determine the actual effluent exposure concentrations. An on-site experiment allowed for control of effluent concentrations to which mussels were exposed. Therefore, concentration-response relationships could be determined and potentially extrapolated to the receiving environment. In the on-site experiment, the potential effects of confounding factors, such as other discharges to Discovery Passage, and natural differences in oceanography and effluent exposure in the local receiving environment, were reduced or eliminated. This approach also allowed the use of ambient receiving water as the control and dilution water, which would not have been possible in a laboratory setting without the costly transportation of receiving environment water and effluent. In addition, the temperature and photo-period reflected the natural environment. The use of a controlled field study attempted to replicate more "real-world" conditions than a laboratory bioassay. Laboratory studies tend to overestimate potential effects of a chemical if the actual exposure concentrations change rapidly in the receiving environment (Graney et al. 1995). In contrast, effects of chemicals that require metabolic activation, are chronically toxic, and/or bioaccumulate, will be underestimated in acute laboratory bioassays (Graney et al. 1995), due to the short-term nature of most standard laboratory toxicity tests. Development of a novel, shore-based method to assess the possible effects of pulp mill effluent on marine bivalves allowed environmental monitoring of chronic, whole-organism effects in a controlled setting which was not confounded by laboratory constraints and used waters from the local environment. The use of caged mussels, placed at the study site, had several advantages from analysing field-collected mussels. The use of caged organisms reduces variability by using organisms of similar size and genetic history, increasing the discriminating power of the test (Salazar and Salazar 1995). Secondly, the use of transplanted organisms is environmentally relevant and also has more experimental control, similar to that in laboratory bioassays (Salazar and Salazar 1995). 39 Thirdly, repeated, non-destructive measurements allow estimates of temporal and spatial variability and this increase the statistical power of the test (Salazar and Salazar 1995). Studies using caged mussels also allow the concentration and/or dose-response relationships and the exposure period to be well defined. (Salazar and Salazar 1995). In addition, it has been suggested that mussels are suitable to determine the effects of pulp mill effluents (Salazar and Salazar 1997). 1.5.3 Null Hypotheses The following null hypotheses were tested in this study: (1) There were no differences in survival and/or growth between the control mussels and those exposed to 0.23 to 4.6% v/v effluent during the 89-d exposure period. Growth was measured by changes in whole animal weights and shell length over the exposure period; (2) There were no differences in condition index, lipid and moisture content between the mussels at the beginning of the study (T = 0) and at the end of the 89-d study; and (3) There were no differences in condition index, lipid and moisture content between the control mussels and those exposed to the effluent concentrations during the 89-d exposure period. 40 CHAPTER 2 MATERIALS AND METHODS 2.1 Study Design The draft Guide for Conducting Field Bioassays with Marine, Estuarine and Freshwater Bivalves (Environment Canada 1997) was followed with some major modifications. An exposure time of 90 d was chosen this exposure period was recommended by Environment Canada (1997) because tissue concentrations for most chemicals reach equilibrium and it allows for the measurement of adverse effects on survival and growth. Five nominal concentrations of effluent were used for exposure to mussels: 0.22, 0.46, 1.00, 2.20, and 4.60% v/v. Control and dilution water consisted of seawater pumped from Discovery Passage. Lower concentrations in the dilution series were chosen to reflect possible effluent concentrations in the receiving environment, as the maximum concentration of effluent in the environment is predicted to be approximately 1% within 250 m of the diffuser (Hatfield Consultants Ltd. 1994). This estimate is a conservative estimate of the actual effluent concentrations in the receiving environment, as it includes a safety factor (Hatfield Consultants Ltd. 1994). In total, 2800 mussels were used in the experiment, (5 concentrations x 400 mussels = 2000, 1 control x 400 mussels = 400, 1 T = 0 x 400 mussels = 400; total 2800 individuals) including 400 mussels that were sacrificed previous to effluent exposure, to determine their baseline condition (T = 0). The mussels were suspended in 220-L barrels on PVC pipe in 4 groups. Each group (A, B, C, D) included 7 bags of mussels: the first 6 bags contained 15 mussels 41 each and bag 7, contained 10 mussels (100 in total). The mussels within one group were in separate bags due to depth restrictions of the test vessels. The study was designed to enable tracking of each individual mussel through the entire experiment through the placement of the mussels into individual compartments, separated by cable ties, in the oyster netting bags. To measure the effect of effluent concentrations on mussel growth, shell length and whole weight of each individual mussel were measured at the onset and termination of the experiment. The change in shell length or whole weight was calculated. Wet tissue and shell weights were measured at test termination and used to calculation condition index. Mussel survival was also recorded. At study termination, tissue from all surviving mussels in each group was composited to form one sample for lipid and moisture analysis. In total, 28 tissue samples were collected, including the baseline mussels (T = 0). For the resin acid analysis, one composite sample per treatment was analysed, for a total of 6 samples. 2.2 Experimental Set-Up A schematic of the experimental design is outlined in Figure 2-1. A marquee was installed to shade the entire experimental area (Figure 2-2). Electricity, to power the pumps, was obtained from the mill. 42 Figure 2-1 Schematic of Seawater and Effluent Flow Routes and Arrangement of Treatments, Used in the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 43 Figure 2-2 Experimental Set-Up of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 2.3 Seawater Seawater was pumped from Discovery Passage, using two pumps connected in series, into a 850-L (225 gal) headtank (Nalge Nunc International, Rochester, NY) (Figure 2 - 3 ) . Two pumps were necessary to overcome the nine metre head between the study site and the water. Seawater was pumped through a screened intake that was placed at least one metre below the lowest tide level. A foot valve and 51-mm wire-reinforced rubber suction hose were attached to the intake. Seawater was delivered by the pumps to the headtank using 51 mm rubber discharge hose and a length of schedule 40 51-mm PVC pipe. The headtank was placed in a plywood box to contain overflow that was drained back down the cliff through PVC pipes, away from the intake source. A manifold, constructed of schedule 40 51 mm PVC pipe and 25-mm hose barbs, was 44 attached to the headtank. Lengths of 25-mm hose were attached to each of the barbs; seawater flowed by gravity through the hose into each of the six 20-L white plastic mixing buckets. A globe valve, used to adjust seawater flows, was installed in the side of the 20-L mixing buckets. 2.4 Effluent The combined effluents from the kraft and TMP treatment systems were used in this study. Effluent was obtained from the pulp mill discharge immediately before the outfall. The final effluent was gravity fed into a 220-L barrel (Figure 2-4). Effluent was pumped from this barrel into mixing buckets using peristaltic pumps (Model WZ1R031, Universal Electric, Owosso MI) with pumping rate controllers (Stir Pak Solid State Control 4558-03, Cole Parmer, Chicago, IL). Effluent flowed by gravity from the mixing buckets down a 51 mm diameter PVC standpipe-diffuser into the barrels. 2.5 Test Vessels Test vessels were 220-L blue plastic barrels that were approximately 110 cm deep with a 35 cm diameter (Figure 2-5). Each of the barrels had an overflow consisting of PVC pipe drains inserted just below the rim of the barrels. Standpipes and diffusers constructed of 51 mm schedule 40 PVC pipe, were attached to drains (51 mm wide, schedule 40) in the bottom of the mixing buckets. The seawater and effluent flowed upwards through a 6 mm wide slit in the diffuser. 2.6 Bag Construction Mussels were placed into 6 mm oyster netting bags (6 mm mesh tubing, Norplex Industries, Auburn, WA) that were approximately 1.3 m long. Mussels were separated from each other using cable ties. Approximately 4 to 5 cm of space existed between each of the mussels to allow for growth. Each was weighted with a stainless steel nut, which prevented the bags from 45 floating. The bags were labelled with two number code: the first number indicated the concentration and group (1 - 28); the second number referred to the bag number (1 - 7). Figure 2-3 Experimental Set-Up Showing Seawater Headtank and Hoses Attached from Seawater Manifold to the Mixing Buckets for 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 46 Figure 2-4 Barrel and Peristaltic Pumps Used to Hold and Pump Effluent for the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 47 Figure 2-5 Test Exposure Barrels, Mixing Buckets, and Cross-Bars Used for Mussel Bag Attachment in the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 2.7 Mussel Source The mussels, Mytilus edulis, were spawned from certified broodstock at Island Scallops, Bowser, BC in June 1998. The mussels were placed at Kanish Bay Shellfish Farm, Quathiaski Cove, BC, as spat in July 1998. From November 1998 to June 1999, the mussels grew in oyster netting "socks" until harvested for this study. On June 4, 1999, approximately forty-five hundred 2 - 3 cm mussels were obtained for this study. Mussels from these sources have been used in other mussel studies (Salazar 2000, Grout and Levings 2001). M. edulis is most often used in marine environmental studies, using bivalves, and therefore, it was chosen for this study so that it could be directly compared to other studies that 4S used M. edulis. In addition, M. edulis does not experience summer mortality which would have affected the test results if M. trossulus, a west coast species, had been used (Salazar 2000). 2.8 Mussel Sorting and Distribution Mussels were transported on ice packs to the study site. At the site, mussels were transferred into 3 coolers containing seawater, where they remained during the preliminary size sort. During the size sort, mussels were placed into 4-L white plastic buckets which were half-filled with seawater and labelled as 1-mm size classes between <19 and >31 mm. Mussels were measured using plastic calipers. Water in the coolers was changed once during the sorting procedure. Individuals of each size class were placed into oyster netting bags and suspended in the control seawater barrel for overnight storage. The control barrel had a constant flow of fresh seawater. Mussels that were 20 to 27 mm in length were used in the study. Initial length and whole weights were measured between June 5 and 8, 1999, using digital calipers (Digimatic, Mitutoyo America Corporation, Mississauga, ON) and a 0.01 g electronic balance (Mettler PJ3 00, Mettler Toledo Canada, Mississauga, ON). Subsequently, the mussels were distributed into oyster netting bags, starting with bag series #1 on June 5 and ending with #7 on June 8. Mussels were placed into bags starting with the smallest size class (Figure 2-6). Bags were filled such that each concentration would have similar sized mussels, according to the Environment Canada (1997) method. The bags were randomly assigned to the concentrations. During the distribution period, mussels were placed in seawater and the water was changed periodically. Unmeasured mussels were stored in the control barrel during this time. Once installed in the test exposure barrels, mussels were attached to 25-mm PVC pipe crossbars using cable ties (Figure 2-5). 49 2.9 Flow Rates The targeted total flow to each barrel was 620 L/hour, based on an estimated filtration rate of 2.0 L/hour/g of estimated mussel tissue at the onset of the experiment. The estimated filtration rate was based on an average of 15 filtration rates found in the literature for Mytilus edulis. The initial amount of tissue per treatment was estimated at approximately 0.77 g per mussel or a total of 310 g tissue per treatment. However, the estimated amount of wet tissue in each treatment at the beginning of the study was approximately 210 g, as calculated from the tissue in the T = 0 treatment group. At the end of the study, the amount of tissue per treatment ranged from 219.50 g (4.88% v/v) to 285.54 g (2.07% v/v). Therefore, the flow rate in the test vessels was sufficient to supply more than 2.0 L/hour/g wet tissue wet for the mussels. 2.10 Study Initiation On June 9, 1999 at 10:30 h, effluent flow was initiated. Seawater and effluent flow rates were measured and adjusted within 10% of the target values. Dissolved oxygen, temperature, pH, and salinity were measured and recorded. Dissolved oxygen was measured using a dissolved oxygen probe and meter (Model 54, YSI, Yellow Springs, OH). Temperature was measured using a glass thermometer. Salinity was measured using a hand held salinity refractometer (Vista A366ATC, VWR Scientific, Edmonton, AB). pH was measured using a pH probe attached to a pH meter (Porta Mate pH Meter 175, Instrumentation Laboratory, Inc., Lexington, MA). 50 Figure 2-6 Mussels Being Placed into Oyster Netting Bags for the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, Elk Falls Division, Campbell River, BC, June to September 1999 2.11 Daily Measurements and Maintenance On 56 days during the study, dissolved oxygen, temperature, pH, and salinity were measured in each effluent concentration barrel by Norske Canada, Elk Falls Division staff. Effluent and seawater flow rates were measured, using a graduated cylinder and a stopwatch, and adjusted as necessary, to be within 10% of the target values. The bottoms of the barrels were 51 siphoned out on an as-needed basis to prevent sediment accumulation. The peristaltic pumps were oiled and the tubing shifted and cleared regularly to avoid any potential effluent flow problems. 2.12 Baseline Condition of Mussels Four hundred mussels were sacrificed on June 9 and 10, 1999 to determine the baseline condition index and lipid and moisture content (T = 0). Tissue from each group was placed into a 125-ml amber glass jar and frozen at -20°C. This frozen tissue was subsequently thawed and homogenised, using a blender (Osterizer 8, Sunbeam Products, Inc. Boca Raton, FL) for 60 seconds. An aliquot (10 g) of tissue was transferred to a 15-ml polypropylene conical tube for lipid and moisture analyses. The remaining tissue was returned to the amber glass jars. 2.13 Study Termination, Observations, and Measurements The experiment was originally planned to be 90 d, however on day 89, one of the peristaltic effluent pumps failed and effluent flows were terminated. Therefore, mussels were exposed to ambient seawater for approximately 16 hours before they were removed from the barrels and transported to Vancouver, BC on September 7, 1999. At experiment termination, barnacles were growing on the inside walls of all the barrels. Marine worms, shrimp, and other invertebrates were observed in the barrels. Mussels were transferred into coolers containing ice and seawater-saturated burlap bags and paper towels (1 cooler per treatment) and transported to the BC Research facilities, Vancouver, BC. At BC Research, mussels were placed into six 220-L barrels containing aerating seawater. Each barrel contained mussels from one concentration. Seawater was obtained from the Vancouver Aquarium and is routinely used for control/dilution water in marine bioassays conducted at BC Research. Mussels were stored in these barrels during the final measurement period. Dissolved oxygen in the barrels was at or near saturation; salinity was 28 to 30%o; pH 52 values were between 7.7 and 8.1 and temperatures were similar to those at the end of the experiment on-site (12 to 14°C). The photo-period was set as 14 hours light: 10 hours dark, which closely reflected the natural photo-period. All mussels were measured and sacrificed on September 8 and 9, 1999 and length, whole weight, and tissue and shell weights were measured, using digital (Digimatic, Mitutoyo America Corporation, Mississauga, ON) and analytical balances (Mettler PJ300, Mettler PL200, Mettler PI20, Mettler Toledo Canada, Mississauga, ON). Mussels that were missing or dead were recorded as such on data sheets. Any mussels that were found outside the oyster netting bags were discarded without taking measurements, given that their exact identity could not be confirmed. Tissue was absent in the shells of most of the dead mussels, indicating they had been dead for some time. Therefore, this holding period did not likely contribute to the mussel mortality. A total of 86 mussels were missing. More mussels were missing at the lower concentrations and only 4 were missing in the highest concentration. It is not known why there were more mussels missing at the lower effluent concentrations, as compared to the highest effluent concentrations. A possible explanation for the missing mussels could be that they outgrew their compartments and squeezed out of the bags. Several unidentified mussels were found outside the bags; some of these may have been the missing individuals or may have been pumped in as larvae with the seawater. Missing mussels were not included in the data analysis. Mussel tissue from each group was transferred into an amber glass jar and refrigerated (24 samples). The mussel tissues were homogenised in a blender (Osterizer 8, Sunbeam Products, Inc. Boca Raton, FL) for 60 seconds. A 10-g aliquot was transferred into a 15-ml polypropylene conical tube; the remaining tissue was returned to the amber glass jar. Between each group of mussels, the blender was rinsed a minimum of 3 times with deionised water. Tissue samples were 53 subsequently frozen at -20°C until transported to the laboratory for lipid and moisture analysis (ALS (formerly ASL), Vancouver, BC). Samples were transported on ice and were kept frozen until sample preparation occurred. In addition, subsamples from each group of mussels (4 per treatment) were combined (for a total of 6 samples) and transported to ALS, for resin acid analysis. 2.14 Lipid and Moisture Analyses The lipid and moisture analyses were conducted at ALS, Vancouver, BC. Mussel tissue was analysed for lipid content using the Association of Official Analytical Chemists Method 983.23 (1995). The tissues were solvent extracted with 1 part chloroform (40 mL) to 2 parts methanol (80 mL) in the presence of an enzyme (Clarase 40,000). The extract was evaporated to dryness and the residue was determined gravimetrically. For moisture determination, mussel tissue was analysed gravimetrically by drying the sample at 103°C for a minimum of six hours. 2.15 Resin Acid Tissue Concentrations Resin acid concentrations of the mussel tissues were analysed at ALS, Vancouver, BC. The analyses were conducted using a modified version of the BC Ministry of Environment, Lands and Parks (1994) and Environment Canada methods. The method was modified to include the analysis of tissue samples, such as the use of gel permeation chromatography. The tissues were extracted using a Soxhlet apparatus with dichloromethane. Gel permeation chromatography was subsequently used to clean-up the extracts. The extracts were methylated using diazomethane and analysed using capillary gas chromatography with mass spectrometric detection. The detection limits were 0.02 ug/g (wet weight) for abietic, chlorodehydroabietic, dehydroabietic, and dichlorodehydroabietic acids; 0.1 ug/g (wet weight) for pimaric acid; and 0.2 Ltg/g (wet weight) 54 for isopimaric, levopimaric, neoabietic, and sandaracopimaric acids. A method blank and a spiked sample were also analysed. 2.16 Calculations and Statistical Analyses It was intended to have four replicates of mussels for each effluent concentration, with each group of mussels being a replicate. However, due to restrictions on pumping equipment, all four groups were contained in the same test vessel. This is considered to be pseudoreplication, as there were water connections between the four sets of mussels in each treatment (Hurlbert 1984). A better experimental design would have been four replicates of mussels, placed in separate containers, for each effluent concentration. However, due to the large number of mussels in the study, statistically significant differences were still able to be determined for some of the parameters. 2.16.1 Survival/Mortality Percent survival in each group was calculated by dividing the number of surviving mussels by the total number of mussels at the end of the study (alive plus dead, excluding missing mussels). A Z-test to compare proportions (a = 0.05, number of comparisons = 5) was used to determine if there were significant differences in mussel survival between the control and the effluent treatments (Moore and McCabe 1999). This statistical test was chosen as it allowed multiple comparisons of proportional data that had different sample sizes. The no observed effect concentration (NOEC) and the lowest effect concentration (LOEC) for the survival data were also calculated. The NOEC is defined as the highest concentration of effluent in which the observed response is not statistically different from the 55 control response. The LOEC is defined as the lowest concentration of effluent in which the observed response is statistically different from the control response. Effluent concentrations that would be predicted to cause a 20 or 25% reduction in survival (LC20 or LC25) and their associated 95% confidence intervals were calculated using the probit method in ToxCalc™ (Version 5.0.18), a Microsoft Excel-based software application (Tidepool Scientific Software 1994-1997). The data were corrected for the control mortality using Abbott's formula prior to calculating the LC20/25. 2.16.2 Growth and Condition Index Two growth indicators were used to assess the effect of the effluent treatments: length change and weight change. Length and weight changes were calculated as the difference between final and initial shell lengths and final and initial whole weights. In addition, condition index was calculated by dividing wet tissue weight by shell weight (Equation 1, Grout and Levings 2001). Cl = ^ iS^l (Equation 1) w,(g) where; CI = condition index W t = wet tissue weight (g) W s = shell weight (g) Shapiro-Wilk's and Bartlett's tests were used to determine whether the growth and condition index data were normally distributed and that the variances were homogenous. For the initial data, the Kolmogorov-Smirnoff-Lillifors (KSL) test was used to determine whether the data were normally distributed and was used as the sample size was greater than 2000. These tests were used to determine whether the data met the assumptions for a one-way analysis of variance (ANOVA). A one-way ANOVA (a = 0.05) was performed on the initial weight and length data, 56 on the length and weight change data, and on the condition index data. Bonferroni's test was used to identify the treatments that were statistically different from the control treatment (Moore and McCabe 1999). In addition, Bonferroni's test was used to determine whether the condition indices in the control and the effluent treatment were significantly different from the T = 0 condition indices. Bonferroni's test was used as it allowed for multiple comparisons to be made between the five treatment groups and the control, which had different sample sizes, to determine significant differences. 2.16.3 Lipids and Moisture Shapiro-Wilk's and Bartlett's tests were used to determine whether the lipid and moisture data were normally distributed and had equality of variances, respectively. A one-way A N O V A (a = 0.05) was performed on the lipid and moisture data and a least significance difference test (Steel and Torrie 1960) was used to determine which effluent concentrations had significant differences in lipids and moisture, as compared to the control and the T = 0 treatments. A least significant difference test was used to make multiple comparisons between the effluent concentrations and the control, which had the same sample sizes, to determine significant differences. All ANOVAs, and Shapiro-Wilk's, KSL, and Bartlett's tests were conducted using JMP, Version 4.0.2 (SAS Institute Inc. 1989-2000). The other statistical tests, unless otherwise noted, were calculated using the formulae found in the cited references and using Microsoft Excel 97 SR-2 (Microsoft Corporation 1985 - 1997) to do the calculations. 57 CHAPTER 3 RESULTS 3.1 Effluent Characteristics The effluent characteristics during the study are summarised in Table 1-6. 3.2 Seawater and Effluent Flow Rates Total (seawater and effluent) and effluent flow rates were measured on 56 d and adjusted, if >10% deviation from nominal concentrations had occurred. The flowrates were adjusted accordingly and subsequently re-measured (Table 3-1 and Table 3-2). Hence, the number of data points differs for each concentration. The mean total flow rates were within 1.3% of the targeted flow of 620 L/hour (Table 3-1). The effluent concentrations were calculated by dividing the effluent flow rate by total flow rate (Table 3-2). The mean measured concentrations were within 7% of the nominal treatment concentrations. The measured concentrations were used in the statistical analyses. 58 Table 3-1 Total Flow Rates (L/hour) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E l k Falls Division, Campbell River, B C , June to September 1999 Mean Measured Mean Total SD n Min imum Maximum 10% Target Effluent Flow Rate Range (% v/v) Concentration (% v/v) (L/hour) 0 (Control) 619 28 78 554 789 558 - 682 0.23 616 23 92 553 672 558 -682 0.46 612 36 88 410 676 558 - 682 1.01 624 22 87 549 681 558 -682 2.07 614 26 85 508 660 558 -682 4.88 620 23 85 528 689 558 -682 Table 3-2 Mean Effluent Concentrations (% v/v) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Nominal Mean Measured SD n Min imum Maximum 10% Target Effluent Effluent Range (% v/v) Concentration Concentration (% v/v) (% v/v) 0 (Control) 0.00 0.00 78 0.00 0.00 0.00 - 0.00 0.22 0.23 0.02 92 0.19 0.28 0.20 - 0.24 0.46 0.46 0.06 88 0.37 0.77 0.41 -0.51 1.0 1.01 0.10 87 0.67 1.28 0.90- 1.10 2.2 2.07 0.13 85 1.65 2.49 1.98-2.42 4.6 4.88 0.31 85 3.84 5.68 4.14-5.06 3.3 Water Quality Measurements Dissolved oxygen, temperature, pH, and salinity were measured in each treatment on 56 d during the experiment. 3.3.1 Dissolved Oxygen Mean dissolved oxygen concentrations were at or near saturation in all test treatment concentrations and were relatively stable during the study (Table 3-3 and Table 3-4). The highest dissolved oxygen concentrations were observed in the control and decreased with increasing effluent concentration. All treatments had mean DO saturation of >90%. However, the mean dissolved oxygen concentration in the 4.88% v/v treatment differed from the control by less than 59 1 mg/L or <5% saturation. The range of dissolved oxygen concentrations within each treatment was 1 mg/L or <20% saturation. Table 3-3 Dissolved Oxygen (DO) Concentrations (mg/L) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (n = 56) Effluent Concentration Mean SD Min imum Maximum (% v/v) 0 (Control) 8.5 0.2 7.9 8.9 0.23 8.3 0.2 8.0 8.8 0.46 8.3 0.2 7.9 8.7 1.01 8.2 0.2 7.8 8.8 2.07 8.1 0.2 7.7 8.7 4.88 7.9 0.2 7.5 8.4 Table 3-4 Dissolved Oxygen (DO) Concentrations (% Saturation) during the 89-d O n -Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (n = 56) Effluent Concentration Mean SD Min imum Maximum (% v/v) 0 (Control) 97.5 2.7 85.1 102.5 0.23 96.6 2.2 88.9 99.6 0.46 96.3 2.3 87.1 100.3 1.01 95.6 2.3 87.7 101.0 2.07 94.6 2.9 84.4 101.8 4.88 92.6 2.9 83.9 98.1 3.3.2 Temperature Water temperatures were relatively stable during the experiment (Table 3-5). Higher temperatures were observed with increasing effluent concentrations, however, the overall difference between the mean temperatures in the control and the 4.88% v/v treatment was 0.8°C. 60 Table 3-5 Test Temperatures ( °C ) Measured during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E l k Falls Division, Campbell River, B C , June to September 1999 (n = 56) Effluent Concentration (% v/v) Temperature SD Min imum Maximum 0 (Control) 12.6 1.1 10.3 14.3 0.23 12.7 1.1 10.5 14.3 0.46 12.9 1.0 10.5 14.5 1.01 13.0 1.1 10.5 14.5 2.07 13.1 1.1 10.5 14.5 4.88 13.4 1.1 10.5 15.3 3.3.3 p H pH measurements exhibited highest values in the control and decreased with increasing effluent concentration (Table 3-6). An average difference in 0.6 pH units between the control and the highest concentration was observed. During one measurement day, pH was not measured and therefore the number of measurements is 55 for pH, while for the other 3 measurements, there are 56 data points. Table 3-6 p H Measurements during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (n = 55) Effluent Concentration (% v/v) Mean SD Min imum Maximum 0 (Control) 7.6 0.2 .7.2 8.0 0.23 7.5 0.2 7.0 7.9 0.46 7.4 0.2 7.0 7.9 1.01 7.3 0.3 6.8 7.8 2.07 7.2 0.3 6.7 7.7 4.88 7.0 0.2 6.5 7.5 3.3.4 Salinity Salinity was also relatively stable during the study (Table 3-7). An average of a 3%o difference between the control and the 4.88% v/v treatment was observed, with higher effluent concentrations exhibiting slightly lower salinities. 61 Table 3-7 Salinity Measurements (%o) during the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (n = 56) Effluent Concentration (% v/v) Mean SD Min imum Maximum 0 (Control) 32 1 28 35 0.23 32 1 29 35 0.46 31 1 30 35 1.01 31 1 29 35 2.07 30 1 29 34 4.88 29 1 28 33 3.4 Musse l Data 3.4.1 Initial Lengths and Weights No statistically significant differences between treatments were observed at the commencement of the test in shell lengths and whole weights, including the T = 0 treatment (length: F 6 , 2 7 9 3 = 0.02, p = 0.99; weight F 6 ,2793 = 0.06, p = 0.99) (Table 3-8 and Table 3-9). The initial length and weight data had equal variances (Bartlett's test; p = 0.99) and were non-normally distributed (KSL test p < 0.05). Table 3-8 Mussel Lengths at Test Initiation of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Effluent n Mean S E M Min imum Maximum Concentration (% v/v) Length (mm) (mm) (mm) 0 (Control) 400 22.94 0.08 20.00 26.68 0.23 400 22.92 0.08 20.02 26.53 0.46 400 22.95 0.08 20.05 26.98 1.01 400 22.91 0.08 20.01 26.52 2.07 400 22.93 0.08 20.05 26.19 4.88 400 22.94 0.08 20.06 26.89 T = 0 400 22.93 0.08 20.00 26.51 6 2 Table 3-9 Mussel Weights at Test Initiation of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Effluent n Mean S E M Min imum Maximum Concentration (% v/v) Weight (g) (g) (g) 0 (Control) 400 1.22 0.01 0.73 1.93 0.23 400 1.21 0.01 0.70 1.96 0.46 400 1.22 0.01 0.75 1.97 1.01 400 1.21 0.01 0.71 1.90 2.07 400 1.21 0.01 0.69 2.10 4.88 400 1.21 0.01 0.62 2.05 T = 0 400 1.22 0.01 0.73 1.94 3.4.2 Survival / Mortality Several mussels were missing at the end of the experiment. Therefore, survival was calculated from the number of surviving mussels in each treatment divided by the number of mussels present at the end of the exposure period (Table 3-10). The mortality in the control at the end of the 89-d exposure period was 9% (Table 3-10, Figure 3-1). Mortality in mussels exposed to pulp mill effluent ranged from 15% in the lowest effluent concentration (0.23% v/v) to 31% in the highest effluent concentration (4.88% v/v) (Table 3-10, Figure 3-1). Statistically significant decreases in survival were observed at all effluent concentrations tested, as compared to the control, with the exception of the 0.23% v/v concentration (Z = 3.10 (0.46% v/v) to 7.43 (4.88% v/v; p < 0.05). However, the survival in 0.46 to 2.07% v/v effluent concentrations were >75%. The effluent concentration predicted to cause a 20% increase in mortality was estimated to be 3.80%, with confidence intervals of 2.09 to > 4.88% v/v (Table 3-11). The NOEC and LOEC, based on statistically significant differences from the control, were 0.23%> and 0.46% v/v effluent, respectively (Table 3-11). 63 Table 3-10 Summary of Survival and Mortality of Mussels at Test Termination of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Effluent Number at Number Number Number Survival Mortality Concentration Test Alive Dead Missing ± S E M ± S E M (% v/v) Termination (%) (%) 0 (Control) 381 346 35 19 91 ± 1.5 9 ± 1.5 0.23 376 321 55 24 85 ± 1.8 15 ± 1.8 0.46 383 319 64 17 83 ± 1.9f 17 ± 1.9 1.01 389 304 85 11 7 8 ± 2 . 1 f 22 ± 2 . 1 2.07 389 310 79 11 80 ± 2.0f 20 ± 2 . 0 4.88 396 275 121 4 6 9 ± 2 . 3 f 31 ± 2 . 3 ' Indicates a statistically significant difference from the control (Z= 3.10 (0.46% v/v) to 7.43 (4.88% v/v; p <0.05). Table 3-11 89-d L C 2 0 and L C 2 5 Values, 95% Confidence Intervals and N O E C and L O E C for the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 L C 2 0 95% Confidence L C 2 5 N O E C L O E C Intervals 3.80% v/v 2.09 - > 4.88% v/v > 4.88% v/v 0.23% v/v 0.46% v/v 64 Figure 3-1 Mortality of Mussels Exposed from the 89 d, June to September 1999 of the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C (mean ± S E M ; see Table 3-10 for n) (Hatched bars indicate a statistically significant difference as compared to the 0% v/v (Z= 3.10 (0.46% v/v) to 7.43 (4.88% v/v; p < 0.05)) 35% -| 30% 1 25% , rh T O 20% 1 i——i -0 0.23 0.46 1.01 2.07 4.88 Effluent Concentration (%v/v) 3.4.3 Growth 3.4.3.1 Growth, Based Changes in Length All of the treatments, with the exception of the highest effluent concentration, exhibited greater growth, based on differences in final and initial lengths, as compared to the control. This growth data had unequal variances (Bartlett's test; p = 0.0003) and were non-normally distributed (Shapiro-Wilk's Test,/? < 0.01). Statistically significant increases in growth between the 1.01 and 2.07% v/v treatments were observed (F5, , 8 6 9 = 7.49, p < 0.0001; Bonferroni's test p < 0.05) (Table 3-12, Figure 3-2). It was not possible to calculate an IC20/25, as a reduction in growth was only observed in the highest effluent concentration and the percentage difference as compared to 65 the control was only 1.3%. Therefore, the 89-d IC20 or 1C25 for growth, based on changes in length, is estimated to be > 4.88% v/v. 3.4.3.2 Growth, Based on Changes in Whole Weight The results for growth, based on changes in whole weight, were similar to the results observed for the changes in length. The growth data, based on whole weight data had unequal variances (Bartlett's test; p = 0.0023) and were non-normally distributed (Shapiro-Wilk's Test, p < 0.01). Statistically significant increases in growth between 0.23, 1.01, and 2.07% v/v treatments were observed (F5, i 8 6 9 = 8.42, p < 0.0001; Bonferroni's test p < 0.05) (Table 3-12, Figure 3-3). It was not possible to calculate an IC20 or IC25, based on a reduction in whole weight growth, as a reduction in growth was only observed in the highest effluent concentration and the difference as compared to the control was only 3.7%. Therefore, the 89-d IC20 or IC25 for growth, based on changes in whole weight, is estimated to be > 4.88% v/v. Table 3-12 Summary of Growth in Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Effluent Concentration (% v/v) n Change in Length Mean ± S E M (mm) Change in Whole Weight Mean ± S E M (g) 0 (Control) 346 5.20 ±0 .15 1.07 ± 0 . 0 3 0.23 321 5.76 ± 0 . 1 6 1.21 ± 0 . 0 4 T 0.46 319 5.36 ± 0 . 1 6 1.13 ± 0 . 0 4 1.01 304 6.31 ± 0 . 1 9 T 1 . 3 2 ± 0 . 0 4 f 2.07 310 5 . 8 4 ± 0 . 1 7 f 1.23 ± 0 . 0 4 f 4.88 275 5.13 ± 0 . 1 6 1.03 ± 0 . 0 4 Indicates a statistically significant difference from 0% v/v. 66 Figure 3-2 Growth, Based on Change in Length, in Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (mean ± S E M ; see Table 3-12 for n) Hatched bars indicate a statistically significant difference as compared to 0% v/v (F5, m 9 = 7.49,p < 0.0001; Bonfcrroni's test/; < 0.05) 7.00 0.23 0.46 1.01 Effluent Concentration (%v/v) 2.07 4.88 67 Figure 3-3 1.60 Growth, Based on Change in Whole Weight, in Mussels from the 89-d O n -Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (mean ± S E M , see Table 3-12 for n) Hatched bars indicate a statistically significant difference as compared to 0% v/v (F5, 1 8 6 9 = 8.42p < 0.0001; Bonferroni'sp < 0.05) 0.23 0.46 1.01 Effluent Concentration (%v/v) 2.07 4.88 3.4.4 Condition Index There were two less condition index data points, one each for the 0.46% v/v and 4.88% v/v treatments, because the shells from these two mussels were not measured and therefore it was not possible to calculate the condition indices. All of the treatments had a greater condition index, including the T = 0 treatment, as compared to the control. The condition index data had unequal variances (Bartlett's test; p = 0.0001) and were non-normally distributed (Shapiro-Wilk's Test,/? < 0.01). Statistically significant differences in condition index between all of the effluent treatments and the control were observed (F5, i 8 6 7 = 15.0/? < 0.0001; Bonferroni's test p < 0.05) (Table 3-13, Figure 3-4). In addition, the condition index in the T = 0 was statistically higher than the condition index of all the treatments at the end 68 of the 89-d exposure (F6, 2 2 66 = 140.74, p < 0.0001; Bonferroni's test, p < 0.05). It was not possible to calculate an IC20 or IC25 for condition index, as all of the treatments had higher condition indices than the control. Therefore, the 89-d IC20 or IC25 for condition index is estimated to be > 4.88% v/v. Table 3-13 Summary of Condition Index of Mussels from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Effluent Concentration (% v/v) n Condition Index Mean ± S E M 0 (Control) 346 0.98 ±0 .01* 0.23 321 1.04 ± 0.011"* 0.46 318 1 . 0 4 ± 0 . 0 1 f * 1.01 304 1 .09±0 .01 f * 2.07 310 1 .06±0 .01 t * 4.88 274 1 . 0 5 ± 0 . 0 1 r T = 0 400 1.30 ± 0.01T ' Indicates a statistically significant difference from 0% effluent * Indicates a statistically significant difference from T = 0. 69 Figure 3-4 Mussel Condition Index of Mussels Exposed from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (mean ± S E M ; see Table 3-13 for n) Hatched bars indicate a statistically significant difference as compared to 0% v/v (Bonferroni's test, p < 0.05) Asterisks indicate a statistically significant difference as compared to the T = 0 (Bonferroni's test,/> < 0.05) 1.40 0 0.23 0.46 1.01 2.07 4.88 T=0 Effluent Concentration (%v/v) 3.5 Lipids and Moisture 3.5.1 Lipids Lipid data, excluding the T = 0 treatment, were normally distributed (Shapiro-Wilk's p = 0.38) and exhibited equality of variances (Bartlett's test p = 0.60). Statistically significant decreases in lipid content between the 1.01, 2.07, and 4.88% v/v effluent treatments and the control were observed (F5, , 8 = 26.89,p < 0.0001; LSD testp < 0.05) (Table 3-14, Figure 3-5). The lipid data, including the T = 0 treatment were non-normally distributed (Shapiro-Wilk's p < 0.0001) and exhibited equality of variances (Bartlett's test p = 0.04). The lipid concentrations in all of the treatments were significantly lower than those observed in the T = 0 70 treatment (F6,21 = 146.71, J O < 0.0001; LSD test p < 0.05). The reduction in lipid concentration during the experiment ranged from 39% in the control to 51% in the 4.88% v/v effluent concentration. Table 3-14 Mean Lip id and Moisture Concentrations in Mussel Tissues including Baseline Mussels (T = 0), from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Effluent Concentration (% v/v) Lipids (%) a S E M Moisture (%) b S E M 0 (Control) 1.99T 0.04 81.8n 0.17 0.23 1.94* 0.02 81.8** 0.29 0.46 1.97t 0.03 81.4ft 0.32 1.01 1.83** 0.02 81.9** 0.09 2.07 1.84** 0.02 81.5" 0.25 4.88 1.58" 0.03 81.9Tt 0.39 T = 0 3.24 0.09 77.7 0.35 a Wet weight basis b Dry weight basis * Statistically significant difference from T = 0 (F 6 2 i = 146.71, LSD test p < 0.05) * Statistically significant difference from 0% v/v (F5 > 1 8 = 26.89, LSD test p < 0.05) ** Statistically significant difference from T = 0 (F6,2| = 28.69, LSD testp < 0.05) 71 Figure 3-5 Mean L ip id Concentrations in Mussel Tissues, including Baseline Mussels (T = 0) from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (% Wet Weight) (mean ± S E M , n = 4) Hatched bars indicate a statistically significant difference from 0% v/v (F5 j l 8 = 26.89; LSD test p < 0.05) Asterisks indicate a statistically significant difference from T = 0 (F 6 , 2i = 146.71; LSD test/) < 0.05) 0 0.23 0.46 1.01 2.07 4.88 T=0 Effluent Concentration (%v/v) 3.5.2 Moisture The moisture data, excluding the T = 0 group, were normally distributed (Shapiro-Wilk's p = 0.20) and exhibited equality of variances (Bartlett's p = 0.34). No statistically significant differences in moisture content were found between treatments (excluding T = 0) (F 5 i ] 8 = 0.62, p = 0.69). Increased moisture content in the control and the effluent treatments at the end of the experiment was statistically significant relative to the T = 0 treatment (F6,21 = 28.69, p < 0.0001; LSD testp < 0.05) (Table 3-14, Figure 3-6). 72 Figure 3-6 Mean Moisture Concentrations in Mussel Tissues, including Baseline Mussels (T = 0), from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 (mean ± S E M , n = 4) Asterisks indicate a statistically significant difference from T = 0 ( F W i = 28.69; LSD testp < 0.05) 0.23 0.46 1.01 2.07 Effluent Concentration (%v/v) T=0 3.6 Resin Acid Tissue Concentrations Resin acid concentrations in the mussel tissues were measured and only dehyroabietic acid was detectable in the 0.23, 1.01, 2.07, and 4.88% v/v effluent concentrations (Table 3-15). No resin acids were detected in the method blank. Method spike recoveries ranged from 48% for levopimaric acid to 124% for isopimaric acid. The recovery for dehydroabietic acid was 115%. Levopimaric and neoabietic acids can undergo thermal rearrangement during the analysis to palustric and abietic acid and therefore, the results for levopimaric and neoabietic may be lower than expected, while the results for abietic acid may be higher than expected (ASL 2000). 73 Table 3-15 Resin Ac id Concentrations in Mussel Tissues (ug/g wet weight basis), from the 89-d On-Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk September 1999 Falls Division, Campbell River, B C , June to Effluent Concentration 0% v/v 0.23% 0.46% 1.01% 2.07% 4.88% Resin Ac id (Control) v/v v/v v/v v/v v/v Abietic <0.02 <0.02 <0.02 <0.02 <0.02 <0.02 Chlorodehydroabietic <0.02 <0.02 <0.02 <0.02 <0.02 <0.02 Dehydroabietic <0.02 0.02 <0.02 0.09 0.08 0.15 Dichlorodehydroabietic <0.02 <0.02 <0.02 <0.02 <0.02 <0.02 Isopimaric <0.4 <0.2 <0.5 <0.4 <0.4 <0.4 Levopimaric <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 Neoabietic <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 Pimaric <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 Sandaracopimaric <2 <2 <2 <2 <2 <2 3.7 Quality Assurance/Quality Control 3.7.1 Mussel Measurements To determine and ensure the precision of measurements, Environment Canada (1997) recommends that 5% of the test organisms be re-measured. A ± 5% difference in the re-measured data is acceptable (Environment Canada 1997). At the onset of experiment, some of the mussels in the 0.46% and 1.01 % treatments were re-measured, which was approximately 7% of all the test organisms. At the end of the experiment, the first and eighth mussel in each bag were re-measured for length, whole weight, and shell weight, for a potential total of 336 (14%) mussels being re-measured. However, some of these mussels were dead or missing. Tissue weights were not re-measured due to the nature of the sample. All re-measured initial and final shell length data were within 1.2% (Table 3-16). Of the initial whole weight measurements, three of the duplicate measurements were >5%. Of the final whole weight measurements, seven of the duplicate measurements were >5%. For the final shell weights, four duplicate measurements were >5%. All of the re-measured T = 0 shell weights were 74 within 5% of each other. Therefore, very few data points deviated greatly from the standard of ± 5%. Table 3-16 Re-Measured Mussel Data (% Differences) for Mussels from the 89-d O n -Site Flow-Through Bioassay Using Marine Mussels, Conducted at Norske Canada, E lk Falls Division, Campbell River, B C , June to September 1999 Parameter Initial Shell Initial Final Shell Final Final Shell T = 0 Length Whole Length Whole Weight (g) Shell (mm) Weight (g) (mm) Weight (g) Weight (g) Mean 0.02 1.17 0.04 0.48 0.01 0.34 SD 0.18 1.22 0.15 1.22 2.79 0.94 Minimum -0.52 -2.41 -0.77 -0.32 -32.88 0.00 Maximum 1.17 6.71 0.69 11.41 11.31 3.70 n 199 199 311 256 236 56 3.7.2 Instrument Calibration Balances were calibrated daily using a 1 gram weight and were tared as necessary. Digital calipers were frequently set to zero. Instruments used to take daily measurements were calibrated each day before use. The pH meter was calibrated using pH 7 and 10 buffers; the dissolved oxygen meter was set to 100% saturation according to the temperature and salinity of the seawater; and the refractometer, used to measure salinity, was adjusted to zero using deionised water. 75 CHAPTER 4 DISCUSSION 4.1 Water Quality 4.1.1 Dissolved Oxygen In this study, mean DO concentrations were > 90% saturation. The minimum dissolved oxygen concentrations ranged from 84% saturation in the 4.88% v/v concentration to 89% saturation in the 0.23% v/v concentration. In addition, DO concentrations decreased with increasing effluent concentration. At 60 to 90% saturation, mussels are able to regulate oxygen consumption by increasing the efficiency at which the oxygen is removed from the water (Newell 1989). At dissolved oxygen concentrations decrease to < 90%, anaerobic pathways are used to maintain energy production, as necessary (Newell 1989). On June 10, 1999, all of the DO measurements were less than 90% saturation and this may have been a DO meter calibration problem. On all of the other measurement days, the DO in the 0 to 1.01% v/v treatments were > 90% saturation. There were an additional 3 d during the study that had DO concentrations of < 90% in the 2.07% v/v concentration and an additional 7 d in the 4.88% v/v. It is likely that the mussels used anaerobic pathways when the DO concentrations were < 90% saturation. However, as the majority of the DO concentrations were > 90% during the exposure, the mussels would have mainly used aerobic metabolism during the experiment. 76 4.1.2 Temperature Studies assessing the effect of water temperature on the growth of M. edulis have shown that temperatures between 10 - 2 0 ° C result in optimal mussel growth (Seed 1976). The test temperatures ranged from 10.3 to 15 .3°C and were well within this range for optimal growth. The range of temperatures in the test are more variable than what would be expected in a laboratory bioassay (e.g. ± 1°C); however, this is attributed to changes in ambient water temperatures over the exposure period. The mean temperatures increased with increasing effluent concentrations, which was a result of the incoming effluent temperature, which has an average discharge temperature 3 8 . 8 ° C (Table 1-6). 4.1.3 pH Mean test pH measurements ranged range during the study were 7.0 - 7.6. The pH of bivalve blood ranges from 7.2 - 7.7 and various enzymes have optimum activity between 7.0 - 8.0 (Seed 1976). Therefore, the measured pH values in the study were within the tolerance of the test organisms. The pH of the treatments decreased with increasing effluent concentration, as the average pH of the incoming effluent was 6.7 (Table 1-6). 4.1.4 Salinity Mean test salinity in the treatments ranged from 29 - 3296o. The minimum and maximum values for salinity were 28 - 35%o. The mean salinity of the treatments decreased with increasing effluent concentration. The difference in the minimum and maximum salinity measurement were widest in the control treatment and this would have been affected by dilution of the seawater with fresh water from, for example, freshwater inputs to Discovery Passage or increased precipitation. M. edulis, a euryhaline species, can survive at a wide range of salinities (e.g. 5 to 34%o) (Newell 1989) and similar growth was exhibited by mussels in 28.8%o as compared to 32%o salinity (Seed 7 7 and Richardson 1990). Therefore, the salinity values observed in this study were well within the tolerance of the test organisms. 4.1.5 T r e n d s in W a t e r Q u a l i t y M e a s u r e m e n t s As discussed in the previous sections, trends in the water quality measurements were observed between the treatments. These trends were decreasing DO concentrations, salinity, and pH and increasing temperature, with increasing effluent concentration. The reasons for these trends was that the incoming effluent had different water quality characteristics, such as lower dissolved oxygen, pH, and salinity, and higher temperature, as compared to the control/dilution water. It is not known how the different water quality characteristics may have impacted the survival, growth, condition index, and lipid content of the mussels in this study. However, it is expected that similar differences in the water quality in receiving environment near the effluent discharge point would also be observed. 4.2 Survival / Mortality The mortality in the control did not exceed 15%, as recommended by Environment Canada (1997). The high survival (91%) in the control indicated that the experimental conditions were suitable for mussel survival. The control survival was similar to that which is obtained in laboratory based toxicity tests. Typically, short-term (e.g. 48 h to 7 d) laboratory toxicity tests require minimum survival of 80 to 90% in the control to meet test validity criteria. In this study, a trend of increasing mortality was observed, with increasing effluent concentration (with the exception of the 2.07% v/v treatment). Environment Canada (1998) states that environmental samples should be considered to fail a laboratory toxicity test when there is a significant difference in survival in the control and the treatment groups and when the survival in the treatment group is greater than 20% different 78 from the control survival. Therefore, based on this criteria, only the highest effluent concentration showed an adverse effect on mussel survival, as the survival in this treatment was 69%. The maximum effluent concentration in the receiving environment within a 250 m radius of the effluent diffuser was conservatively estimated to be equal to or less than 1% (Hatfield Consultants Ltd. 1994). Therefore, reduced mussel survival may occur outside of 250 m of the effluent diffuser, if the effluent concentrations are approximately 0.5 to 1.0% v/v effluent. Most other long-term mussel studies using pulp mill effluent have shown high mussel survival. For example, Burggraaf et al. (1996) had 2% mortality in mussels (H. menziesi) transplanted for a maximum of 28 d near the outfall of a kraft pulp and paper mill. M. edulis survival was 95% in a 68 d experiment that involved placing mussels at various distances from the outfall of a sulphite pulp and paper mill (Salazar 2000). Similar survival was also observed in MytUus edulis after a 90 d exposure to sites downstream of a pulp and paper mill (St. Jean et al. 2003). Survival of mussels (Anodonta cygnea) transplanted downstream of a pulp and paper mill for 28 and 91 d had mortality of 7.5% and 4%, respectively (Hayer and Pihan 1996, 1998). Mussel survival was high after a 56 d exposure to 10, 20, 40, and 80% v/v pulp and papermill effluent in an outdoor, flow-through, experiment exposure (Kernaghan et al. 1999b). In addition, mussel survival was > 90% for a 28-d study using transplanted A. cygnea, U. pictorum, and D. polymorpha near the outfall of a pulp and paper mill (Hayer et al. 1996). Only one study was found where there was significant mortality in adult mussels exposed to pulp mill effluent (Wu and Levings 1976). However, the mussels in this experiment were exposed to untreated effluent, which is significantly more toxic than effluent that has undergone secondary treatment. 79 4.3 Growth The mussels in all concentrations, expect for the highest concentration, exhibited greater growth (based on changes in length and weight) than the control. The growth was significantly higher in the 1.01 and 2.07% v/v concentrations, relative to the control. In addition, the growth (based on whole weight) was also significantly higher in the 0.46% v/v treatment. The percentage increases in growth (based on length) ranged from 3% (0.46% v/v) to 21% (1.01% v/v) and the increases for growth (based on weight) were 6% (0.46%) to 23% (1.01%). This type of effect is a recognised response to low levels of toxic substances in bioassays (McNaught 1989, Moore and Farrar 1996, Calabrese and Baldwin 1998a & b, Calabrese 1999) and is known as hormesis. Hormesis is thought to be a short-term compensation of homeostatic regulatory mechanisms in response to an external challenge (Moore and Farrar 1996). However, at higher concentrations this effect is negated. Also, at lower concentrations, exposure to pulp mill effluent can result in increased growth and reproduction in Daphnia magna and Ceriodaphnia dubia (Kinnee, pers. obs.). Therefore, the increased growth observed in the pulp mill effluent exposed mussels could be also due to a fertiliser effect, as the pulp mill effluent can be a source of additional nutrients (e.g. algae, bacteria, suspended solids). These additional nutrients may have resulted the increases in mussel growth and condition index in some of the effluent treatments relative to the control. No effects on growth (based on length) were observed in freshwater mussels after 56 d exposure to 0 to 80% papermill effluent (Kernaghan et al. 1999b). However, the soft tissue index (% tissue as a % of total weight) was significantly lower in mussels placed on sediments and exposed to 80% effluent. However, some significant differences in dry weight of mussels near the pulp mill, as compared to the upstream site, were observed (Hayer and Pihan 1996). In addition, the whole weights and growth rates of mussels closest to the pulp mill were significantly lower in the mussels 0.3 km from the outfall, as compared to those at 3 and 10 km away, at the end of the exposure period (Salazar 2000). St. Jean et al. (2003) found increased growth in transplanted 80 mussels at the site closest to a pulp mill than mussels at the four stations which were further away from this outfall. It was suggested that the nutrients in the pulp mill effluent may explain the increased growth in the station closest to the outfall. 4.4 Condition Index and Lipid Concentrations Condition index is the ratio of tissue weight to shell weight, length, or volume and is indicative of the nutrient state of bivalves. It can be influenced by water temperature, season, food availability, and reproductive stage (Gabbott 1976, Bayne et al. 1985). High water temperatures and low food availability can lead to decreases in condition index (Gabbott 1976, Bayne et al. 1985). Condition index increases during energy storage and gametogenesis and decreases upon spawning (Bayne et al. 1985, Gosling 2003). Typically, condition index is low in the early months of the year, increases during the spring, and then decreases in the fall (Bayne et al. 1985). Condition index decreases when the metabolic rate exceeds that of the ingested food, and to compensate for this carbohydrate reserves (e.g. glycogen) are metabolised, followed by lipid and protein reserves (Gabbott 1976). A reduction of condition index can be a response to stress (Dame 1996). There was a significant decrease in the condition index and in the lipid content of the mussels over the experimental period. As the temperatures were within the tolerance ranges of the M. edulis, it is unlikely that it was a temperature mediated effect. Food availability may have been a limiting factor and the mussels may have used either their lipid or protein reserves to obtain energy (Gabbott 1976). The decrease in lipids during the study may also indicate that sufficient food was not available. However, as growth was observed in the mussels, limited food may not have caused the decrease in the condition index and lipid concentration. In addition, condition indices are reduced when spawning occurs (Bayne et al. 1985, Gosling 2003). Also, during spawning, up to 40% of the soft tissue mass can be lost in mussels (Cossa 1989) and 81 during gametogenesis, lipid reserves increase (Gabbott 1976). Some spawning was observed at the beginning of the experiment and some of the T = 0 mussels had ripe gonads, the reduction in condition index is likely due to spawning. In addition, it is possible that the experimental conditions, such as the density of mussels and the flow rates in the experimental units, may not have been the ideal for the mussels and hence is a reflection of a potential stressor. All of the treatments had increased condition indices as compared to the control at the end of the exposure period. Therefore, a hormetic or fertiliser effect was also observed for condition index, as it was with the growth endpoints. There was a significant decrease in the lipid content of the mussels exposed to 1.01 to 4.88% v/v effluent as compared to the control. The reduction in lipids ranged from 8.0% in 1.01% v/v effluent to 21% in 4.88% v/v effluent. Significant reductions in lipids could result in decreased gamete production and/or decreased survival, as mussels would have less lipid reserves from which to obtain energy for reproduction and during times of low food availability. However, it is not expected that decreased lipid concentrations would be observed in mussels in Discovery Passage, greater than 250 m from the diffuser, as the expected effluent concentrations outside of this radius, are less than 1%. No significant differences were found in physical condition or tissue lipids between mussels transplanted near pulp mill outfalls exposed for 28 to 91 d (Burggraaf et al. 1996, Hayer and Pihan 1996, 1998, St. Jean et al. 2003). No effects on condition index (total wet weight vs. shell length ratio) were observed after a 56 d exposure to a maximum concentration of 80% papermill effluent (Kernaghan et al. 1999b). Similar to this study, Salazar (2000) found significantly reduced lipid concentrations and condition indices in the mussels placed 0.3 and 3 km from the outfall of a sulphite pulp mill compared to those at the site farthest (10 km) from the pulp mill after a 68 d exposure period. Hayer et al. (1996) also observed decreased lipid 82 concentrations in mussels transplanted close to a pulp and paper mill. In addition, significant decreases in the lipid content of the gills and gonads of Anodonta cygnea were observed after a 91 d exposure near a pulp mill. 4 . 5 Resin Acid Tissue Concentrations Only dehydroabietic acid was in the mussel tissues. There was a slight increasing trend in the dehydroabietic acid concentration in the mussel tissues. The concentrations in the control and three lowest effluent concentrations were at or below the detection limit. The observed dehydroabietic acid concentrations were similar in the 1.01 and 2.07% v/v concentrations. The dehydroabietic acid concentration in the highest effluent concentration (4.88% v/v) was approximately twice as high at that measured in the 1.01 and 2.07% v/v concentrations. The presence of dehydroabietic acid in the mussel tissue is logical, as it is the most abundant resin acid (Leach and Thakore 1976, Leppanen et al. 1998). Burggraaf et al. (1996) observed dehydroabietic acid concentrations of 0.231 ± 0.044 ug/g (wet weight, ± SD) after a 28-d exposure to pulp mill effluent. Dehydroabietic acid was present in the mussel tissue at the highest concentration, as compared to five other resin acids (pimaric, isopimaric, abietic, dehydroabietic, 14-chlorodehydroabietic, and dichlorodehydroabietic acid). The concentration of these resin acids ranged from < 0.0033 |j.g/g (wet weight) for dichlorodehydroabietic acid to 0.176 ug/g (wet weight) for pimaric acid. The concentrations of resin acids in the mussel tissues at the end of the experiment may have been affected by the depuration period that occurred prior to the mussels being shucked. On day 89 of the experiment, at least one of peristaltic pumps that were used to deliver the effluent into the barrels, stopped working. The mussels were removed from the barrels on day 90, as scheduled, and transported back to Vancouver for processing. Upon arrival in Vancouver, the 83 mussels were placed into seawater from the Vancouver Aquarium until they were processed, which took two days. Therefore, the mussels were in seawater for three to four days prior to shucking. However, it is not recommended to depurate mussels whose tissues that will be analysed for resin acids, as the half-life of resin acid is approximately 3 d (Burggraaf et al. 1996). Therefore, the resin acid concentrations in the tissues may have been greater than two times higher than those observed, if the tissues were sampled immediately after the effluent exposure was terminated. 4.6 Improvements to Study Design There are several improvements that could be made to the overall design of this study that should be considered, if a similar experiment was to be conducted in the future. Firstly, the use of more than one experimental unit per treatment would allow statistical comparisons to be made both within treatments and between treatments. By having replicated treatments, the number of mussels per test vessel could also be reduced, reducing the loading density of the mussels in the test vessels. Secondly, the overall number of mussels used could be reduced in future experiments, as absolute differences of <10% in survival and growth resulted in statistically significant differences, as compared to the control group. St. Jean et al. (2003) showed that the use of 78 mussels per treatment would detect a 10% difference in growth. The processing of the mussels at test initiation and termination was very labour intensive, which required three people three days at test initiation and about 9 people over two days at test termination to process all of the mussels. Therefore, using fewer mussels would reduce the amount of time needed to measure the mussels at test initiation and termination. In addition, mussels who were not going to spawn during the exposure period should be used in future studies, so that major changes in mussel condition would not occur during the study. 84 Several different endpoints could also be measured in future studies to determine whether exposure to pulp mill effluent had for example, molecular, cellular, immunological, and reproductive effects on marine mussels. The induction of MFO in fish after exposure to pulp mill has been measured (Hewitt et al. 1996, Hodson 1996) and the use of these enzymes has been proposed as biomarker in mussels exposed to anthropogenic chemicals (Livingstone et al. 2000). At the cellular level, histological examination of bivalve tissues for changes after exposure to pulp mill effluent, as damage in digestive cells after exposure to a mixture of resin acids has been observed in a laboratory experiment (Fahraeus-Van Ree and Payne 1999). Immune system effects could be determined by measuring phagocytic activity, lysosome retention, and conducting bacterial challenge tests (St. Jean et al. 2003). Reproductive endpoints that have been used in other mussel studies include sex steroid analysis and counting the number of gravid females (Kernanghan et al. 1999b). It may also be useful to determine the effects of pulp mill effluent on juvenile bivalves, as one laboratory study showed reduced survival of juvenile bivalves after exposure to pulp mill effluent (McKinney and Wade 1996). To confirm actual exposure and to relate these exposures to effects, concentrations of various pulp mill effluent constituents such as plants sterols, such as campesterol (Salazar 2000), EOX, and resin acids and their degradation products, such as retene and fichtelite (Burggraaf et al. 1996) could be measured. No depuration period should be used as many pulp mill effluent constituents have relatively short half-lives (Makela et al. 1991, Burggraaf et al. 1996). 4.7 Conclusions The survival in the controls at the end of the study was >90%, which indicates that the study design was suitable for mussel survival. Significantly reduced survival was observed in effluent concentrations of 0.46 and 1.01% v/v. No reductions in growth or condition index, as compared to the control were observed during the 89-d study. In fact, increases in growth and 85 condition index were observed in some of the effluent treatments, as compared to the control mussels. The reductions in the condition index and lipid content of the mussels at the beginning of the study, as compared to the control mussels at the end of the study, were likely due to the mussels spawning during the exposure period. The lipid content of the mussels exposed to 1.01 to 4.88% v/v effluent were significantly lower, relative to the control mussel lipid content. Based on the results of this study, survival and lipid content were the most sensitive parameters measured, as significant reductions in both parameters were observed as compared to the control organisms. This pulp mill effluent discharge may have an adverse effects on the long-term health of marine mussels, based on observed reductions in survival, if they are continually exposed to effluent concentrations of > 0.5% v/v. It is not expected that reduced lipid content in native mussels in Discovery Passage would be observed, as effluent concentrations exceeding 1% are not likely to occur outside of a 250 m radius of the effluent discharge. 86 LITERATURE CITED Almada-Villela, PC, Davenport, J and Gryffydd, LD. 1982. The effects of temperature on the shell growth of young MytUus edulis L. Journal of Experimental Marine Biology and Ecology 59:275-288. ASL. 2000. Elk Fall E E M Tissue Analysis Report. EF716. ASL File No. L3416. Vancouver, BC. Association of Official Analytical Chemists Methods. 1995. Fat in foods. Chloroform-methanol extraction method. Volume 45.4.02, Chapter 45, p. 64. Bailey, HC, Elphick, JR, Potter, A, Chao, E, Konasewich, D, and Zak, JB. 1999. Causes of toxicity in stormwater runoff from sawmills. Environmental Toxicology and Chemistry 18(7):1485-1491. Bayne, BL, Brown, DA, Burns, K, Dixon, DR, Ivanovici, A, Livingstone, DR, Lowe, DM, Moore, MN, Stebbing, ARD, and Widdows, J. 1985. The effects of stress and pollution on marine animals. Praeger Scientific, New York, NY, USA. Bayne, BL, Hawkins, AJS, Navarro, E and Iglesias, JIP. 1989. Effects of seston concentration on feeding, digestion, and growth in the mussel, MytUus edulis. Marine Ecology Progress Series 55:47-54. BC Ministry of Environment, Lands and Parks. 1994. British Columbia Field Sampling Manual -for Continuous Monitoring and the Collection of Air, Air-Emission, Water, Wastewater, Soil, Sediment and Biological Samples. 1994 Ed. Laboratory Services, Environmental Protection Department, BC Ministry of Environment, Lands and Parks, and the BC Quality Assurance Users Committee, Victoria, BC. Borton, DL, Streblow, WR, Bradley, WK, Bousquet, T, Van Veld, PA, Wolke, RE, Walsh, A H . 1996. Survival, growth, production and biomarker responses of fish exposed to high-substitution bleached kraft mill effluent in experimental streams. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 473-482. Broman, D. and Ganning, B. 1985. Bivalve mollusks (MytUus edulis and Macoma balticd) for monitoring diffuse oil pollution in a Baltic archipelago. Ambio 14:23-28. Burggraaf, S, Langdon, A G , Wilkins, A L , and Roper, DS. 1996. Accumulation and depuration of resin acids and fichtelite by the freshwater mussel, Hydridella menziesi. Environmental Toxicology and Chemistry 15(3):369-375. 87 Calabrese, EJ and Baldwin, LA. 1998a. A general classification of U-shaped dose-response relationships in toxicology and their mechanistic foundations. Human and Experimental Toxicology, 17:353-364. Calabrese, EJ and Baldwin, LA. 1998b. Hormesis as a biological hypothesis. Environmental Health Perspectives, 106(Suppl. l):357-362. Calabrese, EJ. 1999. Evidence that hormesis represents an "overcompensation" response to a disruption in homeostasis. Ecotoxicology and Environmental Safety, 42:135-137. Carlberg, GE and Stuthridge, TR. 1996. Environmental fate and distribution of substances. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 169-178. Chemical Land 21. Tall Oil. Viewed on May 3, 2005. http://www.chemicalland21.com/ arokorhi/specialtychem/finechem/TALL%20OIL.htm. Cossa, D, Bourget, D, Pouliot, D, Puize, J, and Chanut JP. 1980. Geographical and seasonal variation in the relationship between trace metal mussel content and body weight in MytUus edulis. Marine Biology 58:7-14 Cossa, D. 1989. A review of the use of MytUus sp. as quantitative indicators of cadmium and mercury contamination in coastal waters. Oceanologica Acta 12(4):417-432. Craig, GR, Orr, PL, Robertson, JL, and Vrooman, WM. 1990. Toxicity and bioaccumulation of AOX and EOX. Pulp and Paper Canada 91(9):39-45. Dame, RF. 1996. Ecology of Marine Bivalves: An Ecosystem Approach. CRC Press, Boca Raton, FL. Davies, IM and Pirie, JM. 1978. The mussel MytUus edulis as a bioassay organism for mercury in seawater. Marine Pollution Bulletin 9:128-132. De Kock, WC. 1983. Accumulation of cadmium and polychlorinated biphenyls by MytUus edulis transplanted from pristine water into pollution gradients. Canadian Journal of Fisheries and Aquatic Sciences 40(Suppl. 2):282-294. Dwernychuk, LW. 1990. Effluent, receiving water, bottom sediments and biological tissues: a baseline organochlorine survey. January/February 1990. Prepared for Fletcher Challenge Canada Ltd., Elk Falls Pulp and Paper, Campbell River, BC, Hatfield Consultants Ltd., West Vancouver, BC. As cited in: Hatfield Consultants Ltd. 1994. Elk Falls Environmental Effects Monitoring (EEM) Pre-Design Reference Document. Hatfield Consultants Ltd., West Vancouver, BC. Eklund, B, Linde, M, and Tarkpea, M. 1996. Comparative assessment of the toxic effects from pulp and paper mill effluents to marine and brackish water organisms. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 95-106.. 88 Environment Canada. 1997. Draft guide for conducting field bioassays with marine, estuarine & freshwater bivalves, Applied Biomonitoring, Kirkland WA, USA and EVS, Seattle, WA. Environment Canada. 1998. Biological Test Method: Reference Method for Determining Acute Lethality of Sediment to Marine or Estuarine Amphipods. Report EPS l/RM/35. Method Development and Application Section, Environmental Technology Centre, Environment Canada. Ottawa, ON. Environment Canada. 2003. National Assessment of Pulp and Paper Environmental Effects Monitoring Data: A Report Synopsis. National Water Research Institute, Burlington, ON. NWRI Scientific Assessment Report Series No. 2. 28 p. Environment Canada. The National Environmental Effects Monitoring Office, viewed March 26, 2005. http://www.ec.gc.ca/eem/English/PulpPaper/data/millresults.cfm Fahraeus-Van Ree, GE and Payne, JF. 1999. Enzyme cytochemical responses of mussels (Mytilus edulis) to resin acid constituents of pulp mill effluents. Bulletin of Environmental Contamination and Toxicology 63:430-437. Foe, C and Knight, A. 1987. Assessment of the biological impact of point source discharges employing Asiatic clams. Archives of Environmental Contamination and Toxicology 16(l):39-52. Foster, RB and Bates, JM. 1978. Use of freshwater mussels to monitor point sources industrial discharges. Environment Science and Technology. 12:958-961. Gabbott, PA. 1976. Energy Metabolism. In Bayne, BL, eds, Marine Mussels: Their Ecology and Physiology, International Biological Programme 10. Cambridge University Press, Cambridge, England, pp. 293 - 356. Geyer, H, Sheehan, P, Kotzias, D, Freitag, D, and Kortez, F. 1982. Prediction of ecotoxicological behaviour of chemicals: relationship between physio-chemical properties and bioaccumulation of organic chemicals in the mussel, Mytilus edulis. Chemosphere 11:1121-1134. Gifford, JS. 1996. Recent advances in environmental fate of chemicals from pulp mills. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 271-282. Gilek, M., M . Bjork, and C. Naf. 1996. Influence of body size on the uptake, depuration, and bioaccumulation of polychlorinated biphenyl congeners by Baltic Sea blue mussels, Mytilus edulis. Marine Biology. 125:499-510. Goldberg, ED, Bowen, VT, Farrington, JW, Harvey, G, Martin, JH, Parker, PL, Risebrough, RW, Robertson, W, Schneider, E and Gamble, E. 1978. The mussel watch. Environmental Conservation 5:101-125. Gordon, M , Knauer, G. and Martin, JH. 1980. Mytilus californianus as a bioindicator of trace metal pollution: variability and statistical considerations. Marine Pollution Bulletin. 11:195-198. 89 Gosling, EM. 2003. Bivalve Molluscs: Biology, Ecology and Culture. Fishing News Books, Oxford, UK. 443 p. Government of BC. Ministry of Sustainable Resource Management. Free Stuff, Map of BC, viewed March 26, 2005. http://srmwww.gov.bc.ca/bmgs/2mil/bcmap.gif. Government of BC. Ministry of Water, Land and Air Protection. Industrial Waste. Pulp, Paper And Lumber. Map of Pulp Mills in BC. Viewed March 26, 2005. http://wlapwww.gov.bc.ca/epd/epdpa/ industrial_waste/forestry/map_pmills.html. Government of Canada. 1992. Fisheries Act: Pulp and Paper Effluent Regulations. Updated to August 31, 2004. Viewed May 1, 2005. http://laws.justice.gc.ca/en/F-14/SOR-92-269/121652.html Graney, RL, Giesy, JP, and Clark, JR. 1995. Field Studies. In Rand, GM. Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment. 2n d Ed. Taylor and Francis, Washington, DC. Grout, JA and Levings, CD. 2001. Effects of acid mine drainage from an abandoned copper mine, Britannia Mines, Howe Sound, British Columbia, Canada, on transplanted blue mussels (Mytilus edulis). Marine Environmental Research 51:265-288. Gustavson, K and Jonsson, P. 1999. Some halogenated organic compounds in sediments and blue mussel (Mytilus edulis) in Nordic Seas. Marine Pollution Bulletin 38(8):723-736. Hall, TJ, Haley, RK, Borton, DL and Bousquet, T M. 1996. The use of chronic bioassays in characterizing effluent quality changes for two bleached kraft mills undergoing process changes to increased chlorine dioxide substitution and oxygen delignification. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 53-68. Hatfield Consultants Ltd. 1994. Elk Falls Environmental Effects Monitoring (EEM) Pre-Design Reference Document. Hatfield Consultants Ltd., West Vancouver, BC. Hatfield Consultants Ltd. 2000. Fletcher Challenge Canada Ltd., Elk Falls Pulp and Paper. Environmental Effects Monitoring (EEM) Cycle Two Interpretative Report, 1997 to 2000. Hatfield Consultants Ltd., West Vancouver, BC. Hayer, F and Pihan, JC. 1996. Accumulation of extractable organic halogens (EOX) by the freshwater mussel, Anodonta cygnea L. exposed to chlorine bleached pulp and paper mill effluents. Chemosphere 32(4):791-803. Hayer, F, Wagner, P, and Pihan, JC. 1996. Monitoring of extractable organic halogens (EOX) in chlorine bleached pulp and paper mill effluents using four species of transplanted aquatic mollusks. Chemosphere 33(11): 2321-2334. Hayer, F and Pihan, JC. 1998. Accumulation of extractable organic halogens (EOX) in different organs of the freshwater mussel, Anodonta cygnea L. exposed in situ to chlorine bleached pulp and paper mill effluents. Annales de Limnologie 34(4):375-386. 90 Hemingway, RW and Greaves, H. 1973. Biodegradation of resin acid sodium salts. Tappi 56(12):189-192. Herve, S. Heinonen, P, Paukku, R, Knuutila, M, Koistinen, J and Paasivirta, J. 1988. Mussel incubation method for monitoring organochlorine pollutants in watercourses. Four-year application in Finland. Chemosphere 17(10): 1945-1961. Herve, S, Prest, H, Heinonen, P, Hyorylainen, Y, Koistinen, J, and Paasivirta, J. 1995. Lipid-filled semipermeable membrane devices and mussels as samplers of organochlorine compounds in lake water. Environmental Science and Pollution Research 2(l):24-30. Herve, S, Heinonen, P, and Paasivirta, J. 1996. Monitoring trends in chlorophenolics in Finnish pulp mill recipient watercourses by bioaccumulation in incubated mussels. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental fate and effects of pulp and paper mill effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 335-340. Herve, S, Paasivirta, J, and Heinonen, P. 2001. Trends of organochlorine compounds in Finnish inland waters: results of mussel incubation monitoring 1984 - 1998. Environmental Science & Pollution Research 8(1): 19-26. Herve, S, Heinonen, P, and Paasivirta, J. 2002. Survey of organochlorines in Finnish watercourses by caged mussel method. Resources, Conversation and Recycling 35:105-115. Hewitt, L M , Carey, JH, Dixon, DG, and Munkittrick, KR. 1996. Examination of bleached kraft mill effluent fractions for potential inducers of mixed function oxygenase activity in rainbow trout. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 79-94. Hickey, CW, Buckland, SJ, Hannah, DJ, Roper, DS, and Stuben, K. 1997. Polychlorinated biphenyls and organochlorine pesticides in the freshwater mussel Hyridella menziesi from the Waikato River, New Zealand. Bulletin of Environmental Contamination and Toxicology 59:106-112. Hodgins, D.O. and Knoll, M. 1991. Effluent dispersion study for the Elk Falls Pulp and Paper mill in Discovery Passage, British Columbia. Prepared for Fletcher Challenge Canada, Elk Falls Pulp and Paper, Campbell River, BC Seaconsult Marine Research Ltd., Vancouver, BC. As cited in Hatfield Consultants Ltd. 1994. Elk Falls Environmental Effects Monitoring (EEM) Pre-Design Reference Document. Hatfield Consultants Ltd., West Vancouver, BC. Hodson, PV. 1996. Mixed function oxygenase induction by pulp mill effluents: Advances Since 1991. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 349-358. Holmbom, B. 1977. Improve gas chromatographic analysis of fatty and resin acid mixtures with special reference to tall oil. Journal of the American Oil Chemists Society 54:289-293. 91 Hurlbert, SH. 1984. Pseudoreplication and the design of ecological field experiments. Ecological Monographs 54(2): 187-211. Kallqvist, T, Carlberg, GE, and Kringstad, A. 1989. Ecotoxicological characterization of industrial wastewater - sulphite pulp mill with bleaching. Ecotoxicology and Environmental Safety 18:321-336. Kauss, PB and Hamdy, YS. 1985. Biological monitoring of organochlorine contaminants in the St. Clair and Detroit Rivers using introduced clams, Elliptio complanatus. Journal of Great Lakes Research. 11(3):247-263. Kernaghan, NJ, Ruessler, DS, Miles, CJ, and Gross, TS. 1999a. Bioaccumulation of methyl mercury by the freshwater mussel, Elliptio buckleyi. Paper presented at the SETAC Meeting, Philadelphia, PA. November 1999. Kernaghan, NJ, Ruessler, DS, Wiebe, JJ, Wieser, C M , Quinn, BP, Holm, S and Gross, TS. 1999b. An evaluation of the potential effects of papermill effluents on freshwater mussels. Paper presented at the SETAC Meeting, Philadelphia, PA. November 1999. Koehn, RK. 1991. The genetics and taxonomy of species in the genus Mytilus. Aquaculture 94(2/3): 125-145. Kovacs, TG, Gibbons, JS, Tremblay, LA, O'Connor, BI, Martel, PH, and Voss, RH. 1995. The effects of a secondary-treated bleached kraft mill effluent on aquatic organisms as assessed by short-term and long-term laboratory tests. Ecotoxicology and Environmental Safety 31:7-22. Kovacs, T, Martel, P, O'Connor, B and Gibbons, S. 1997. Effluent-related benefits derived from the process and treatment changes implemented by the Canadian Pulp and Paper Industry in the 1990s. Miscellaneous Report MR367, Pulp and Paper Research Institute of Canada, Pointe-Claire, QC, Canada. Kraak, MHS, Toussaint, M, Lavy, D, and Davids, C. 1994. Short-term effects of metals on the filtration rate for the zebra mussel, Dreissena polymorpha. Environmental Pollution. 84:139-143. LaFleur, LE. 1996. Sources of pulp and bleaching derived chemicals in effluents. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 21-32. Landner, L, Grahn, O, Hardig, J, Lehtinen, KJ, Monfelt, C and Tana, J. 1994. A field study of environmental impacts at a bleached kraft pulp mill site on the Baltic Sea coast. Ecotoxicology and Environmental Safety 27:128-157. Latouche, YD, and Mix, MC. 1982. Effects of depuration size and sex on trace metals levels in bay mussels. Marine Pollution Bulletin 13:27-39. Lawrence, A.J and Nicholson, B. 1998. The use of stress proteins in Mytilus edulis as indicators of chlorinated effluent pollution. Water Science and Technology 38(7):253-261. 92 Leach, JM and Thakore, AN. 1976. Toxic constituents in mechanical pulping effluents. Tappi 59(2):129-132 Leach, JM and Thakore, AN. 1977. Compounds toxic to fish in pulp mill waste streams. Progress in Water Technology 9:787-798. Lee, HB and Peart, TE. 1990. Gas chromatographic and mass spectrometric determination of some resin and fatty acids in pulpmill effluents as their pentafluorobenzyl ester derivatives. Journal of Chromatography 498:367-379. Lee, HB and Peart, TE. 1992. Supercritical carbon dioxide extraction of resin and fatty acids from sediments at pulp mill sites. Journal of Chromatography 594(l/2):309-315. Lee, RF. 1981. Mixed function oxygenases (MFO) in marine invertebrates. Marine Biology Letters 2:87-105. Lehtinen, KJ. 1996. Biochemical responses in organisms exposed to effluents form pulp production: are they related to bleaching? In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 359-368. Leppanen, H, Martinen, S and Oikari, A. 1998. The use of fish bile metabolite analyses as exposure biomarkers to pulp and paper mill effluents. Chemosphere 36(12):2621-2634. Leppanen, H and Oikari, A. 1999. Occurrence of retene and resin acids in sediments and fish bile from a lake receiving pulp and paper mill effluents. Environmental Toxicology and Chemistry 18(7):1498-1505. Li, K, Chen, T, Bicho, P, Breuil, C and Saddler, JN. 1996. A comparison of gas chromatographic and immunochemical methods for quantifying resin acids. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental fate and effects of pulp and paper mill effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 119-127. Livingstone, DR and Pipe, RK. 1992. Mussels and environmental contaminants: molecular and cellular aspects. In: Gosling, E. 1992. The Mussel MytUus: Ecology, Physiology, Genetics, and Culture. Elsevier Science Publishers, Amsterdam, Netherlands. Livingstone, DR, Chipman, JK, Lowe, DM, Minier, C, Mitchelmore, CL, Moore, MN, Peters, LD, and Pipe, RK. 2000. Development of biomarkers to detect the effects of organic pollution on aquatic invertebrates: recent molecular, genotoxic, cellular and immunological studies on the common mussel (MytUus edulis L.) and other mytilids. International Journal of Environment and Pollution 13(l-6):56-91. Mahood, HW and Rogers, IH. 1975. Separation of resin acids from fatty acids in relation to environmental studies. Journal of Chromatography 109(2):281-286. Makela, P, and Oikari, AOJ. 1990. Uptake and body distribution of chlorinated phenolics in the freshwater mussel, Anodonta anatina L. Ecotoxicology and Environmental Safety 20:354-362. 93 Makela, TP, Petanen, T, Kukkonen, J, and Oikari, AOJ. 1991. Accumulation and depuration of chlorinated phenolics in the freshwater mussel (Anodonta anatina L.). Ecotoxicology and Environmental Safety. 22:153-163. McDowell Capuzzo JM, Farrington, JW, Rantamaki, P, Clifford, CH, Lancaster, BA, Leavitt, DF, Jia, X. 1989. The relationship between lipid composition and seasonal distribution of PCBs in Mytilus edulis L. Marine Environmental Research 28:259-264. McKinney, AD and Wade, DC. 1996. Comparative response of Ceriodaphnia dubia and juvenile Anodonta imbellicis to pulp and paper mill effluents discharged to the Tennessee River and its tributaries. Environmental Toxicology and Chemistry 15(4):514-517. McLeay, D and Associates Ltd. 1987. Aquatic toxicity of pulp and paper mill effluent: a review. Report EPS 4/PF/l. Environment Canada, Fisheries and Oceans Canada, Canadian Pulp and Paper Association, and Ontario Ministry of Environment. McLeese, DW, Zitko, V and Sergeant, DB. 1980. Uptake and excretion of aminocarb, nonylphenol and pesticide diluent 585 by mussel (Mytilus edulis). Bulletin of Environmental Contamination and Toxicology 24:575-581. McNaught, DC. 1989. Functional bioassay utilizing zooplankton: a comparison. Hydrobiologia, 188/189:117-121. Metcalfe, J.L. and M.N. Charlton. 1990. Freshwater mussels as biomonitors for organic industrial contaminants and pesticides in the St. Lawrence River. The Science of the Total Environment. 97/98:595-615. Microsoft Corporation. 1985-1997. Microsoft® Excel 97 SR-2. Microsoft Corporation, Redmond, WA. Miettinen, V, Lonn, BE and Oikari, A. 1982. Effects of biological treatment on toxicity for fish of combined debarking and kraft pulp bleaching effluent. Paperi Puu 64(2):251-254. Mikac, N, Kwokal, Z, Martincic, D, and Branica, M. 1996. Uptake of mercury species by transplanted mussels Mytilus galloprovincialis under estuarine conditions (Krka river estuary). The Science of the Total Environment 184L 173-182. Moles, A and Hale, N. 2003. Use of physiological responses in Mytilus trossulus as integrative bioindicators of sewage pollution. Marine Pollution Bulletin 46:954-958. Moore, DS and McCabe, GP. 1999. Introduction to the Practice of Statistics. 3rd Ed. WH Freeman and Company, New York, NY. Moore, DW and Farrar, JD. 1996. Effect of growth on reproduction in the freshwater amphipod Hyalella azteca (Saussure). Hydrobiologia, 328:127-134. Morales, A., Birkholz, DA, and Hrudey, SE. 1992. Analysis of pulp mill effluent contaminants in water, sediment, and fish bile-fatty and resin acids. Water Environment Research 64(5):660-668. 94 Morton, B. 1992. The evolution and success of the heteromyarian form in the Mytiloida. In: Gosling, E. 1992. The Mussel MytUus: Ecology, Physiology, Genetics, and Culture. Elsevier Science Publishers, Amsterdam, Netherlands. Muir, DCG and Servos, MR. 1996. Bioaccumulation of Bleached Kraft Pulp and Paper Mill Related Organic Chemicals by Fish. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 283-296. Muncaster, BW, Innes, DJ, Hebert, PDN, and Haffher GD. 1989. Patterns of organic contaminant accumulation by freshwater mussels in the St. Clair River, Ontario. Journal of Great Lakes Research. 15(4):645-653. Muncaster, BW, Hebert, PDN, and Lazar, R. 1990. Biological and physical factors affecting the body burden of organic contaminants in freshwater mussels. Archives of Environmental Contamination and Toxicology. 19:25-34. Newell, R.I.E. 1989. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (North and Mid-Atlantic) - blue mussel. US Fish and Wildlife Service Biological Report 82(11, 102) US Army Corps of Engineers, TR El-82-4. 25 pp. Oikari, A and Nakari, T. 1982. Kraft pulp mill effluent components cause liver dysfunction in trout. Bulletin of Environmental Contamination and Toxicology 28:266-270. Oikari, A, Holmbom, B, and Bister, H. 1982. Uptake of resin acids into tissues of trout (Salmo gairdneri Richardson). Annales Zoologici Fennici 19:61-64. Oikari, A, Anas, E, Kryzynski, G, and Holmbom, B. 1984a. Free and conjugated resin acids in the bile of rainbow trout, Salmo gairdneri. Bulletin of Environmental Contamination and Toxicology 33:233-240. Oikari, A, Nakari, T. and Holmbom, B. 1984b. Sublethal actions of simulated kraft pulp mill effluents (KME) in Salmo gairdneri: residues of toxicants, and effects on blood and liver. Annales Zoologici Fennici 21:45-53. Oikari, A, and Kunnamo-Ojala, T. 1987. Tracing of xenobiotic contamination in water with the aid of fish bile metabolites: a field study with caged rainbow trout (Salmo gairdneri). Aquatic Toxicology 9:327-341. Oikari, A, Fragoso, N, Leppanen, H, Chan, T, and Hodson, PV. 2002. Bioavailability to juvenile rainbow trout (Oncorhynchus mykiss) of retene and other mixed-function oxygenase-active compounds from sediments. Environmental Toxicology and Chemistry 21:121-128. Owens, JW. 1991. The hazard assessment of pulp and paper effluents in the aquatic environment: a review. Environmental Toxicology and Chemistry 10:1511-1540. Pellinen, J, Kukkonen, J, Herb, A, Makela, P and Oikari, A. 1993. Bioaccumulation of pulp mill effluent-related compounds in aquatic animals. The Science of the Total Environment, Supplement 499-510. 95 Peven, CS, Uhler, AD, and Querzoli, FJ. 1996. Caged mussels and semipermeable devices as indicators of organic contaminant uptake in Dorchester and Duxbury Bays, Massachusetts. Environmental Toxicology and Chemistry 15(2): 144-149. Pillai, MC, Blethrowb, H, Higashi, RM, Vines, CA and Cherr, GN. 1997. Inhibition of the sea urchin sperm acrosome reaction by a lignin-derived macromolecule. Aquatic Toxicology 37: 139-156. Prest, HF. Richardson, BJ, Jacobson, LA, Vedder, J and Martin, M . 1995. Monitoring organochlorines with semi-permeable devices (SPMDs) and mussels (Mytilus edulis) in Corio Bay, Victoria, Australia. Marine Pollution Bulletin 30(8), 543-554. Priha, MH. 1996. Ecotoxicological impacts of pulp mill effluents in Finland. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 637-650. Pruell, RJ, Lake, JL, Davis, WR, and Quinn, JG. 1986. Uptake and depuration of organic contaminants by blue mussels (Mytilus edulis) exposed to environmentally contaminated sediments. Marine Biology. 91:497-507. Pugsley, CW, Hebert, PDN, Wood, GW, Brotea, G, and Obal, TW. 1985. Distribution of contaminants in clams from the Huron-Erie corridor: PCBs and octachlorostyrene. Journal of Great Lakes Research. 11(3):275-289. Rantio, T., Koistinen, J. and Paasivirta, J. 1996. Bioaccumulation of pulp chlorobleaching-originated aromatic chlorohydrocarbons in recipient watercourses. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental fate and effects of pulp and paper mill effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 341-345. Roberts, D. 1975. Sub-lethal effects of chlorinated hydrocarbons on bivalves. Marine Pollution Bulletin. 6:20-24. Robinson, RD, Carey, JH, Solomon, Kr, Smith, IR, Servos, MR, and Munkittrick, KR. 1994. Survey of receiving environmental impacts associated with discharges from pulp mills. 1. Mill characteristics, receiving-water chemical profiles and lab toxicity tests. Environmental Toxicology and Chemical 13(7): 1075-1088. Saint-Jean, SD, Courtenay, SC, and Parker, RW. 2003. Immunodulation in blue mussels (Mytilus edulis) exposed to a pulp and paper mill effluent in Eastern Canada. Water Quality Research Journal of Canada 38(4):647-666. Salanki, J, Farkas, A, Kamardina, T and Rozsa, KS. 2003. Molluscs in biological monitoring of water quality. Toxicology Letters 140-141:403-410. Salazar, M H and Salazar, SM. 1991. Assessing site-specific effects of TBT contamination with mussel growth rates. Marine Environmental Research. 32:131-150. Salazar, MH and Salazar, SM. 1995. In-situ bioassays using transplanted mussels: I. Estimating chemical exposure and bioeffects with bioaccumulation and growth. Environmental 96 Toxicology and Risk Assessment-3 volume, ASTM STP 1218, Hughes, JS, Biddinger, GR and Mones, E, eds. American Society for Testing and Materials, Philadelphia, PA. pp. 216-241. Salazar, MH, Duncan, PB, Salazar, SM and Rose, KA. 1995. In-situ bioassays using transplanted mussels: II. Assessing contaminated sediment at a Superfund site in Puget Sound. Environmental Toxicology and Risk Assessment-3rd volume, ASTM STP 1218, Hughes, JS, Biddinger, GR and Mones, E, eds. American Society for Testing and Materials, Philadelphia, PA. pp. 242-263. Salazar, M H and Salazar, SM. 1997. Using caged bivalves to characterize exposure and effects associated with pulp and paper mill effluents. Water Science and Technology 35(2-3):213-220. Salazar, MH. 2000. Caged Mussel Pilot Study Port Alice Mill, Vancouver Island E E M Program. Regional Manuscript Report: MS 00-01. Submitted to Environment Canada, Pacific and Yukon Region, North Vancouver, BC. Sandstrom, O. 1996. In Situ Assessments of the Impact of Pulp Mill Effluent on Life-History Variables in Fish. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 449-458. SAS Institute. 1989 - 2000. JMP Statistical Discovery Software. Version 4.0.2. SAS Institute Inc., Cary, NC, USA. Scroggins, RP, Miller, A, Borgmann, A l and Sprague, JB. 2002. Sublethal toxicity findings by the pulp and paper industry for Cycles 1 and 2 for the Environmental Effects Monitoring Program. Water Quality Research Journal of Canada 37(1): 21-48. Seed, R. 1976. Ecology. In Bayne, BL, eds, Marine mussels: their ecology and physiology, International Biological Programme 10. Cambridge University Press, Cambridge, England, pp. 13-65. Seed, R. and Richardson, CA. 1990. MytUus growth and its environmental responsiveness. In Stefano, GB, ed. Neurobiology of MytUus edulis. Manchester University Press, Manchester, England. Seed, R and Suchanek, TH. 1992. Population and community ecology of MytUus. In Gosling, E. 1992. The mussel MytUus: ecology, physiology, genetics, and culture. Elsevier, Amsterdam, Netherlands. Pp. 87-169. Servizi, JA, Martens, DW, Gordon, RW, Kutney, JP, Singh, M , Dimitriadis, E, Hewitt, G M , Salisbury, PJ, Choi, LSL. 1986. Microbiological detoxification of resin acids. Water Pollution Research Journal of Canada. 21(1):119. Sjostrom, E. 1981. Wood Chemistry Fundamentals and Applications. Academic Press, New York, NY. Smook, GA. 1992. Handbook for Pulp and Paper Technologists, 2n d ed. Angus Wilde Publications, Vancouver, BC, Canada. 97 Steel, RGD and Torrie, JH. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York, NY. Stuijfzand, SC, Drenth, A, Helms, M, and Kraak, MHS. 1998. Bioassays using the midge Chironomus riparius and the zebra mussel Dreissena polymorpha for evaluation of river water quality. Archives of Environmental Contamination. 34:357-363. Suntio, LR, Shiu, WY, and Mackay, D. 1988. A review of the nature and properties of chemical present in pulp mill effluents. Chemosphere 17:1249-1290. Swanson, SM, Holley, K, Bouree, GR, Pryke, DC, and Owens, JW. 1996. Concentrations of chlorinated organic compounds in various receiving environment compartments following implementation of technological changes at a pulp mill. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 253-260. Tavendale, M H , Wilkins, A L , Langdon, AG, Mackie, KL, Stuthridge, TR and McFarlane PN. 1995. Analytical methodology for the determination of freely available bleached kraft mill effluent-derived organic constituents in recipient sediments. Environmental Science and Technology 29(5)1407-1414. Tavendale, MH, Hannus, IM, Wilkins, AL, Langdon, A G , Mackie, KL, and McFarlane, PN. 1996. Bile analysis of goldfish (Crassius auratus) resident in a New Zealand hydrolake receiving a bleached kraft mill discharge. Chemosphere 33(11):2273-2289. Tidepool Scientific Software. ToxCalc™. 1994-1997. Comprehensive Toxicity Data Analysis and Database Software. Version 5.0.18. Tidepool Scientific Software, McKinleyville, CA, USA. Veldhuizen-Tsoerkan, MB, Holwerda, DA, de Bont, AMT, Smaal, A C , and Zandee, DI. 1991. A field study on stress indices in the sea mussel, Mytilus edulis: application of the "stress approach" in biomonitoring. Archives of Environmental Contamination and Toxicology. 21:497-504. Verta, M , Ahtiainen, J , Nakari, T, Langi, A, and Talka, E. 1996. The effect of waste constituents on the toxicity of TCF and ECF pulp bleaching effluents. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 315-326. Walden, CC and Howard, TE. 1977. Toxicity of pulp and paper mill effluents: a review of regulations and research. Tappi 60(1): 122-125. Whyte, JJ, Jung, RE, Schmitt, CJ, and Tillitt, D. 2000. Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Critical Reviews in Toxicology. Volume 30(4):347-570. Wu, RSS and Levings, CD. 1980. Mortality, growth and fecundity of transplanted mussel and barnacle populations near a pulp mill outfall. Marine Pollution Bulletin. 11:11-15. 98 Yunker, MB and Cretney, WJ. 1996. Dioxins and furans in crab hepatopancreas: use of principal components analysis to classify congener patterns and determine linkages to contamination sources. In Servos, MR, Munkittrick, KR, Carey, JH, and Van Der Kraak, GJ. 1996. Environmental Fate and Effects of Pulp and Paper Mill Effluents. St. Lucie Press, Delray Beach, FL, USA. pp. 315-326. Zhou, S, Ackman, RG, and Parsons, J. 1996. Very long-chain aliphatic hydrocarbons in lipids of mussels (Mytilus edulis) suspended in the water column near petroleum operations off Sable Island, Nova Scotia, Canada. Marine Biology. 126: 499-507. 99 APPENDICES 100 Appendix A - Summary of Mussel Survival and Mortality 101 Summary of Mortality, Survival and Missing Mussels (2-51-902) Summary of Mortality, Survival and Missing Mussels (2-51-902) Mortalities Group Concentration (%v/v) Total 0.00% 0.23% 0.46% 1.01% 2.07% 4.88% A 12 15 15 21 20 29 112 B 12 14 16 25 16 26 109 C 6 12 15 17 24 35 109 D 5 14 18 22 19 31 109 Mean 8.8 13.8 16.0 21.3 19.8 30.3 110 S D 3.8 1.3 1.4 3.3 3.3 3.8 1.5 Minimum 5 12 15 17 16 26 109 Maximum 12 15 18 25 24 35 112 Sum 35 55 64 85 79 121 525 Missing Group Concentration (%v/v) Total 0.00% 0.23% 0.46% 1.01% 2.07% 4.88% A 6 7 6 2 5 2 28 B 6 6 5 5 3 1 26 C 4 6 5 2 2 0 19 D 3 5 1 2 1 1 13 Mean 4.8 6.0 4.3 2.8 2.8 1.0 22 S D 1.5 0.8 2.2 1.5 1.7 0.8 6.9 Minimum 3 5 1 2 1 0 13 Maximum 6 7 6 5 5 2 28 Sum 19 24 17 11 11 4 86 Total Number Group Concentration (%v/v) Total 0.00% 0.23% 0.46% 1.01% 2.07% 4.88% A 94 93 94 98 95 98 572 B 94 94 95 95 97 99 574 C 96 94 95 98 98 100 581 D 97 95 99 98 99 99 587 Mean 95.3 94.0 95.8 97.3 97.3 99.0 2314 S D 1.5 0.8 2.2 1.5 1.7 0.8 6.9 Minimum 94 93 94 95 95 98 572 Maximum 97 95 99 98 99 100 587 Sum 381 376 383 389 389 396 2314 TN+Miss 400 400 400 400 400 400 2400 Mortalities (% ) Group Concentration (%v/v) 0.00% 0.23% 0.46% 1.01% 2.07% 4.88% A 12.8 16.1 16.0 21.4 21.1 29.6 B 12.8 14.9 16.8 26.3 16.5 26.3 C 6.3 12.8 15.8 17.3 24.5 35.0 D 5.2 14.7 18.2 22.4 19.2 31.3 Mean 9.2 14.6 16.7 21.9 20.3 30.5 SD 4.1 1.4 1.1 3.7 3.4 3.6 Minimum 5.2 12.8 15.8 17.3 16.5 26.3 Maximum 12.8 16.1 18.2 26.3 24.5 35.0 102 Summary of Mortality, Survival and Missing Mussels (2-51-902) Survival (%) Group Concentration (%v/v) 0.00% 0.23% 0.46% 1.01% 2.07% 4.88% A 87.2 83.9 84.0 78.6 78.9 70.4 B 87.2 85.1 83.2 73.7 83.5 73.7 C 93.8 87.2 84.2 82.7 75.5 65.0 D 94.8 85.3 81.8 77.6 80.8 68.7 Mean 90.8 85.4 83.3 78.1 79.7 69.5 S D 4.1 1.4 1.1 3.7 3.4 3.6 Minimum 87.2 83.9 81.8 73.7 75.5 65.0 Maximum 94.8 87.2 84.2 82.7 83.5 73.7 Survival (%) Group Concentration (%v/v) 0.00% 0.23 0.46 1.01 2.07 4.88 A 87.2 83.9 84.0 78.6 78.9 70.4 B 87.2 85.1 83.2 73.7 83.5 73.7 C 93.8 87.2 84.2 82.7 75.5 65.0 D 94.8 85.3 81.8 77.6 80.8 68.7 Mean 90.8 85.4 83.3 78.1 79.7 69.5 S D 4.1 1.4 1.1 3.7 3.4 3.6 Minimum 87.2 83.9 81.8 73.7 75.5 65.0 Maximum 94.8 87.2 84.2 82.7 83.5 73.7 Missing and Mortalities Group 0.00% 0.23% 0.46% 1.01% 2.07% 4.88% Total A 18 22 21 23 25 31 B 18 20 21 30 19 27 C 10 18 20 19 26 35 D 8 19 19 24 20 32 Total 54 79 81 96 90 125 525 Number Alive Group 0.00% 0.23% 0.46% 1.01% 2.07% 4.88% Total A 82 78 79 77 75 69 B 82 80 79 70 81 73 C 90 82 80 81 74 65 D 92 81 81 76 80 68 . • ., . , Total 346 321 319 304 310 275 1875 Grand Total 400 400 400 400 400 400 2400 103 Appendix B Statistical Calculations 104 Myti lus e d u l i s 90 day-Surv iva l Start Date: 6/9/1999 Test ID: 2-51-902D Sample ID: 0 1 4 - F C C - E l k Falls End Date: 9/7/1999 Lab ID: BCRI-Toxicology Lab Sample Type: 014-Final Mill Effluent Sample Date: Protocol: E C 1997-Draft Test Spec ies : ME-Myti l is edulis Comments: C o n c - % 1 Control 0.9081 0.23 0.8537 0.46 0.8329 1.01 0.7815 2.07 0.7969 4.88 0.6944 T r a n s f o r m : U n t r a n s f o r m e d N u m b e r Tota l C o n c - % M e a n N - M e a n M e a n M i n M a x C V % N R e s p N u m b e r Control 0.9081 1.0000 0.9081 0.9081 0.9081 0.000 1 35 381 0.23 0.8537 0.9401 0.8537 0.8537 0.8537 0.000 1 55 376 0.46 0.8329 0.9172 0.8329 0.8329 0.8329 0.000 1 64 383 1.01 0.7815 0.8605 0.7815 0.7815 0.7815 0.000 1 85 389 2.07 0.7969 0.8775 0.7969 0.7969 0.7969 0.000 1 79 389 4.88 0.6944 0.7647 0.6944 0.6944 0.6944 0.000 1 121 396 Auxi l i ary T e s t s Statistic Cr i t i ca l S k e w Kurt Normality of the data set cannot be confirmed Equality of variance cannot be confirmed M a x i m u m Like l ihood-Prob i t Parameter V a l u e S E 95% F i d u c i a l L i m i t s C o n t r o l C h i - S q Cr i t i ca l P-va lue M u S i g m a Iter Slope 0.59775 0.12934 0.34424 0.85127 Intercept 3.81184 0.08775 3.63986 3.98383 T S C R 0.09261 0.01474 0.06372 0.1215 Point Probi t s % 95% F i d u c i a l L i m i t s EC01 2.674 0.01247 0.00021 0.06765 E C 0 5 3.355 0.17218 0.01899 0.45541 E C 1 0 3.718 0.69785 0.19377 1.35449 E C 1 5 3.964 1.79401 0.81499 3.22037 E C 2 0 4.158 3.79954 2.09005 7.8283 E C 2 5 4.326 7.23318 3.97213 19.7989 E C 4 0 4.747 36.6328 14.7688 278.253 E C 5 0 5.000 97.2079 30.4528 1457.9 E C 6 0 5.253 257.948 61.8284 7757.74 E C 7 5 5.674 1306.39 197.568 126860 E C 8 0 5.842 2486.98 312.375 385661 E C 8 5 6.036 5267.19 532.147 1411292 E C 9 0 6.282 13540.8 1038.75 7229968 E C 9 5 6.645 54880.9 2793.59 8.2E+07 E C 9 9 7.326 757726 17799.1 4.9E+08 0.09186 3.71572 7.81472 0.29 1.9877 1.67293 1.0 $ 0.6 10-5 8 0.4 * 0.3 If / If / I Jr 0.2 j 0.1 \ 0.0 0.0001 0.1 100 100000 1E+08 1E+11 D o s e % Page 1 ToxCalc v5.0 105 Reviewed by:. Myt i lus edu l i s 90 day-Surv iva l Start Date: 6/9/1999 Test ID: 2-51-902D Sample ID: 0 1 4 - F C C - E l k Fal ls End Date: 9/7/1999 Lab ID: BCRI-Toxicology Lab Sample Type: 014-Final Mill Effluent Sample Date: Protocol: E C 1997-Draft Test Spec ies : ME-Myti l is edulis Comments: D o s e - R e s p o n s e Plot Page 2 ToxCalc v5.0 106 Reviewed by:. Z-Test for Survival Treatment rii # Alive (X) p h a , (X /n) pooled p h a t 1-Phat 1/n 1/n + 1/n Phat*1-Phat*1/n + 1/n S E Z Signif icant 0 381 346 0.9081365 - - 0.0026247 - - - -0.23 376 321 0.8537234 0.881109643 0.11889 0.002660 0.0052842 0.000553554 0.02353 2.31 No 0.46 383 319 0.8328982 0.870418848 0.129581 0.002611 0.0052356 0.000590527 0.0243 3.10 Yes 1.01 389 304 0.781491 0.844155844 0.155844 0.0025707 0.0051954 0.000683485 0.02614 4.84 Yes 2.07 389 310 0.7969152 0.851948052 0.148052 0.0025707 0.0051954 0.000655305 0.0256 4.34 Yes 4.88 396 275 0.6944444 0.799227799 0.200772 0.0025253 0.005150 0.000826371 0.02875 7.43 Yes a^/m a 0.05 m 5 number of comparisons (n-1) Therefore Z 0 0 i 2.33 (df = infinity; from Table B.3, p. 485 Zar) o Bonferroni's Test for Length, Weight and Condition Index C D =(ta / 2 / m).sqrt((MSe/n i) + (MSJn)) t a / 2 / 5 = 2.58 t a / 2 / 6 = 2.64 Treatment n - L & W n-CI MSe Length MSe Weight MSe CI I MSe CI II 0 346 346 8.413 0.43314 0.026778 0.0275 0.23 321 321 8.413 0.43314 0.026778 0.0275 0.46 319 318 8.413 0.43314 0.026778 0.0275 1.01 304 304 8.413 0.43314 0.026778 0.0275 2.07 310 310 8.413 0.43314 0.026778 0.0275 4.88 275 274 8.413 0.43314 0.026778 0.0275 T=0 400 400 - - 0.026778 0.0275 MSe/ni Treatment Length Weight CI I CI II 0 0.024315 0.001252 0.0000773931 0.0000794798 0.23 0.0262087 0.001349 0.0000834206 0.0000856698 0.46 0.026373 0.001358 0.0000842075 0.0000864780 1.01 0.0276743 0.001425 0.0000880855 0.0000904605 2.07 0.0271387 0.001397 0.0000863806 0.0000887097 4.88 0.0305927 0.001575 0.0000977299 0.0001003650 T=0 - 0.0000687500 sqrt(MSe/ni) + (MSe/nj) Treatment Length Weight CI I CI II 0.23 0.2247749 0.051002 0.012681231 0.012851053 0.46 0.2251401 0.051085 0.012712223 0.012882459 1.01 0.2280118 0.051736 0.012863848 0.013036115 2.07 0.2268342 0.051469 0.01279741 0.012968787 4.88 0.234324 0.053169 0.013233404 0.01341062 T=0 - - - 0.012174965 (ta/2/m)*sqrt((MSe/ni) + (MSe/nj)) 2.58 2.64 Treatment Length Weight CI I CI II 0.23 0.58 0.13 0.033 0.034 0.46 0.58 0.13 0.033 0.034 1.01 0.59 0.13 0.033 0.034 2.07 0.59 0.13 0.033 0.034 4.88 0.60 0.14 0.034 0.035 T=0 - 0.032 Means Treatment Length Weight CI I CI II 0.0 5.20 1.07 0.98 0.98 0.23 5.76 1.21 1.04 1.04 0.46 5.36 1.13 1.04 1.04 1.01 6.31 1.32 1.09 1.09 2.07 5.84 1.23 1.06 1.06 4.88 5.13 1.03 1.05 1.05 T=0 1.30 108 Bonferroni's Test for Length, Weight and Condition Index Mean Differences Treatment Length Weight Condition Index I Condition II 0.0 0.00 0.00 0.000 0.320 0.23 0.56 0.14 0.060 0.260 0.46 0.16 0.06 0.060 0.260 1.01 1.11 0.25 0.110 0.210 2.07 0.64 0.16 0.080 0.240 4.88 0.07 0.04 0.070 0.250 T=0 0.980 0.000 Significance Treatment Length Weight Condition Index I Condition II 0.0 No No No Yes 0.23 No Yes Yes Yes 0.46 No No Yes Yes 1.01 Yes Yes Yes Yes 2.07 Yes Yes Yes Yes 4.88 No No Yes Yes T=0 No 109 Least Significant Difference Test lsd(0.05) = to.osSd t, Error df tn.05 0^.01 r 18 1.734 2.552 4 0.05 Sd s 2 r tabular value of t for error df s d = sqrt(2s2/r) error variance number of observations per mean lsd(0.05) - Moisture 0.66 lsd(0.01) - Moisture 0.98 Concentration Mean Error Mean Differences Signifcant at Significant at (%v/v) Moisture Variance @ 0 0.05 @ 0 0.01 @ 0 0 81.8 0.292917 0.0 0.23 81.8 0.292917 0.0 No No 0.46 81.4 0.292917 0.4 No No 1.01 81.9 0.292917 -0.1 No No 2.07 81.5 0.292917 0.3 No No 4.88 81.9 0.292917 -0.1 No No lsd(0.05) - Lipid 0.07 lsd(0.01) - Lipid 0.11 Concentration Mean Error Mean Differences Signifcant at Significant at (%v/v) Lipid Variance @ 0 0.05 @ 0 0.01 @ 0 0 1.99 0.00339 0.00 0.23 1.94 0.00339 0.05 No No 0.46 1.97 0.00339 0.02 No No 1.01 1.83 0.00339 0.16 Yes Yes 2.07 1.84 0.00339 0.15 Yes Yes 4.88 1.58 0.00339 0.41 Yes Yes 110 Least Significant Difference Test lsd(0.05) = t 0 .05S d t0.05 tabular value of t for error df s d s d = sqrt(2s2/r) s 2 error variance r number of observations per mean Error df 21 to.os 1-721 t-0.01 2.518 r 4 lsd(0.05) - Moisture 0.69 lsd(0.01) - Moisture 1.01 Concentration Mean Error Mean Differences Signifcant at Significant at (%v/v) Moisture Variance @ T=0 0.05 @ T=0 0.01 @ T=0 T=0 77.7 0.3206 0 81.8 0.3206 -4.1 Yes Yes 0.23 81.8 0.3206 -4.1 Yes Yes 0.46 81.4 0.3206 -3.7 Yes Yes 1.01 81.9 0.3206 -4.2 Yes Yes 2.07 81.5 0.3206 -3.8 Yes Yes 4.88 81.9 0.3206 -4.2 Yes Yes lsd(0.05) - Lipid 0.11 lsd(0.01)- Lipid 0.16 Concentration Mean Error Mean Differences Signifcant at Significant at (%v/v) Lipid Variance @ T=0 0.05 @ T=0 0.01 @ T=0 T=0 3.24 0.00797 0 1.99 0.00797 1.25 Yes Yes 0.23 1.94 0.00797 1.30 Yes Yes 0.46 1.97 0.00797 1.27 Yes Yes 1.01 1.83 0.00797 1.41 Yes Yes 2.07 1.84 0.00797 1.40 Yes Yes 4.88 1.58 0.00797 1.66 Yes Yes 111 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092115/manifest

Comment

Related Items