An Effects-Based Assessment of the Health of Fish in a Small Estuarine Stream Receiving Effluent from an Oil Refinery

An effects-based assessment of the health of fish in a small estuarine stream receiving effluent from an oil refinery

Geneviève Vallières

B.Sc. Biology, Université de Sherbrooke, 1998

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

Master of Science

In the Graduate Academic Unit of Biology

Supervisors: Kelly Munkittrick, Ph.D. Department of Biology Deborah MacLatchy, PhD. Department of Biology

Examining Board: Kenneth Sollows, Ph.D., Department of Engineering, Chair Simon Courtenay, Ph. D. Department of Biology

External Examiner: Kenneth Sollows, Ph.D., Department of Engineering

This thesis is accepted by the Dean of Graduate studies.

THE UNIVERSITY OF NEW BRUNSWICK

May, 2005

ABSTRACT

A large oil refinery discharges its effluent into Little River, a small estuarine stream entering Saint John Harbour. An effects-based approach was used to assess the potential effects of the oil refinery effluent on fish and fish habitat. The study included a fish community survey, a sentinel species survey, a fish caging experiment, and a water quality survey. The study showed that the fish community and the sentinel species, the mummichog (Fundulus heteroclitus), were impacted in the stream receiving the oil refinery effluent. Lower abundance and species richness were found downstream of the effluent discharge whereas increased liversomatic index and MFO (females only) were measured in fish collected in Little River. Water quality surveys demonstrated that the receiving environment is subjected to extended periods of low dissolved oxygen levels downstream of the effluent discharge. The anoxic periods correlated with the discharge of ballast water through the waste treatment system.

ACKNOWLEDGMENTS

I would like to thank my supervisors, Kelly Munkittrick and Deborah MacLatchy, for their support as well as the independence from which I benefited during the entire project. This experience would not have been so positive and formative without them. I would like the acknowledge Louise Stewart and Rick Russell, from the Irving Oil refinery, without whom the project would not have been possible. Funding came from the Industrial Postgraduate Scholarships program from the Natural Sciences and Engineering Research Council of Canada as well as Irving Oil Ltd.

I would like to thank all the graduate students in the Munkittrick and MacLatchy laboratories for their help in so many ways. Thank you for sharing the good and bad moments, for making the unending learning curve easier and of course babysitting the girls. A particular thanks to Kate Frego, Rémy Rochette and Matt

Litvak for their help with fish community analysis and statistics. Finally, I would like to thank my family for their support and their encouragement, particularly my husband Franck for being an extraordinary friend, husband, and father as well as my daughters, Raphaëlle and Élodie, for being my sunshine.

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TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGMENTS ...... iii

TABLE OF CONTENTS ...... iv

LIST OF TABLES ...... vii

LIST OF FIGURES ...... ix

LIST OF ABBREVIATIONS ...... xiii

1 GENERAL INTRODUCTION ...... 1

1.1 Background Information...... 4

1.1.1 EEM Methodologies and Challenges with Monitoring Small

Estuarine Streams ...... 4

1.1.2 Toxicity of Oil Refinery Waste Waters ...... 10

1.2 Statement of the Problem and Study Area ...... 12

1.2.1 Oil Refinery...... 20

1.3 Previous Studies in the Area...... 23

1.4 Organization of Thesis...... 25

1.5 Objectives and Hypothesis ...... 26

2 FISH COMMUNITY SURVEY...... 27

2.1 Introduction...... 27

2.2 Methodology ...... 28

2.2.1 Fishing with Minnow Traps ...... 28

2.2.2 Seining Trials...... 29

2.2.3 Statistics ...... 30

2.3 Results...... 32

2.3.1 Fishing with Minnow Traps ...... 32

2.3.2 Seining ...... 53

2.4 Discussion ...... 56

2.4.1 Seining Versus Trapping ...... 61

3 FISH HABITAT ASSESSMENT ...... 66

3.1 Introduction...... 66

3.2 Methodology ...... 67

3.3 Results...... 71

3.4 Discussion ...... 93

4 RESPONSE OF WILD MUMMICHOG TO THE EFFLUENT

DISCHARGE 104

4.1 Introduction...... 104

4.2 Methodology ...... 108

4.2.1 Statistics ...... 109

4.2.2 Mixed Function Oxygenase Activity...... 110

4.3 Results...... 112

4.4 Discussion ...... 121

5 FISH CAGING EXPERIMENT ...... 126

5.1 Introduction...... 126

5.2 Methodology ...... 127

5.2.1 Mixed Function Oxygenase Activity...... 133

5.2.2 Testosterone Production ...... 133

5.3 Results...... 135

5.4 Discussion ...... 139

6 GENERAL CONCLUSION...... 146

7 REFERENCE...... 150

8 APPENDICES...... 159

LIST OF TABLES

Table 1. Measured endpoints for the fish survey and their indicator of performance in terms of reproduction, growth, energy storage and survival...... 6 Table 2. Habitat characteristics of studied sites: Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek WC). Sampling seasons from June to November 2003 and May to September 2004...... 19 Table 3. Total number of fish caught per site (Marsh Creek Upstream [MCU], Marsh Creek Downstream [MCD], Little River Upstream [LRU], Little River Downstream [LRD], Hazen Creek Upstream [HCU], Hazen Creek Downstream [HCD], and West Quako Creek [WC]). Sampling from July to November 2003 and May to September 2004...... 36 Table 4. Number of species and abundance per trap for the 2003 and 2004 sampling seasons and comparison between metal and plastic-coated traps. Mean ± standard error (sample size). Different letters represent statistical differences...... 54 Table 5. Abundance and percentage (in parentheses) of fish caught using a beach seine at Little River upstream site. July 22, 2003...... 55 Table 6. Total number of recording hours from hydrolab sondes deployed in LRD and LRU and percentage of hours recording dissolved oxygen below 2 mg/L and 0.5 mg/L. Results from 2003 (A) and 2004 (B)...... 75 Table 7. Maximum temperature recorded during extended sonde deployments in Little River Downstream (LRD) and Little River Upstream (LRU) in 2003 and 2004...... 80 Table 8. Atlantic Coastal Action Program surface water quality monitoring program. Means, maximum and minimum values for sampling summer 2003 in Marsh Creek upstream and downstream (MC up & dw), Little River upstream and downstream (LR up & dw), and Hazen Creek upstream and downstream (HC up & dw)...... 82 Table 9. Atlantic Coastal Action Program surface water quality monitoring program. Means, maximum and minimum values for sampling summer 2004 in Marsh Creek upstream and downstream (MC up & dw), Little River upstream and downstream (LR up & dw), and Hazen Creek upstream and downstream (HC up & dw)...... 83 Table 10. Definition of fish parameters used in the EEM Fish Survey...... 107 Table 11. Total mass (g), total length(cm), gonad mass(g) and liver mass(g) of male and female mummichog collected in each site in July and August 2003. Means ± standard errors (sample size)...... 113 Table 12. R2, slope with standard deviation, intercept with standard deviation, and sample size for liver mass in relation with total mass. Data for male mummichog collected in July 2003 at each site...... 119

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Table 13. Mixed-function oxygenase (MFO) activity (pmol/min/mg) for mummichog collected in each site in July 2003. Values are mean ± standard error (sample size). Different letters represent statistical differences (p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 120 Table 14. Caging experiment designs. Starting date, duration of experiment, number of fish per cage, fish sex, sites used, and type of cages used...... 129 Table 15. Means and standard deviation for total mass and total length for each caging experiment...... 132

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LIST OF FIGURES

Figure 1. Map of the Bay of Fundy area, Canada...... 13 Figure 2. Map of the sampling sites in the Saint John area. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek downstream (HCD)...... 14 Figure 3. Map of the sampling site in the St. Martin’s area (approximately 55 km east of Saint John). West Quako Creek (WC)...... 18 Figure 4. Relative abundance (%) of each species collected by minoow traping per site from July to November 2003. Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC). ..33 Figure 5. Relative abundance (%) of each species collected by minnow trapping per site from May to September 2004. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek Downstream (HCD)...... 34 Figure 6. Total abundance (A) and total number of fish species (B) per month per site. Results from 10 traps per day per month, June to November 2003. Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD) and West Quako Creek (WC)...... 37 Figure 7. Total abundance (A) and total number of fish species (B) per month per site. Results represent data from 10 traps per day per month, May to September 2004. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek Downstream (HCD)...... 38 Figure 8. Frequency of empty traps. Results from 10 traps per month, from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors(n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 40 Figure 9. Total abundance per month per site from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors (n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 41

Figure 10. Total number of species per month per site from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors (n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 42 Figure 11. Shannon biodiversity index per month per site from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors (n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 43 Figure 12. Dendrogram of the ordination according to similarity among months and sites, from June to November 2003. Labels represent site and date (month, year). E.g., LRU0703 stands for Little River Upstream July 2003...... 45 Figure 13. Dendrogram of the ordination according to similarity among months and sites, from June to November 2004. Labels represent site and date (see previous figure). LRD0704 is missing as no fish were collected...... 46 Figure 14. Dendrogram of the ordination according to similarity among months and sites, from June to November 2003 and May to September 2004. Compilation of four common sites of both sampling years. Labels represent site and date (see previous figures)...... 47 Figure 15. Multidimensional scaling of Bray-Curtis similarity per month and site, from June to November 2003. Labels represent site and date (see previous figures). Bubble sizes indicate tidal influence (small circle= no influence; big circle= strong tidal influence)...... 48 Figure 16. Multidimensional scaling of Bray-Curtis similarity per month and site, from May to September 2004. Labels represent site and date (e.g. LRU0504 stands for Little River Upstream May 2004). Bubble sizes indicate tide influence (small circle= no influence; big circle= high tide influence). LRD0704 is missing as no fish were collected...... 50 Figure 17. Multidimensional scaling of Bray-Curtis similarity per month and site, from June to November 2003 and May to September 2004. The symbols represent the four common sites of both sampling years. Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), and Hazen Creek Downstream (HCD)...52 Figure 18. Position of ACAP sampling sites for water quality monitoring program. Marsh Creek Upstream and Downstream (MU and MD), Little River Upstream and Downstream (LU and LD), and Hazen Creek Upstream and Downstream (HU and HD)...... 68 Figure 19. Little River Downstream site. Effluent plume and effluent concentration at low, high and highest tides...... 72

Figure 20. Results of extended sonde deployments in Little River Downstream (LRD) and Little River Upstream (LRU) in 2003 (A) and 2004 (B), including total recording hours, number of hours recording dissolved oxygen lower than 2 mg/L and 0.5 mg/L, and duration of the longest continuous hypoxic (<2 mg/L) period...... 74 Figure 21. Salinity (ppt) and dissolved oxygen (mg/L) recorded in Little River Downstream site, from June 18–25, 2003 (A) and from August 29 to September 10, 2003 (B)...... 76 Figure 22. Salinity (ppt) recorded in Little River Downstream site with tidal cycle, from June 18 to 24, 2003. Tidal cycle 0= low tide and 1= high tide. (Source: Tourism Saint John 2004)...... 77 Figure 23. Salinity (ppt) for upstream site (UP3), downstream site (DWN5) and effluent. Data from the refinery’s summer water monitoring, from May to August 2003 (A) and June to August 2004 (B)...... 78 Figure 24. Water temperature (oC) recorded in Little River Upstream (LRU) and Downstream (LRD) sites, and difference between the two sites (LRD - LRU). September 24-30, 2003 (A) and June 1-21, 2004 (B)...... 81 Figure 25. Dissolved oxygen (DO), temperature (oC), and pH for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2003...... 85 Figure 26. Fecal coliforms (#/0.1 L) and turbidity (NTU) for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2003...... 86 Figure 27. Dissolved oxygen (DO), temperature (oC), and pH for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2004...... 87 Figure 28. Fecal coliforms (#/0.1 L) and turbidity (NTU) for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2004...... 88 Figure 29. Combination of Irving Oil Little River summer monitoring (IOL), ACAP and sonde data for temperature (A), salinity (B), and dissolved oxygen (C), from July 22 to August 11, 2003...... 90 Figure 30. Combination of IOL, ACAP and sonde data for temperature (A), salinity (B), and dissolved oxygen (C), from June 1 to 21, 2004...... 91 Figure 31. Combination of IOL, ACAP and sonde data for temperature (A), salinity (B), and dissolved oxygen (C), from July 1 to 8, 2004...... 92 Figure 32. Liversomatic index (LSI) for male and female mummichog (Fundulus heteroclitus) collected in July 2003 at each site. Values are means with standard errors (7≤n≤28). Different letters indicate statistical differences among sites (p<0.05). Statistical analysis could not be completed for males as interactions were found (ANCOVA p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 114

Figure 33. Log liver in relation to log mass, female and male mummichog (Fundulus heteroclitus) collected in July 2003 at each site. Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 115 Figure 34. Gonadosomatic index (GSI) for male and female mummichog (Fundulus heteroclitus) collected in July 2003 at each site. Values are means with standard errors (7≤n≤28). Different letters indicate statistical differences among sites (p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 116 Figure 35. Condition factor (K) for male and female mummichog (Fundulus heteroclitus) collected in July 2003 at each site. Values are means with standard errors (7≤n≤28). Different letters indicate statistical differences among sites (p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC)...... 117 Figure 36. Cage dimensions for both cage types...... 130 Figure 37. In vitro testosterone production in male mummichog (Fundulus heteroclitus) after caging, October 2004. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek Downstream (HCD)...... 136 Figure 38. Mixed-function oxygenase (MFO) activity in male mummichog (Fundulus heteroclitus) after caging experiment, October 2004. Different letters indicate statistical differences among sites (p<0.05).Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), negative (Cx) and positive (BNF) controls...... 138 Figure 39. Suggestions for design of improved mummichog cages...... 144

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LIST OF ABBREVIATIONS

α Type I error, probability to reject Ho when Ho is true

β Type II error, probability to accept Ho when Ho is false

7ER 7-ethoxyresorufin

ACAP Atlantic Coastal Action Program

ANCOVA Analysis of covariance

ANOVA Analysis of variance

BOD Biological oxygen demand

BSA Bovine serum albumin

COD Chemical oxygen demand

DO Dissolved oxygen

DMSO Dimethyl sulfoxide

DPM Disintegrations per minute

EEM Environmental effects monitoring

FSEWG Fish survey expert working group

GSI Gonadosomatic index

HCD Hazen Creek Downstream

HCU Hazen Creek Upstream

IBMX 3-Isobutyl-1-methylxanthine

IOL Irving Oil Limited

K Condition factor

LRD Little River Downstream

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LRU Little River Upstream

LSI Liver somaticindex

MCD Marsh Creek Downstream

MCU Marsh Creek Upstream

MDS Multidimensional scaling

MFO Mixed-function oxygenase

NADPH Nicotinamide adenine dinucleotide phosphate (reduced form)

NSB Non-specific binding

PAH Polycyclic aromatic hydrocarbon

PPER Pulp and paper effluent regulations

PCB Polychlorinated biphenyl

RIA Radioimmunoassay

TCR Total counts reference

TMS Tricaine methane sulfonate

WC West Quako Creek

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1 GENERAL INTRODUCTION

Canadian industrial effluents discharged into surface waters are controlled by

provincial and federal regulations. These regulations have been instrumental in

improving the quality of receiving environments, and have usually been in the

form of standards that are uniformly applied on an industry-wide basis. Many

effluent discharge regulations have included biological testing or monitoring

components (varying from acute lethality tests to monitoring field effects) since

the early 1970s (Putman 1989; Sergy & Scroggins 1991; Environment Canada

2004).

The effects of oil refinery effluent on fish have rarely been studied and existing studies usually demonstrate no responses or responses suggesting toxicity or enrichment (King & Hite 1993; Krause 1994; Kuehn et al. 1995; Ostrander et al.

1995). In Canada, oil refineries must pass acute toxicity tests every month, which are rarely failed and thus suggest no acute impacts on fish (St-Cyr, 1995). These tests are limited in their ability to assess long-term effects on fish populations and communities. The absence of major acute toxicity issues has lessened regulatory interest in oil refinery effluent receiving environment studies.

A large oil refinery in Saint John, New Brunswick, discharges its effluent into a small coastal stream and meets current requirements under the petroleum

refinery liquid effluent regulations. Because the refinery was built before 1973,

but expanded its capacity after 1973, it is under extended guideline status (St-

Cyr 1995). In 1992, the compliance for toxicity testing was 91.75% (one test

failure) and there was one exceeded discharge amount for oil and grease,

ammonia nitrogen and phenols during that year. All other parameters (sulphide,

total suspended matter and pH) were within the guidelines (St-Cyr 1995). In

2001, 2003, and 2004, Irving Oil Limited complied with all requirements for

deleterious substance deposits, pH level and toxicity testing. In 2002, the

company complied 99.4% of the time with the phenols and total suspended

matter requirements (both had one exceeded deposit during the year) and the

company demonstrated 91.75% compliance with rainbow trout (Salmo Gairdneri) toxicity test (one test failure). All other parameters (oil and grease, ammonia,

sulphide, and pH) had 100% compliance (Irving Oil Limited 2001, 2002, 2003,

2004).

Over the past several decades, there has been a gradual shift in the emphasis of

environmental monitoring from end-of-pipe assessments to biologically-based

receiving environment assessments focused on the site-specific ability of

receiving environments to assimilate wastes. Recent amendments to the

Fisheries Act for both pulp and paper (Pulp and Paper Effluent Regulations,

1994) and metal mining (Metal Mining Liquid Effluent Regulations [MMLER,

2001]) included requirements for environmental effects monitoring (EEM). The

EEM program is a cyclical monitoring requirement designed to determine

whether a receiving environment is protected when the facility is in compliance

with its effluent discharge regulations. While these effluent monitoring

requirements were developed specifically for pulp and paper and metal mining

discharges, there have been attempts to develop similar monitoring requirements

for freshwater aquaculture facilities and sewage discharge facilities (Walker et al.

2003). The principles and technology are applicable to a wide range of effluent

situations.

The impact assessment of discharges needs to be inclusive and site-specific, as

the effects of a discharge are not only dependent on the effluent composition but

also on the assimilative capacity of the receiving environment (Munkittrick et al.

2000). Specific characteristics of the receiving environment, including all

anthropogenic activities, need to be considered when designing and interpreting

the potential impacts of a point source discharge.

Monitoring in complex freshwater environments receiving multiple discharges can

be challenging. However, assessing the effects of discharges on fish in marine

and brackish environment has been even more problematic (Courtenay et al.

2002). Marine and estuarine environments are more complex than freshwater systems, with more mobile fish populations and inconsistent exposure scenarios, increasing the difficulty in linking potential effects on fish health to effluent

exposure. There is a need for evaluating the effectiveness of monitoring

methodologies developed for freshwater systems when used in small coastal

streams, nearshore areas and estuaries.

1.1 Background Information

In the early 1990s, studies reported that fish exposed to pulp and paper mill

effluent had reproductive and growth alterations suggesting that the Pulp and

Paper Effluent Regulations (PPER) under the Fisheries Act were not adequately

protecting fish communities and habitat (Walker et al. 2002). Consequently,

amendments to the regulations in 1992 included a new EEM program applied

throughout Canada to examine the ability of the regulations to protect fish and

fish habitat (Walker et al. 2002).

1.1.1 EEM Methodologies and Challenges with Monitoring Small Estuarine

Streams

The EEM program is designed to be scientifically defensible and cost-effective. It

focuses on the assessment of the effects rather than on the identification of the

pollutants and aims to provide ecologically-relevant results for decision makers

(Environment Canada 1998). Monitoring frequency varies with the specific

program, but for pulp and paper, the results are to be reported every 3-4 years with the first cycle completed in 1996 for the pulp and paper industries. The EEM includes three components: 1) a fish survey to assess effects on fish; 2) a benthic invertebrate community survey to assess effects on fish habitat; and 3) a tainting study and tissue analysis when necessary to assess effects on fisheries

resources (Environment Canada 1998). There are supporting information

requirements for sublethal toxicity testing and water or sediment chemistry.

For the fish survey, the methodology uses a sentinel species approach. During

predesign surveys, a fish community survey is recommended to identify potential

sentinel species in the receiving environment. The EEM program uses a survey

of benthic invertebrate species as an indicator of the habitat quality.

The fish survey measures endpoints related to age distributions, energy use

(growth, reproductive investments) and energy storage (liver size, condition) as

indicators of performance (Table 1). These indicators were selected because

they integrate physiological changes that are relevant at the population level.

These endpoints are also at the interface between the individual and the

population levels, and are a level considered to be high enough to be relevant

ecologically, but not too high to prevent corrections being possible before lost of

biodiversity occurs (Environment Canada 2003).

Energy use is assessed through measurements of growth (or size-at-age) and reproductive potential. The reproductive potential of fish populations is assessed under EEM protocols through relative gonad size (gonadosomatic index; GSI), egg size and fecundity. Energy storage can be assessed through relative liver size (liversomatic index; LSI) and relative mass

Table 1. Measured endpoints for the fish survey and their indicator of performance in terms of reproduction, growth, energy storage and survival. Measured endpoints Indicators Gonad mass Gonadosomatic index (GSI) Egg mass Egg size Reproduction Total number of eggs Fecundity per female Length Condition factor (K) Growth and Total body mass Liversomatic index (LSI) energy storage Mass of liver Age Age distribution Survival Mean age (Environment Canada 1998; Munkittrick et al. 2000)

compared to fish length (condition factor; LSI). Finally, survival can be estimated

with mean age and age distributions (Table 1).

When integrated together and compared to reference sites, these indicators give

information on population performance. When performance is lower in exposed

than reference sites, the response pattern gives clues to the cause. Response patterns found downstream of pulp and paper mills include nutrient enrichment

and endocrine disruption (Munkittrick et al. 2000).

The EEM program requires that careful consideration be given to the selection of sentinel species to ensure that the data are meaningful. Sentinel species should be selected so they can reflect the overall condition of the aquatic environment in which the fish reside (Environment Canada 2003). Consequently, sentinel species should have biological characteristics linking effects to the habitat

(Munkittrick et al. 2000). The most important considerations are that the potential sentinels be abundant, exposed to the stressors of interest, and have life history and performance characteristics that are quantifiable. The species and sampling time should be selected to maximize effluent exposure (Munkittrick et al. 2000).

Depending on the receiving environment, and the degree to which movements are restricted, larger fish species are more likely to have a large home range and a lower growth rate, limiting the ability to see differences (Munkittrick et al. 2000).

The sample size for designing the study is a critical component. The power to

detect differences is affected by variability, sample size, and the significance

levels set for both α and β. Determining the sample sizes required is a function

of the effect size, which represents the level of difference at which it is

considered that there is an impact. Effect size is essential to determine the

appropriate number of fish to be collected at a determined power. The power was

initially set at 1 – β = 0.80 for pulp and paper mills (Cycle 1), whereas for the

mining industries α was set equal to β (both set at 0.10) (Glozier et al. 2002).

Subsequent to Cycle 1, to determine sample size for the fish survey in the EEM

for pulp and paper mills, effect sizes were set at 20-30% for GSI. The fish survey

expert working group (FSEWG) considered the reproduction as the most

sensitive and important parameter to measure effects of the pulp and paper mills

effluent on fish abundance (Munkittrick et al. 1997). The new target effect size was based on the first EEM cycle results and was based on: 1) natural variance

for GSI was the highest of all parameters and can be as high as 20% at

reference sites; 2) the selection of a smaller effect size would have implied an

unreasonably large sample size; 3) too high effect size would not assess the

efficiency of the new regulation (Munkittrick et al. 1997). The analysis of Cycle 2

results suggested that effect size should be set at 25% for GSI and LSI and 10%

for K (Lowell et al. 2003).

The program has improved over the cycles in response to the problems

encountered and advances in scientific knowledge. The main challenges related

to the fish survey for Cycle 1 and 2 were:

1) difficulty in finding appropriate reference sites;

2) inadequate number of fish or fish species;

3) uncertain exposure to the effluent ; and

4) difficulty in using alternative methods. (Courtenay et al. 2002)

The difficulties associated with the fish survey were more frequent and more severe in marine systems and estuaries than in fresh water (Courtenay et al.

2002). These areas were difficult for a variety of reasons. Monitoring approaches are complicated in many nearshore areas by a relatively low species diversity as well as tidal and seasonal movements of fish, thus limiting the availability of suitable sentinel species (Courtenay et al. 2002). The absence of a unidirectional flow in the receiving environment causes additional problems in finding proper reference sites, as a simple upstream - downstream model is not always applicable. The selection of reference sites can also be complicated by the high variability of habitat among estuaries. The inconsistency of effluent dispersion increases the difficulty in linking potential effects on fish health to effluent exposure. Finally, a lack of basic biological information on fish species inhabiting these environments complicates the selection of adequate sentinel species and sampling periods (Courtenay et al. 2002).

1.1.2 Toxicity of Oil Refinery Waste Waters

Since 1973, petroleum refineries in Canada have been regulated for discharge of their liquid effluent (Environment Canada 2004b). The regulations limit the amount of deleterious substances discharged into the receiving environment and define these substances as phenols, total suspended matter, sulphide, ammonia nitrogen, oil and grease and any substances that can alter the pH of the effluent

(Environment Canada 2004b). The discharge amount allowed is determined

based on the starting date of operation and the total production of the refinery.

Except for pH, the regulations are based on total amount discharged per day and

per month rather than their concentrations in the effluent or in the receiving

environment (St-Cyr 1995). The potential effects on the aquatic habitat and

aquatic life can consequently vary tremendously depending on the total effluent

dilution as well as the characteristics of the receiving environment. As a result,

compliance with the regulations does not guarantee the absence of an impact on

fish and fish habitat.

The toxicity of refinery effluent to fish and other biological components was

particularly studied in the 70’s and 80’s (Cote 1976; Buikema et al. 1981; Rowe

et al. 1983a,b; Saha & Konar 1984). Since then, government reports have shown a considerable reduction in the amount of deleterious substances in petroleum refinery effluent (Environment Canada 1995). It is assumed that the improvement in effluent quality has caused an improvement in the health of the environment,

but only a few studies have looked at the health of fish, biodiversity and system productivity since the 1980s (Krause 1994; Sherry et al. 1994; Bleckmann et al.

1995; Khan 1998). These studies have shown that responses of fish are variable and no general trends can be observed, which confirms the specificity of each situation. There have been reports of both increases and decreases in diversity, abundance, condition factor, liver and gonadosomatic indices, age structure and fecundity when exposed fish are compared to reference fish (King & Hite 1993;

Krause 1994; Kuehn et al. 1995; Ostrander et al. 1995; Knudsen et al. 1997).

All oil refineries in Canada are regulated under the Petroleum Refinery Liquid

Effluent Regulations. The refineries built after 1973 must comply with the regulations, whereas pre-existing refineries are subject to different guidelines

(Environment Canada 2004). The monthly maximum values allowed for each parameter depend on the amount of crude oil treated and include an average monthly amount, one-day amount and a maximum daily amount. In addition to

the maximum allowable deposits, a monthly 96-hour toxicity test on rainbow trout

(Oncorhychus mykiss) is required (St-Cyr 1995). The regulations are stricter than the guidelines, except for the pH level and the fish toxicity test requirements, which are the same in both cases (St-Cyr 1995). The 96-hour static toxicity test is

done once a month and the effluent concentration used for testing is based on

the refinery effluent flow rate during the month (Environment Canada 1974).

1.2 Statement of the Problem and Study Area

The oil refinery facility is located in the city of Saint John, New Brunswick, along

the Bay of Fundy (Figure 1). Regardless of compliance with regulatory

guidelines, there is a concern about whether the discharge of the Saint John oil refinery is within the assimilative capacity of Little River, the estuarine stream receiving the discharge (Figure 2). There was little background data available to help design a study looking at the health of Little River. The assessment was designed as an effects-based evaluation to determine whether fish populations were performing to a level that would be expected in a small coastal, tidal stream. However, because the study area is an estuary, many complications were expected in terms of fish movement, reference sites, and exposure to the effluent. The study therefore provided an opportunity to evaluate monitoring methodologies in small coastal stream environments.

Little River, the receiving stream for the effluent, is an 8-km long stream that

flows into the Saint John harbour. The effluent from the oil refinery is discharged into the river approximately 1.7 km from the river’s mouth (Figure 2). The studied area was within a 1.5-km section and contained two sites: upstream (400 m) and downstream (700 m) of the effluent (Figure 2). Little River Upstream (LRU) and

Little River Downstream (LRD) are separated by 500 m.

Figure 1. Map of the Bay of Fundy area, Canada.

Figure 2. Map of the sampling sites in the Saint John area. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek downstream (HCD).

The flow of the river is approximately 2.3 m3/s upstream of the effluent discharge.

The upstream site consists of a main stream within a marsh, mainly composed of

cattail (Thypha sp.) and a bottom with abundant soft substrate. The stream is

largely colonized with macrophytes including water-milfoil (Myriophyllum sp.),

American duck weed (Elodea canadensis), and common pondweed

(Potamogeton epihydrus). At LRD, the river has a narrower stream with faster current speed and a hard bottom. The shore is colonized mainly with grass

(Gramineae family). No significant amounts of macrophytes grow within the stream in this section.

Little River is affected by many anthropogenic activities or infrastructures. Two retention dams are upstream of the actual effluent discharge, and are located above the studied section. In addition to the effluent discharge, there is a runoff water discharge that drains the water falling outside of the refinery limits. This discharge is not measured for chemical contents or volume.

Other inputs in the section of the river include a paper mill built along the river and two outfalls of untreated sewage from the city. Until fall 1995, the paper mill was partially treating its effluent for solids before discharge into the river. At that time, Little River had high levels of suspended matter and resin acids. Since

November 1995, the paper mill effluent receives secondary treatment and is discharged directly into the harbour (Leblanc 1998).

Wastewater discharges from the municipality include residential and industrial wastes, including heated water from the power plant (though not the condenser cooling water). Combined municipal outfalls represent a discharge volume of 110 m3/day (0.0013 m3/s) (Peter Hanlon, Manager, City of Saint John, Water &

Sewerage Services; pers.comm.). Although these are confounding factors, the main discharge comes from the oil refinery (24 274 m3/day; 0.28 m3/s or about

12% of the flow) and consequently is presumed to be the major input to the

stream.

Finally, there are a few culverts along Little River, including one between the

upstream and downstream sites and one within the downstream site. During fall

of 2003, beavers used the entrance of the first culvert to build their dam. The

beaver dam was destroyed during the winter and was rebuilt in early summer

2004. It was also destroyed during heavy rain events and again rebuilt within few

days.

In addition to the sites on Little River (LRD the exposed site, LRU a reference

site), other sites were added to the study. In 2003, three sites within three other

small coastal streams were used to evaluate the relevance of the differences

seen in Little River. West Quako Creek (WC) and Hazen Creek Downstream

(HCD) were used as reference sites (Figures 2-3). Marsh Creek Downstream

(MCD), contaminated with creosote and untreated municipal waste water, was

used as another contaminated site (Figure 2).

The 2003 studies showed that LRD was not as influenced by tide as previously

thought. In order to reflect this situation, the reference sites were changed in

2004. An upstream-downstream design was adopted and two upstream sites

were added in Hazen Creek (HCU) and Marsh Creek (MCU) (Figure 2). West

Quako Creek (WC) had the most noticeable differences in habitat characteristics

(background salinity and substrate type), as well as species presence. It also had

no proper upstream reference site. It was consequently removed from the 2004

study.

All upstream sites are fresh water with no known anthropogenic discharges except for MCU, which is subject to storm water runoff from adjacent parking lots and a concrete fabrication facility, All downstream sites, including WC, are estuaries. At low tide, maximum depth averages 1 m or less and all streams were fresh water with salinities close to zero (Table 2). Bottom salinity in all streams was under 1 ppt at low tide, except for Little River Downstream (Table 2).

Salinities were higher in the two sites receiving discharges (Table 2). Except for

Hazen Creek and West Quako Creek, all downstream sites have been impacted by some waste water and or contamination (Table 2).

Figure 3. Map of the sampling site in the St. Martin’s area (approximately 55 km east of Saint John). West Quako Creek (WC).

Table 2. Habitat characteristics of studied sites: Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek WC). Sampling seasons from June to November 2003 and May to September 2004. Site Sediment type Depth Width Salinity Tide Year (m) (m) (ppt) influence used MCU fine substrate, 0.7 11 0.1 none 2004 organic matter MCD fine substrate, 0.9 ~ 13 0.6 high 2003-04 organic matter LRU organic matter, 0.7 12 0.1 none 2003-04 sand LRD sand, fine gravel, 1.0 15 0.5 – 13.0 small 2003-04 organic matter HCU fine substrate, 0.5 > 20 0.1 none 2004 organic matter (marsh) HCD fine gravel, sand, 0.7 7 0.1 moderate 2003-04 organic matter WC gravel, sand, fine 0.9 8 0 moderate 2003 substrate

1.2.1 Oil Refinery

The Irving Oil refinery was built in 1960 and started operating July 20th of the same year. The initial production capacity was 40,000 barrels a day. Two expansions took place following the initial construction (Irving Oil Limited 2004b).

A first expansion in 1971 increased the production to 120,000 barrels per day, and a second expansion in 1974 increased production to 250,000 barrels per day

(Irving Oil Limited 2004b). The refinery has the largest crude oil capacity in

Canada (St-Cyr 1995). The refinery produces gasoline, diesel, fuel oil, asphalt, butane, and propane. The refinery modernized its facility in the 80’s and 90’s, with a major upgrade from 1998 to 2000. This renovation included the addition of five new modules for environmental controls and replacement of old units for oil product processing (Irving Oil Limited 2004b).

The transformation of crude oil into final hydrocarbon products has many steps.

The refining process starts with fractionation, where crude oil is heated to separate lighter components, which vaporise, from heavier components. Non- vaporized amalgam can be collected at the bottom and heated at higher temperatures to separate it into different compounds (Irving Oil Limited 2004b).

Once vaporized, the compounds are sent to the distillation tower where they

condense in different trays depending on the specific boiling point of each substance (Coyote Oil 2004). Vaporization and condensation take place numerous times to purify the different products before sending them to the cracking unit. There, the hydrocarbon molecules are broken down and reorganized into more valuable substances (Coyote Oil 2004). The last step is the blending process, where hydrocarbons are mixed according to special formulas and additives can be incorporated (Irving Oil Limited 2004). At each step, different substances can be added or removed that will end up in the waste water. This is the major source of water contamination. Oil and grease, phenols, ammonia, suspended and dissolved matter and substances that contribute to chemical oxygen demand (COD) and biological oxygen demand (BOD) are the main contaminants. Other substances such as heavy metals, cyanides and fluorides can also be found in the waste water in smaller quantities (St-Cyr 1995).

In addition to water contamination from oil processing activities, the refinery’s waste water comes from four sources: intake water, storm water, sanitary wastes, and ballast water (St-Cyr 1995). The intake water is necessary in boilers and in cooling towers. Depending on its origin and quality, treatment may be needed prior to its use. Storm water is the result of precipitation and runoff from the refinery propriety. It can be contaminated with oil and grease, phenols and suspended matter. The regulations and guidelines allow additional deposit amounts of those substances when storm water is processed with waste water

(St-Cyr 1995). Sanitary wastes are from employee buildings and laboratories.

The contaminants are mainly suspended and dissolved matter and represent a small portion of total waste water (St-Cyr 1995). Seawater is used as ballast in tankers to ship stability and to clean tanks and compartments. It can be contaminated with oil, phenols, chlorides, and suspended and dissolved matters.

The Irving Oil Refinery processes ballast water separately from the other sources

of waste water. It is stored in specific tanks and processed periodically.

Processing include skimming for surface oil and grease and aeration before it is

mix into final effluent and discharged.

The refinery discharges approximately nine million cubic meters (9,000,000 m3) of treated effluent a year (Irving Oil Limited 2001, 2002, 2003, 2004). No information on the wastewater treatment facilities is available before the mid- seventies. However, at that time, waste water received secondary treatment

(Richard Russell; pers. comm.). The wastewater unit had a major upgrade in the mid-nineties, which doubled its capacity. The actual wastewater treatment facilities include a primary treatment for gravity removal of floatable oil and settling of sludge, and secondary treatment with long-term aeration for toxicity removal, with a retention time of approximately four to five days (Richard Russell; pers. comm.).

1.3 Previous Studies in the Area

Prior to this study, the fish community in Little River was rarely studied. A fish survey (1998-2000) was done upstream of the area selected for this study prior to the installation of a retention dam by Ducks Unlimited (Joe Harvey, Habitat

Specialist Ducks Unlimited Canada; pers. comm.). The main species collected in the fall surveys were suckers (Catostomus spp.) and American eel (Anguilla rostrata) (Joe Harvey, Habitat Specialist, Ducks Unlimited Canada; pers. comm.). In 1995-96, mummichog (Fundulus heteroclitus) were exposed in a lab study to Little River water (Leblanc 1998). In 1995, the paper mill was still discharging its effluent in Little River. A 96-hour exposure determined that the concentration of river water where 50% mortality was recorded (LC50) was 23%

(LeBlanc 1998). In 1996, the paper mill discharged its effluent in the harbour and no mortality was recorded even at 100% Little River concentration. A 12-day exposure at sublethal concentrations was also performed on mummichog and

goldfish (Carassius auratus) although no consistent impacts were found in

plasma and in vitro gonadal steroid levels (LeBlanc 1998).

Marsh Creek has been studied much more than any of the other streams in Saint

John as it is one of the city’s “hot spots” and has more public access. Studies

have mainly been undertaken by Atlantic Coastal Action Program (ACAP) Saint

John, a non-profit organization working on sustainable development. A study

done in 2000 assessed Marsh Creek habitat, water quality, benthos community

and lethal and sublethal effects on fish (Astephen 2000). The studied area was approximately 6 km long, along Marsh Creek and its main tributary with nine sites selected (Astephen 2000). All sites were in fresh water so MCD was not included in this study. A total of nine different fish species were found by electrofishing, with an average of five species per site. Fish species collected were white sucker (Catastomus commersoni), banded killifish (Fundulus

diaphanus), threespine stickleback (Gasterosteus aculeatus), brook stickleback

(Culaea inconstans), ninespine stickleback (Pungitius pungitius), American eel

(Anguilla rostrata), creek chub (Semotilus atromaculatus), blacknose dace

(Rhinichthys atratulus) and rainbow trout (Oncorhynchus mykiss) (Astephen

2000). The benthic community survey indicated poor condition in the lower section of Marsh Creek (including MCU and upstream of MCD) as more than

50% of the community was composed of oligochaetes and chironomids

(Astephen 2000). Sediments in Marsh Creek were shown to be toxic to mummichog in a lab experiment and mixed-function oxygenase (MFO) induction was recorded even at the lowest concentration (0.5 g of sediment in 12 L aquaria) (Astephen 2000).

ACAP assesses water quality in different areas in the vicinity of Saint John, including upstream and downstream of the section of interest in Little River, in

MCD, HCU and MCU. The oil refinery also has a water quality survey conducted on six different sites along its property, including the studied area. The results of

those studies will be discussed in context, in chapter 3.

More studies have been carried out in the Saint John Harbour including studies

on water and sediment quality, invertebrates and fish. Invertebrates collected in

the Saint John harbour had metal and polychlorinated biphenyls (PCB) levels

correlated with exposure to high discharges of municipal sewage (Brillant 1999).

Rock gunnel (Pholis gunnelus) inhabiting areas of high contamination had higher

condition factors, larger liver sizes and lower numbers of juveniles than fish

collected in reference areas (Vallis 2003). LeBlanc (1998) caged mummichog in

different areas in the harbour and found that male mummichog in the most

contaminated areas had altered reproductive endocrine functions.

1.4 Organization of Thesis

The document is divided into six chapters. The first chapter presents general

information. Each of the four other chapters represents a different experiment:

fish community survey, water quality survey, sentinel species survey, and caging

experiment. Each chapter has an introduction, and methods, results and

discussion sections. The last chapter is an overall discussion synthesizing results

and presenting future directions.

1.5 Objectives and Hypothesis

There have been few studies on the effects of oil refinery effluent. When effluent is discharged into an estuary, the effects can be concealed by multiple challenges affecting interpretation, such as fish movement, inappropriate reference sites and inconsistent exposure. This study, therefore, has two main objectives. The first is to assess potential effects of the oil refinery effluent discharge on fish and fish habitat in Little River. The hypotheses are:

HO: Fish living downstream of the oil refinery effluent discharge are not

performing differently than fish in reference sites.

H1: Fish living downstream of the oil refinery effluent discharge have a

different performance than fish in reference sites.

HO: Fish habitat, in terms of water quality, is not impaired downstream of the

oil refinery discharge site.

H1: Fish habitat, in terms of water quality, is different downstream of the oil refinery discharge site.

The second objective is to evaluate the methodology used in the EEM program in a small coastal stream situation.

2 FISH COMMUNITY SURVEY

2.1 Introduction

The relative abundance of potential sentinel fish species was not known for Little

River. A fish survey was conducted to assess seasonal changes in the relative abundance of fish species to identify potential sentinel candidates. The main objectives were to identify proper reference sites, suitable sentinel species, and the best time to collect them. As the project evolved, the importance of the fish community survey became apparent and it was used to assess potential effects of the refinery of the refinery.

The natural features of Little River allowed few fishing gear options. The very shallow water in some areas combined with many small culverts and limited access made a canoe the only boat usable on the river. Electrofishing was impossible since some sites were too deep or had a high turbidity. Passive, large equipment, such as trap nets or hoop nets, were also avoided as three of the sites are easily accessible by the public and there were concerns about interference. Beach seining was believed to be inappropriate because of soft or irregular bottoms, and/or undercut banks, and/or absence of banks free from vegetation. Consequently, minnow traps seemed to be the best gear to capture the fish in the streams.

Trapping permitted the capture of many species of fish but there were concerns regarding efficiency and whether the data were representative of the fish community. The two main biases in using minnow traps are their species selectivity and their variable catch efficiency (Rozas & Minello 1997). Therefore, beach seining (traditional and using a canoe) was utilized to evaluate whether the trapping was missing any information.

2.2 Methodology

2.2.1 Fishing with Minnow Traps

The community survey was initiated in November 2002 in Little River, upstream

and downstream of the effluent discharge. Minnow traps were set in the

downstream area for the months of November, December and February.

Upstream, the traps were only set in November for the upstream site as the

water froze in this section in early December of 2002. Because of low catch

success, trapping was stopped until the ice cover melted in mid-April 2003.

The community survey was initiated again in May 2003 and additional sites were added during that month at three reference sites (LRU, HCD, WC) and two contaminated sites (LRD and MCD). LRD received oil refinery effluent and MCD received untreated municipal sewage and had creosote contamination. After the

fish survey of June, the Marsh Creek downstream (MCD) site was moved about

1.2 km downstream to include the potential effects of seven untreated sewage outfalls from the city of Saint John. For six months, from June to November, monthly community surveys were conducted. Surveys were initiated again in

2004 from May to September.

Each month, 10 minnow traps (baited with dog food) were set per site, usually in pairs and fixed to a buoy. Minnow traps were cylindrical (42 cm long and 23 cm at their largest diameter), with funnel openings on each side (2.5 cm diameter).

Two types of minnow traps were used: galvanized and plastic coated, both having similar mesh size (5 mm). An effort was made to set an equal number of each type of traps per site, as well as tying different types of traps to the same buoy. Traps were removed after 24 hours and information regarding abundance, number of species, number of dead fish, and the type of trap was recorded. Lost or slightly opened traps were also noted. Once the information was recorded, fish were released in the area they were caught. When a fish could not be identified on site, one or two specimens were brought back to the laboratory for identification (Scott & Crossman 1998).

2.2.2 Seining Trials

In June 2003, beach seining was attempted by wading and the seine was

brought as ashore where possible. Sampling was repeated in July, this time

using a canoe to set and haul the seine. Beach seine measured 50 feet long and

six feet high (9-11 mm mesh size). Three hauls where performed at each site per month. Each haul was done in a different zone usually upstream of the previous one.

2.2.3 Statistics

The statistical software programs SYSTAT 9 (Levene’s test and anaylsis of

variance [ANOVA]) and Sigma Stat 3.0 (Kruskal-Wallis) were used. Primer 5.0

was used for community indices (Bray Curtis similarity and cluster analyses and

Shannon biodiversity index). The data were analyzed per site for the total

abundance per year, total abundance per month, total number of species per

month, frequency of empty traps per month, total number of dead, Bray-Curtis

similarity index and Shannon biodiversity index.

Analysis per month (abundance per month, number of species per month,

frequency of empty traps per month, and the community indices) regrouped the

data from 10 minnow traps rather than a mean by trap for each sampling month.

Consequently for a given month and a given site, the result compile the sum of

the fish collected from 10 minnow traps. During a monthly sampling, some traps

were lost or found open. When the total number of minnow traps per site per

month was less than 10, the measured abundance was adjusted to reflect the

abundance for 10 traps by using the following formula:

Adjusted result = Result found X 10 Number of actual traps

The data collected in MCD in June 2003 could not be used for the community

analysis as the site was moved downstream after the monthly sampling. The final

MCD site was downstream of many outfalls from the city as well as legacy creosote deposition. It was eliminated from the analysis because it was assumed

that the fish collection 1.2 km downstream would have been different. Because the analysis per month used all the months to estimate the mean and the standard deviation, it was not possible to include the June survey in the 2003 analysis. For the similarity analyses, each month was taken separately and

compared to the other months. Consequently, it was possible to present results from June except for MCD, which started in July 2003.

For data sorted per month, a Levene’s test was done to assess for homogeneity of residual variances. If data were homogeneous, ANOVA was used. If not, transformation was attempted to meet the assumptions for ANOVA. If transformation of the data did not correct the situation, the Kruskal-Wallis test for non parametric data was used. Pairwise multiple comparison tests were done using the Holm-Sidak method or Dunn’s method depending if data were analysed with ANOVA or Kruskal-Wallis, respectively. Also, tests were done to ensure that the type of trap was not a confounding factor. When no differences would be

found among treatment, the minimum detectable difference was calculated using

the formula:

δ= √(2ks2Φ2/n) δ= Minimum detectable difference

k= Number of treatments or groups

s2= Sample variance

Φ= Value related to noncentrality

parameter used to determine power

n= Sample size (replication)

(Source: Zar 1984).

For the minimum detectable difference, two levels of ∝ and β were applied, ∝ =

β = 0.05 and ∝ = β = 0.10.

2.3 Results

2.3.1 Fishing with Minnow Traps

The community survey using minnow traps found a total of 12 species and more than 2500 fishes in 2003. The most common and abundant fish was the

mummichog (Figure 4). Mummichog were found from June to November at

almost all of the sites. It was the most frequent fish in two sites (LRD and HCD)

and the second most abundant fish in two sites (MCD and WC; Figure 4). In

2004, almost 2800 fishes and 10 species were trapped. Once again the most

common and abundant fish was the mummichog (Figure 5).

Figure 4. Relative abundance (%) of each species collected by minoow traping per site from July to November 2003. Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

Figure 5. Relative abundance (%) of each species collected by minnow trapping per site from May to September 2004. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek Downstream (HCD).

In some sites, mummichog represented more than 50% of the community (MCD

58%, HCU 63.6%, HCD 81.7 %). The fourspine stickleback (Apeltes quadracus)

was the second most abundant fish in 2004, representing 69.4% in MCU, 35.4%

in MCD and 10.1% in HCD (Figure 5).

Little River downstream scored lowest for total abundance in 2003 and second

lowest in 2004 with a total catch of 286 fishes in 2003 and 182 fishes

respectively. The main species found in LRD were the mummichog (75.6%) in

2003 and the threespine stickleback (Gasterosteus aculeatus) in 2004 (69%)

(Figures 4-5). MCU scored lowest in 2004 with 157 fishes caught (Table 3). In

comparison, Hazen Creek downstream and Little River upstream had the highest

numbers in 2003 and 2004, respectively (Table 3). HCD was dominated by the

mummichog and LRU was dominated by the blacknose shiner (Notropis

heterolepis) (Figure 5).

Sampling took place from June to November 2003, with MCD results starting

only in July, and again from May to September 2004 (Figures 6-7). For both

years, LRD had the lowest abundance and number of species per month.

Table 3. Total number of fish caught per site (Marsh Creek Upstream [MCU], Marsh Creek Downstream [MCD], Little River Upstream [LRU], Little River Downstream [LRD], Hazen Creek Upstream [HCU], Hazen Creek Downstream [HCD], and West Quako Creek [WC]). Sampling from July to November 2003 and May to September 2004. MCU MCD LRU LRD HCU HCD WC

2003 501 600 286 752 328

2004 157 288 946 182 718 665

(A) June 250 July Aug 200 Sept Oct 150 Nov

100 Abundance

0 MCD LRU LRD HCD WC

(B) June 9 July 8 Aug 7 Sept 6 Oct 5 Nov

3 Number of species 2

0 MCD LRU LRD HCD WC

Figure 6. Total abundance (A) and total number of fish species (B) per month per site. Results from 10 traps per day per month, June to November 2003. Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD) and West Quako Creek (WC).

(A) May June 500 450 July 400 August 350 Sept. 300 250 200 Abundance 150 100 50 0 MCU MCD LRU LRD HCU HCD

(B) May 9 June 8 July 7 August 6 Sept. 5

3 Number of species 2

0 MCU MCD LRU LRD HCU HCD

Figure 7. Total abundance (A) and total number of fish species (B) per month per site. Results represent data from 10 traps per day per month, May to September 2004. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek Downstream (HCD).

The frequency of empty traps per month in Little River Downstream (LRD) was significantly higher than all other sites in 2003 (ANOVA df=4; p<0.001) (Figure

8A). Similarly, in 2004, LRD had more empty traps per month when compared to other downstream sites as well as when compared to Little River Upstream

(LRU) (ANOVA df=4; p=0.009) (Figures 8B). Reference sites (MCU, LRU, HCU,

HCD, and WC) had a similar frequency of empty traps during each studied year

(ANOVA df=4; p=0.104 or more).

The total abundance per month was less in LRD than in HCD, MCD and LRU in

2003 (ANOVA df=4; p<0.012) (Figure 9A). No significant differences could be found in 2004 (ANOVA df=4; p=0.162) (Figure 9B). The minimum detectable differences for total abundance per month in 2004 were approximately 213 and

159 for α=β=0.05 and α=β=0.10 respectively. The total number of species per month was also lower in LRD for both years when compared to its respective reference sites (Figure 10). In 2003, Little River Downstream also had a lower biodiversity, measured by Shannon-Wiener index, than all sites except HCD

(ANOVA df=4; p=0.010). In 2004, LRD had lower biodiversity compared to LRU as well as other downstream sites (ANOVA df=4; p=0.002)(Figure 11).

A) 9 B 8 7 6 5 A 4 A 3 A

Mean of frequency A 2 1 0 MCD LRU LRD HCD WC

site

B) 9

6 a 5 a 4 a a 3 Mean of frequency 2 a

0 MCU MCD LRU LRD HCU HCD site

Figure 8. Frequency of empty traps. Results from 10 traps per month, from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors(n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

A) A

A A 140

120

100 AB 80

40 B

20 Mean of total abundance per month 0 MCD LRU LRD HCD WC Site

B) 300 a

250 a a 200

150

a b 100 a

50 Mean of total abundance per month 0 MCU MCD LRU LRD HCU HCD Site

Figure 9. Total abundance per month per site from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors (n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

A) A 6 AB 5

B B 4

3 C

1 Mean of total abundance per month 0 MCD LRU LRD HCD WC

Site

B) 8 a

7 a

ab b 5 bc 4 c

Mean of total abundance month per 1

0 MCU MCD LRU LRD HCU HCD Site Figure 10. Total number of species per month per site from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors (n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

1 0.9 0.8 0.7 0.6 A AB AB 0.5 BC value 0.4 C 0.3 0.2 0.1 Mean of Shannon biodiversity index 0 MCD LRU LRD HCD WC Site

B) 1

0.9

0.8

0.7 a ab 0.6 b 0.5 b b 0.4

0.3 c

0.2

0.1

0 MCU MCD LRU LRD HCU HCD Mean of Shannon biodiversity index value Site

Figure 11. Shannon biodiversity index per month per site from July to November 2003 (A) and from May to September 2004 (B). Means with standard errors (n=5). Different letters represent statistical differences. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

For 2003, references sites were very similar in terms of frequency of empty traps,

abundance, number of species and biodiversity. In 2004, MCU was the most

dissimilar site compared to the other upstream sites. It showed decreased

number of species compared to LRU and HCU (ANOVA df=4; p=0.013), and

lower biodiversity compared to LRU and HCU (ANOVA df=4; p<0.001 and

p=0.011, respectively) (Figures 10-11). MCD was similar to reference sites in

2003 and similar to HCD and MCU in 2004 for all the parameters measured

(Figures 8-11).

The cluster analyses present the similarity among each sampling site for each sampling date for 2003, 2004 and a combination of the four sites (MCD, LRU,

LRD, and HCD) that were sampled during the two years of the project (Figures

12-14). A first observation is that, in general, similarity is low among data. The overall data presented a similarity of less than 25%, which would usually

represent the similarity of LRD sampling periods compared to other sites and

dates (Figure 12-14).

The similarity in 2003 was primarily driven by site. Similarity among sampling

period within a site was as high as 50% for LRD and HCD. MCD, LRU and WC

had the lowest similarity among sampling periods with 30% and 25% similarity,

respectively (Figure 12). The multidimensional scaling (MDS) graph shows, similarly, a regrouping of data points for each site (Figure 15).

Figure 12. Dendrogram of the ordination according to similarity among months and sites, from June to November 2003. Labels represent site and date (month, year). E.g., LRU0703 stands for Little River Upstream July 2003.

Figure 13. Dendrogram of the ordination according to similarity among months and sites, from June to November 2004. Labels represent site and date (see previous figure). LRD0704 is missing as no fish were collected.

Figure 14. Dendrogram of the ordination according to similarity among months and sites, from June to November 2003 and May to September 2004. Compilation of four common sites of both sampling years. Labels represent site and date (see previous figures).

Figure 15. Multidimensional scaling of Bray-Curtis similarity per month and site, from June to November 2003. Labels represent site and date (see previous figures). Bubble sizes indicate tidal influence (small circle= no influence; big circle= strong tidal influence).

The grouping is clearer for LRU and LRD, whereas the three other sites tend to group closer and sometimes overlap. These sites are also more influenced by tide compared to LRU and LRD (Figure 15). Each site is temporally stable as

they have similar communities throughout the months. Stress levels for all MDS

figures are bellow 0.2 suggesting that the 2-dimentionnal representations are

satisfactory and a good summary of the sampling period and site relationships

(Clarke & Warwick 2001).

In 2004, the force driving the separation changed and sampling month as well as

upstream-downstream pattern seemed to have more influence on similarity of

populations. No fish were collected in LRD in July. For 2004, similarity among

sites was approximately 45% among sites sampled in May and June, and 42%

for sites sampled from July to September (Figure 13). The influence of the month

is not as well reflected in the MDS graph (Figure 16). All LRU sampling periods

tended to cluster with a similarity around 60% (Figures 13, 16). The upstream

sites had 40% similarity and tended to be all on the upper left section of the

graph and downstream sites are found in the lower right portion of the illustration

(Figure 16).

The LRD fish community found May 2004 is very different from the other months

(Figure 16). This sampling period included the highest abundance and number of

species of LRD in 2004 with five different species and more than 170 fishes

(Figure 7).

Figure 16. Multidimensional scaling of Bray-Curtis similarity per month and site, from May to September 2004. Labels represent site and date (e.g. LRU0504 stands for Little River Upstream May 2004). Bubble sizes indicate tide influence (small circle= no influence; big circle= high tide influence). LRD0704 is missing as no fish were collected.

Finally, when comparing sites common for both years, similarity among LRD

sampling periods was about 40%, and similarity among LRU sampling periods is

just above 35%. The combination of MCD and HCD sampling periods had a

similarity of 40% (Figure 13). There was a high similarity for site and month

between the two years (e.g.., LRU in June [LRU0603 (June 2003), LRU0604

(June 2004)]), in August and in September; LRD in August, HCD in July and in

September; and MCD in June). The MDS presents the same results, with a well-

defined cluster for LRU and LRD and an intermixed cluster of MCD and HCD

(Figure 17). Once again the May 2004 sampling in LRD is more similar to the

other site communities than the other LRD communities and represent the only

outlier of the LRD grouping (Figure 17).

Very little catch mortality was recorded during the study. Overall, the mortality

rate was 1.2% in 2003 and 1.0% in 2004. The highest mortality rate in 2003 was

in LRD with 7.8%, followed by HCD and LRU with 1.5% and 1.2% respectively. In

2004, the highest mortality rate was found in HCD with 2.1% followed by MCU

with 1.3%. No statistical differences were found among sites for mortality per

month in 2003 (ANOVA df=9; p= 0.405). The minimum detectable size in 2003 at

α=β=0.05 was 2.5 dead fish/month and was 1.8 dead fish/month for α=β=0.1. In

2004, the mortality was highest in LRU and HCD with means 2.0 and 2.8 dead

fish/month respectively (ANOVA df=9; p<0.03 for HCD and p<0.049 for LRU).

MCD

LRU

LRD

HCD

Figure 17. Multidimensional scaling of Bray-Curtis similarity per month and site, from June to November 2003 and May to September 2004. The symbols represent the four common sites of both sampling years. Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), and Hazen Creek Downstream (HCD).

An effort was made to place the same amount of plastic and metal traps in each site and as follows an analysis was done to test if type of trap could affect the number of fish and species. For both years, the metal-coated traps collected more species and more individuals than the plastic-coated traps (Kruskal-Wallis

76≤df≤171; p = 0.001). In general, the catch rate of the metal coating is double compared to the plastic coating (Table 4).

2.3.2 Seining

The trials conducted in June 2003 used the traditional beach seining method. At

the upstream site in Little River, accumulation of organic matter on the bottom of

the stream prevented any attempt to walk in the stream. At the downstream site,

the bottom was hard enough to walk on, but the irregularity of the bottom in terms

of depth and obstacles (log and rocks) reduced the speed of the walk, permitting

fish escapes. Moreover, the shore was steep and the vegetation dense so it was

very difficult to bring the seine on shore. No fish were caught in the seine for all

four trials. The trials conducted in July, using a canoe, were more successful and

many fish were caught in the upstream section. No additional species were

captured with beach seine beyong those already captured by the minnow traps

(Table 5). The vast majority of the fish caught in the seine were young of the

year.

No fish were caught downstream of the discharge site for all three hauls.

Table 4. Number of species and abundance per trap for the 2003 and 2004 sampling seasons and comparison between metal and plastic-coated traps. Mean ± standard error (sample size). Different letters represent statistical differences. Metal Plastic Number of 2003 1.60 ± 0.09 (171) 0.86 ± 0.13 (76) species a b 2004 1.81 ± 0.10 (139) 0.90 ± 0.11 (115) A B Abundance 2003 9.90 ± 1.07 (171) 3.32 ± 1.60 (76) a b 2004 11.36 ± 1.46 (139) 4.84 ± 1.61 (115) A B

Table 5. Abundance and percentage (in parentheses) of fish caught using a beach seine at Little River upstream site. July 22, 2003. Fish species Haul 1 Haul 2 Haul 3 Banded killifish (Fundulus diaphanus) 4 (1.8) Blacknose shiner (Notropis heterolepis) 16 (7.3) 6 (4.7) 21 (16.5) Common shiner (Notropis cornutus) 2 (<1) 12 (9.4) Fourspine stickleback (Apeltes quadracus) 64 (29.1) 80 (62.5) 29 (22.8) Mummichog (Fundulus heteroclitus) 2 (<1) 1 (<1) Ninespine stickleback (Pungitius pungitius) 111 (50.5) 38 (29.7) 51 (40.2) Threespine stickleback (Gasterosteus aculeatus) 16 (7.3) 4 (3.1) 13 (10.2) White sucker (Catostomus commersoni) 5 (2.3) Total fish caught 220 (100) 128 (100) 127 (100)

2.4 Discussion

Little River Downstream had a reduced fish community compared to the other sites during the two years of the study. In 2003, LRD was compared to other downstream sites (MCD, HCD and WC) as well as to its upstream site, LRU. In

2004, LRD was compared to HCD and MCD as well as LRU. It had a lower abundance and species richness, higher frequency of empty traps and, consequently, lower biodiversity than the compared sites (Figures 4-11). It also had mortality more than four times larger than the other sites, although the variability was high and the difference was not significant. The difference in the fish community of LRD is also visible when analysing the dendrogram and the

MDS graphs (Figure 12-17).

The main species found in 2003 and 2004 were the mummichog, the threespine stickleback and the ninespine stickleback (Figures 4-5). They represented 98% of the community in 2003 and 96% in 2004 (Figures 4-5). These species have usually a small home range and low mobility, although mobility increases during the spawning season (Scott & Crossman 1998).

The mummichog spawns from May to August in the Maritimes region (Scott &

Crossman 1998). Mummichog migrate in spring to slightly brackish water and even fresh water. Temperature, food availability and current velocity can encourage even further upstream movement (Scott & Scott 1989). This species

usually follows tidal movements and is easier to catch with the rising and ebbing tides as well as slack high tides (Butner & Brattstorm 1960; Kneib & Craig 2001)

For both years, the mummichog was consistently present throughout the months

with two peaks, in July (abundance=19 fishes in 2003) and in October

(abundance=11 fishes in 2003). The constant but low abundance found in many

months could be related to tidal movements. On the other hand, the July peak in

LRD corresponds to the reproductive period of the mummichog and is probably a

consequence of spawning migrations. Overwintering habitats of mummichog are unknown, but the higher abundance in the fall may represent pre-overwintering movements.

The threespine stickleback was also an abundant fish in LRD, representing 69% of the total abundance in 2004 (Figure 5). It is also a spring spawner, spawning in June and July in brackish or fresh water (Scott & Scott 1989). The threespine stickleback was the most abundant fish in 2004 representing 69% of total catch

(abundance=126). However, it was only present in May. Migration from salt

water to spawning site occurs in the spring (Scott & Scott 1989). Males caught

during May had the bright color patterns (blue eyes, pink-red belly and flanks)

found in reproductively active (ripe) males. The same peak of abundance in May

was also recorded in 2003 for this species (data not shown). The presence of the threespine stickleback in LRD seems strongly related to spawning migration.

The ninespine stickleback was the third most abundant species, representing

21% of the total in 2004. This summer spawning species prefers freshwater sites

with vegetation (Scott and Crossman 1998). Once again, the species was only

caught in May, which corresponds to its prespawning period when movement to

spawning sites is high.

The other fish species caught in LRD were all freshwater fishes present in LRU

and were found less than five times over the two years.

The mobility of fish species is also noticeable in other sites. In LRU, mummichog

and threespine stickleback were caught in vast majority between June and

August. These months correspond to their spawning peaks. MCD and HCD are

also suspected to have high fish mobility. The two sites had the highest

similarities, especially when comparing dendrogram and MDS graphs (Figures

13-17). Their resemblances also correspond to their respective proximity to the

harbour and to their high tidal influence.

The effect of tide was more apparent in MCD than in HCD as the section of the

stream used for the study was located between two hills. The window when the

area was not under tidal influence and where site was accessible was

approximately one hour before and after low tide (personal observation). As the

tidal effect was so dominant, fish movement from the harbour into the stream

most likely followed. The species found downstream are mainly species

tolerating a high range of salinity and are capable of living in fresh water and salt

water (Scott, & Scott 1988). Their movements with the tides are also well

documented (Scott & Scott 1988; Kneib & Craig 2001; Teo & Able 2003). It is

highly probable that fish caught in MCD are not continuously living in the area but

rather moving in and out following the tide cycle.

The presence at the same time of Atlantic tomcod (Microgadus tomcod) and the

brook trout (Salvelinus fontinalis) in West Quako Creek could also be related to

fish movement. The coexistence of two species, one freshwater, one saltwater, in the same section of a stream could only be possible if the species are mobile rather than resident (Figure 4).

Fish mobility in brackish and salt water has been one of the major problems in the EEM program for the first two cycles (Courtenay et al. 2002). The mobility limits the capability to confirm exposure and thus the ability to determine the effect of the effluent on fish. Between seasonal and/or reproductive migrations and movement with the tides, it is believed that very few fish species living in estuaries and marine habitats are actually resident enough to be used in environmental assessment (Courtenay et al. 2002).

Although the mobility of fish is apparent in the downstream sites, the low

abundance of fish caught in LRD shows that fishes are avoiding this section of

Little River. The general abundance was low, the frequency of empty trap was

high, and the presence of the different species was sporadic. Both the abundance and the species richness were lower in LRD than at the reference sites. The difference in the fish community found in LRD compared to other site is unquestionable and more investigation will be needed to assess the causes.

If mobility was considered to be a major factor in the downstream sites, the upstream sites should have more resident communities as they were all Fresh water. In general, the upstream sites had similar communities for the majority of the parameters measured. The major differences were in their diversity indices as well as the Bray-Curtis similarities (Figures 11, 13, 16). Each site had a different dominant species, which explain the separation of groupings in the MDS graph (Figures 5, 16).

In 2004, MCU had the highest dissimilarity among upstream sites. Marsh Creek

Upstream had an increased frequency of empty traps and lower abundance than

other upstream sites, although not statistically significant (Figures 8-10). It also

had significantly reduced number of species (Figures 8-10). A survey done by

ACAP in 2001 also shown that the area had lower abundance (Astephen 2000).

Electrofishing permitted to capture a similar number of species (5 fish species)

and less than half the abundance collected in other sites in Marsh

Creek(Astephen 2000). The reduced abundance was attributed to the reduction

of aquatic vegetation in this section of the stream (Astephen 2000).

Also, the absence of mummichog deserves a note (Figure 5). Its absence could be related to: 1) the contaminated area downstream that prevents the fish from moving upstream for spawning, and 2) the distance of MCU from the harbour.

The site was about 4.5 km from the mouth of the stream and mummichog may not move that far inland. The other species absent from MCU but present in the other upstream sites are more often found in weedy ponds, lakes or bays (Scott

& Crossman 1998). As MCU is the only upstream site that was a shallow brook with little submerged vegetation, the habitat characteristics could explain the absence of other species (Scott & Crossman 1998).

Mortality was low for all sites for both years. Mortality was often associated with

American eels in HCD and LRU. The higher abundance of eels in those sites probably explains the significantly increased mortality per month in 2004. In

Marsh Creek Upstream, mortality was related to the accumulation of sediment in the trap. In LRD, mortality could not be attributed to predation or sediment. Death was not associated with any external lesions. It is assumed that mortality was related to water quality (See sections 2.3 and 2.4).

2.4.1 Seining Versus Trapping

The small coastal habitat assessed in this study was not suitable for beach

seining. Even when seining was done from the canoe, multiple factors reduced

the efficiency of the fishing gear. Bringing the two ends of the net together was

slow which permitted the escape of the faster fish. Pulling the seine in the canoe

also increased the loss of some fish as a hole was created between the bottom of the stream and the seine.

The vast majority (more than 95%) of the fish caught were young of the year.

Very few adults were caught which support the hypothesis that there were many

escapes of the faster fish. In addition, many young of the year increased the difficulty in properly identifying the species. More dead specimens were also observed as so many specimens had to be removed from the seine increasing the air exposure period. In addition, the seine moved and retained a lot of soft bottom, which increased stress on the fish. The last two hauls were particularly heavy in sediment and half of the fish caught died before they were released. It was obvious that seining caused perturbation on the fish populations. Concerns were also raised for the safety of the crew as so much sediment had to be manipulated, especially in impacted sites where a lot of potentially toxic sediments were deposited.

The efficiency of minnow traps to assess fish communities have been criticized in the past suggesting that minnow traps constitute a biased sampling technique and should not be used to quantitatively assess estuarine systems (Rozas &

Minello 1997; Layman & Smith 2001). Other studies suggest they can still be used particularly in habitats with inappropriate water depth, dense submerged

vegetation, or where undercut banks prevent the use of any other gear (Ambrose

& Meffert 1999; Halpin 1997).

The beach seining trials in July collected the same species as in the minnow

traps (Figure 4, Table 5). However, the abundance of fish and the presence of

sediment had a heavy impact on fish survival. Moreover, the efficiency of the beach seining can be questioned, particularly as few adult fish were caught.

The study showed that minnow traps can be used to assess effects of a point source discharge and its effect on the fish community in small coastal streams such as those present in the Saint John Harbour area. It was possible to measure an impact downstream of the oil refinery effluent discharge compared to reference sites. However, the high catch variability of the minnow traps did limit

the capacity to see more subtle effects. The catch variability among traps at

each site prevented the use of trap as replicates. The frequent presence of

extreme data (multiple empty traps and some traps containing dozens of fish)

caused a skewed distribution toward the left and a heterogeneous variance among sites. This preventing the use of a two way ANOVA to look at effect of site and time (month) on the fish community. Moreover, the homogeneity of variance was not met even when data were transformed. Although variations among month were present, the variation was more homogeneous and permitted the analysis of variance.

The use of months as replicates had some inconveniences. First, the effect of the

variable “month”, partly caused by fish migrations, could not be assessed. Also,

the variability caused by “month” was added to the actual variability caused by

“site”, reducing the capacity to detect differences among sites. The variability

associated with the month effect probably caused the absence of effects in

abundance per month in 2004 (Figure 9B). When comparison was done among

all sites, no difference was detectable (ANOVA df=4; p=0.175). Even when the

month of May was removed from the analysis, no significant difference could be

found (ANOVA df=3; p=0.119). However, when only comparing the upstream and

downstream site within a stream, LRD had statistically decreased abundance compare to LRU (ANOVA df=9; p=0.012). Nevertheless, the minimum detectable differences for total abundance per month were approximately 106 fish in 2003 and 302 fish in 2004.

In addition to the sources of variability mentioned above, the use of different types of coating on traps was also found to have an effect. Metal minnow traps were more efficient for collecting fish than the plastic-coated traps (Table 4). It is not clear what could have caused this. Plastic-coated traps were more difficult to close properly and more often we would find the plastic trap with a little opening between the two halves (personal observation). This could have led to the escape of some small fishes. The plastic trap is also more visible as the coated mesh is thicker than a simple galvanized metal mesh. A study comparing two types of one-funnel traps, with transparent or opaque funnel openings concluded

that the opaque funnel traps captured less fish than the transparent traps (Culp &

Glozier 1989). The opaque funnel affected fish activity and favoured escapes.

The fact that plastic minnow traps are more visible might have affected the fish behaviour similarly.

It seems evident that the use of minnow traps implies certain compromises, including higher probability of unequal variances among sites and a skewed dataset. In the actual study, the summation of traps content per month in part solved the problem. However, this limited the power of the tests. The use of minnow traps should be considered carefully in study design. Not only does it have variable catch efficiency, but it has a bias for certain species such as the mummichog (Layman & Smith 2001). Nevertheless, minnow traps might often be the only sampling gear usable in complex habitats. The summation of trap content and the use of sub areas within sites (each sub area containing multiple minnow traps) could partially solve the problem of catch variability.

3 FISH HABITAT ASSESSMENT

3.1 Introduction

The EEM methodology for pulp and paper mills uses a benthic community survey to assess effects on fish habitat (Environment Canada 1998). Benthic invertebrates are known to be very sensitive to habitat degradation, whether

through enrichment, toxicity or both. Usually aquatic invertebrate community

studies will favour an upstream-downstream or a gradient design to minimize

changes in habitat characteristics (Hall et al. 1997; Environment Canada 1998;

Glozier et al. 2002). It becomes very difficult to assess the effects of effluent in

complex systems, as changes in communities could also be attributed to

changes in habitat characteristics (Culp et al. 2000).

This study included multiple streams with different substrates and tidal

influences. There was consequently a high risk of confounding factors affecting

the interpretation of an aquatic invertebrate community survey. As an alternative,

it was decided to assess effects on fish habitat, initially through a water quality

survey. Water parameters, such as dissolved oxygen (DO), and biological and/or

chemical oxygen demand (BOD and COD), can provide useful information about

the quality of the habitat. The selection of target levels and endpoints depends on

the study needs (e.g., monitoring for protection of aquatic life, recreational

purposes, drinking water sources) and the type of waste (municipal, agricultural

or industrial) discharged into the environment.

The refinery discharges approximately nine million cubic meters (9,000,000 m3) of treated effluent yearly in Little River (Irving Oil Limited 2001, 2002, 2003,

2004). The effluent is composed of five different sources: processing activities, intake water, storm water, sanitary wastes, and ballast water (St-Cyr 1995). The main deleterious substances discharged and under regulations are phenols, total suspended matter, sulphide, ammonia nitrogen, oil and grease and any substances that can alter the pH of the effluent (Environment Canada 2004b).

3.2 Methodology

The water quality survey took place in various ways and by various parties. The refinery undertakes a weekly summer water-monitoring program upstream and downstream of its Little River outfall for dissolved oxygen (mg/L), salinity (ppt), temperature (oC), pH, faecal coliforms (#/100 ml), and turbidity (NTU). Water

temperature and dissolved oxygen are measured directly on site using a YSI

dissolved oxygen meter model 5100, and water samples are taken from the top

portion of the water column and brought to the lab for other analysis following the

refinery’s Lab Procedures Manual. The refinery also collects information on

monthly liquid discharge for the purpose of monitoring for deleterious substances

as defined under their effluent regulations.

Atlantic Coastal Action Program (ACAP) Saint John conducts a surface water

quality monitoring program throughout the summer months in multiple streams

Figure 18. Position of ACAP sampling sites for water quality monitoring program. Marsh Creek Upstream and Downstream (MU and MD), Little River Upstream and Downstream (LU and LD), and Hazen Creek Upstream and Downstream (HU and HD).

around the city of Saint John. This includes Marsh Creek (upstream and downstream), Hazen Creek (upstream and downstream), as well as upstream and downstream of the studied area in Little River (Figure 18). The monitoring program measures ammonia (total and free ions; mg/L), salinity (ppt), total phosphate (mg/L), dissolved oxygen (mg/L), temperature (oC), pH, faecal

coliform (#/100 ml), and turbidity (NTU). The water samples are taken every two

weeks by taking water samples in the top portion of the water column (ACAP

2001). The sampling is usually done weekly after 17:00.

In addition to these data, YSI 6920 hydrolab sondes were deployed for several

consecutive days in 2003 and 2004 upstream and downstream sites of Little

River. Sondes were attached to an anchor and to a buoy so as to sample the

bottom section of the water column. The sondes recorded the following

parameters every hour: dissolved oxygen (mg/L), salinity (ppt), temperature (oC),

turbidity (NTU), pH, depth, as well as time and date. Calibration following the

manufacturer’s operator manual (YSI environmental Operation Manual for the 6- series, 1999) was completed prior to field deployment, and then every six weeks until removal.

Hydrolab sondes were also used to delineate the plume and determine effluent

dilution at high and low tides using salinity. Every month, from June to November

2003, a sonde was deployed during the slake of highest tide and during low tide.

The sonde was placed in the water beside the canoe approximately 0.7 m into

the water. It was connected to a GPS instrument (Garmin GPS 12) to record

position. The recording started upstream where salinity readings were either

below 0.1 ppt or constant for more than 100 m. The canoe was then slowly

moved downstream in a zigzag movement to cover the maximum surface of the

river. Paddling was done on the opposite side of the sonde or as far as possible

behind the sonde.

Plume dispersion and concentration was calculated by using salinity at the

upstream site as the background salinity and by measuring salinity of the

effluent. Concentration of the effluent at any position in the river was calculated

using the following formula:

Effluent concentration = (Measured salinity - Background salinity ) *100

Effluent salinity

Because salinity was used to track the effluent plume, it was not possible to determine the proportion of salinity attributed to salt water compared to the effluent. However, it was assumed that salt water was coming in the river if the

salinity was higher than the effluent salinity and/or if salinity was increasing as the probe was moving downstream. If salinity in the river was below effluent level and decreasing or stabilizing as it moved downstream, it was assumed that no

salt water was coming in and effluent concentration could be calculated using the above formula.

3.3 Results

The oil refinery effluent dispersion pattern between high and low tide is shown on

Figure 19. During the spring and summer months (May to September),, high tides did not significantly alter the dispersion pattern and the effluent plume reached the first culvert about 100 m upstream of the discharge (Figure 19).

During highest tides of October and November, the presence of salinity higher than baseline salinity was measured up to 400 m upstream of the discharge point. In November 2003, the salinity in Little River constantly increased from 0.1 ppt 400 m upstream of the discharge to 13.9 ppt 800 m downstream of the discharge. Salinity of the effluent was 2.15 ppt. Similar data were collected in

October 2003.

The effluent plume quickly mixes with the stream water and is completely mixed within 400 m (about 100 m before the second culvert). Its dilution did not change dramatically between high and low tides over the sampled summer periods. The effluent concentration reached a minimum of 15%, with an average of 20% even

800 meters downstream of the discharge point (Figure 19).

Figure 19. Little River Downstream site. Effluent plume and effluent concentration at low, high and highest tides.

The most conclusive information derived from the long-term sonde deployments was the presence of extensive periods with low dissolved oxygen level

downstream of the effluent discharge from the oil refinery (Figure 20). Recorded dissolved oxygen in the downstream site reached levels below 2 mg/L during several hours during the summer of 2003 and 2004. The percentage of total recorded hours measuring hypoxic periods (DO < 2 mg/L) reached 21.5% and

40.4 % in July and August 2003, and represented more than 50% of the total recorded time in June 2004 (Table 6).

Levels of extremely low dissolved oxygen (< 0.5 mg/L) were recorded for up to three consecutive days in 2003 and up to 10 consecutive days in 2004. Number of hours recording extremely low dissolved oxygen in LRD represented 15.2% and 21.4% of total recording time in 2003 and 2004 respectively (Table 6). For both years, the periods are frequently correlated to higher salinity (Figure 21).

Whereas the upstream salinity was very stable around 0.1 ppt., the downstream salinity varied from 1 ppt to 13.5 ppt. The increased salinity was not correlated with daily tidal cycle or monthly highest tides (Figure 22). The highest tides of the month for June 2003 were on the 14th and 15th, and constantly decreased in

amplitude the following days (Tourism Saint John 2004). Increased salinity was

not associated with salinity in the upstream site either (Figure 23). The main

contributor to salinity in LRD during these periods is associated with the effluent

outfall (Figure 23).

Number of total recording hours 500 Number of hours with DO <2 mg/L 450 Duration of longest hypoxic (DO <2 mg/L) period 400 Number of hours with DO<0.5 mg/L 350 300 250

Hours 200 150 100 50 0 June 18 - 25, July 22 - Aug.4, Aug. 29 - Sept. Sept. 24 - Oct. Sept. 24 - Oct. 2003 2003 10, 2003 1, 2003 1, 2003

LRD LRU Date and site

600

500

400

300 Hours 200

100

0 May 10-31 June1-21 July 1-8 Aug 18-31 Sept1-17 June1-21

LRD LRU Date and site

Table 6. Total number of recording hours from hydrolab sondes deployed in LRD and LRU and percentage of hours recording dissolved oxygen below 2 mg/L and 0.5 mg/L. Results from 2003 (A) and 2004 (B).

A) Percentage of Percentage of Total recording hours with DO hours with DO Sampling period hours <2 mg/L <0.5 mg/L June 18 - 25, 2003 167 2.4 0 July 22 - Aug.4, 2003 484 21.5 13.0 Aug. 29 - Sept. 10, 2003 312 40.4 34.0 LRD Sept. 24 - Oct. 1, 2003 156 5.1 0.6 LRU Sept. 24 - Oct. 1, 2003 158 0 0

Percentage in LRD for 2003 21.6 15.2

B) Percentage of Percentage of Total recording hours with DO hours with DO hours <2 mg/L <0.5 mg/L May 10 – 31, 2004 517 0 0 June 1 – 21, 2004 492 51.4 45.3 July 1 – 8, 2004 185 0 0 Aug 18 – 31, 2004 320 22.2 4.4 LRD Sept 1 – 17, 2004 396 66.4 43.2 LRU June 1 – 21, 2004 491 0.2 0

Percentage in LRD for 2004 30.7 21.4

A) Salinity

12 DO

DO (mg/L) and Salinity (ppt) 2

6/18/2003 6/19/2003 6/20/2003 6/21/2003 Date 6/22/2003 6/23/2003 6/24/2003 6/25/2003

B) Salinity

10 DO 9 8 7 6 5 4 3 2

DO (mg/L) and Salinity (ppt) 1 0 9/1/2003 9/2/2003 9/3/2003 9/4/2003 9/5/2003 9/6/2003 9/7/2003 9/8/2003 9/9/2003 8/29/2003 8/30/2003 8/31/2003 9/10/2003 Date

Figure 21. Salinity (ppt) and dissolved oxygen (mg/L) recorded in Little River Downstream site, from June 18–25, 2003 (A) and from August 29 to September 10, 2003 (B).

10 Salinity (ppt) 9 Tidal cycle 8 7 6 5 4

Salinity (ppt) 3 2 1 0 6/18/03 6/19/03 6/20/03 6/21/03 6/22/03 6/23/03 6/24/03 Date

Figure 22. Salinity (ppt) recorded in Little River Downstream site with tidal cycle, from June 18 to 24, 2003. Tidal cycle 0= low tide and 1= high tide. (Source: Tourism Saint John 2004).

A) UP3 DWN5 20 18 Effluent 16 14 12 10 8

Salinity (ppt) 6 4 2 0 July 3/03 July 9/03 July Aug 6/03 June 4/03 July 16/03 July 23/03 July 30/03 July Aug 13/03 May 14/03 May 21/03 May 28/03 June 11/03 June 18/03 June 25/03 Date

B) 18 UP3

16 DWN5

14 Effluent 12

Salinity (ppt) 6

0 Aug 4/04 July 08/04 July 14/04 July 21/04 July 28/04 Aug 11/04 June 02/04 June 09/04 June 16/04 June 23/04 June 30/04 Date Figure 23. Salinity (ppt) for upstream site (UP3), downstream site (DWN5) and effluent. Data from the refinery’s summer water monitoring, from May to August 2003 (A) and June to August 2004 (B).

High temperatures have also been recorded in LRD, with a maximum

temperature of 26.6oC (Table 7). The differences between upstream and

downstream temperature would usually be minimal during the summer time,

although some periods recorded differences as high as 5oC (Figure 24).

The pH of water remained within a range of 6.5 - 7.8 throughout the survey. The turbidity varied considerably and was constantly higher downstream of the effluent discharge compared to the upstream site. Turbidity averaged 4 and 30

NTU at the upstream site in 2003 and 2004, respectively, and averaged 52 and

4474 NTU at the downstream site for the same years.

The data collected by ACAP in 2004 show a general high and stable temperature in all three streams (Marsh Creek, Little River and Hazen Creek). The average temperatures for each site vary between 18 and 23 during the summer months

(Tables 8-9). No information on temperature was collected in 2003. For 2003 and 2004, pH is generally around 7 or 8. One exception is in Hazen Creek

Downstream in July 2004 where pH has been as high as 9.2. In 2003, turbidity

was highest in Little River with 1.90 NTU and 0.72 NTU for downstream and

upstream sites, respectively. In 2004, turbidity was highest in LRD (10.7 NTU)

followed by MCD (3.4 NTU). In general, dissolved oxygen increased between

2003 and 2004. Dissolved oxygen levels were between 4 and 15.2 mg/L in 2003

except for LRD where levels were as low as 1.17 mg/L in August. In 2004,

Table 7. Maximum temperature recorded during extended sonde deployments in Little River Downstream (LRD) and Little River Upstream (LRU) in 2003 and 2004.

Maximum temperature Date recorded (oC)

LRU Sept. 24 - Oct. 1, 2003 19.7

June 18 - 25, 2003 25.7

2003 July 22 - Aug.4, 2003 26.6 LRD

Aug. 29 - Sept. 10, 2003 22.6

Sept. 24 - Oct. 1, 2003 23.7

LRU June 1 - 21, 2004 21.5

May 10 - 31, 2004 19.2

June 1 - 21, 2004 26.2 2004 LRD July 1 - 8, 2004 26.4

Aug. 18 - 31, 2004 23.8

Sept. 1 - 17, 2004 21.9

15 LRU

10 LRD LRD - LRU Temperature (oC) 5

B) 09/24/03 09/25/03 09/26/03 09/27/03 09/28/03 09/29/03 09/30/03 Date

LRU 30 LRD 25 LRD - LRU

5 Temperature (oC) 0

-5 6/1/04 6/2/04 6/3/04 6/4/04 6/5/04 6/6/04 6/7/04 6/8/04 6/9/04 6/10/04 6/11/04 6/12/04 6/13/04 6/14/04 6/15/04 6/16/04 6/17/04 6/18/04 6/19/04 6/20/04 Date

Figure 24. Water temperature (oC) recorded in Little River Upstream (LRU) and

Downstream (LRD) sites, and difference between the two sites (LRD - LRU).

September 24-30, 2003 (A) and June 1-21, 2004 (B).

Table 8. Atlantic Coastal Action Program surface water quality monitoring program. Means, maximum and minimum values for sampling summer 2003 in Marsh Creek upstream and downstream (MC up & dw), Little River upstream and downstream (LR up & dw), and Hazen Creek upstream and downstream (HC up & dw). Free Salinity Turbidity DO Fecal colifiorm Ammonia Total PO4- (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) P (mg/L) MC up 0.1 7.2 0.3 8.2 >349 0.00 0.01 Mean MC dw 7.6 7.6 0.4 9.7 2 455 556 0.01 0.09

LR up 0.8 7.4 0.8 8.7 >194,3 0.00 0.00 LR dw 2.6 6.9 2.3 5.5 >122,7 0.02 0.04 HC up 0.3 7.7 0.6 9.1 64 0.00 0.00 HC dw 0.3 7.8 0.6 8.1 >66,43 0.00 0.00 MC up 0.4 7.4 1.0 11.0 >870 0.01 0.02 Max MC dw 16.9 8.5 2.0 15.2 3 600 000 0.05 0.11

LR up 6.0 7.8 2.5 14.4 267 0.00 0.01 LR dw 5.9 8.2 5.3 11.5 270 0.19 0.38 HC up 0.8 7.9 2.0 9.8 176 0.01 0.02 HC dw 0.6 8.7 1.0 12.2 92 0.00 0.01 MC up 0.0 6.9 0.0 6.1 27 0.00 0.01 Min MC dw 0.3 7.1 0.0 4.3 1 050 000 0.00 0.03

LR up 0.0 6.9 0.0 6.0 47 0.00 0.00 LR dw 0.0 6.6 0.0 1.2 24 0.01 0.02 HC up 0.1 7.4 0.0 7.2 15 0.00 0.00 HC dw 0.1 7.1 0.5 4.8 11 0.00 0.00

Table 9. Atlantic Coastal Action Program surface water quality monitoring program. Means, maximum and minimum values for sampling summer 2004 in Marsh Creek upstream and downstream (MC up & dw), Little River upstream and downstream (LR up & dw), and Hazen Creek upstream and downstream (HC up & dw).

Fecal Free Water Salinity Turbidity DO colifiorm Ammonia Total PO4- o Temp ( C) (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) P (mg/L) MC up 18.1 0.1 7.2 1.6 9.4 316 0.00 0.02 Mean MC dw 19.6 0.2 7.3 3.3 9.2 4 379 444 0.02 0.09

lr up 21.4 0.0 7.6 2.5 11.3 59 0.00 0.01

lr dw 22.9 1.1 7.0 10.7 11.2 80 0.02 0.09 hc up 14.4 0.2 7.8 0.3 11.5 114 0.00 0.00 hc dw 21.7 2.6 8.4 1.6 10.3 20 0.02 0.00 MC up 21.0 7.1 7.4 4.3 >870 460 0.03 Max MC dw 23.5 0.2 7.6 6.4 9.6 7 800 000 0.03 0.11

lr up 22.0 6.6 7.7 8.1 >870 61 0.02 lr dw 24.0 4.3 7.2 20.0 14.6 190 0.03 0.18 hc up 15.6 0.4 8.0 1.1 14.6 356 0.00 0.01 hc dw 24.4 11.2 9.2 4.5 14.6 53 0.05 0.01 MC up 0.3 0.1 0.0 0.0 8.9 0 0.00 Min MC dw 17.3 0.2 7.1 0.5 8.5 1 166 667 0.01 0.03

lr up 0.0 -0.1 0.0 0.0 8.5 1 0.00

lr dw 21.5 -0.1 6.7 1.0 8.3 5 0.02 0.05 hc up 13.2 0.0 7.6 0.0 9.9 20 0.00 0.00 hc dw 17.7 0.0 7.7 0.0 8.3 1 0.00 0.00

concentrations were all above 8.0 mg/L including downstream of Little River

(Tables 8-9).

MCD was the only site where the amount of faecal coliforms seems problematic with average amount above two million per 100 mL in 2003 and four million in

2004. Free ammonia had a maximum average of 0.02 mg/L (LRD in 2003-04 and

MCD in 2004) and a maximum level of 0.19 and 0.03 mg/L in 2003 and 2004, respectively (Tables 8-9). Marsh Creek downstream had the highest orthophosphate average with 0.09 mg/L and Little River downstream had the highest level with 0.38 mg/L in 2003. The orthophosphate had its highest average and highest level in MCD for 2004 with 0.17 and 0.26 mg/L, respectively

(Tables 8-9).

Data from the refinery contain similar information as ACAP’s data. Temperature is relatively high for both years during the summer months. The pH is generally around 7 (Figures 25-28). Salinity upstream is constant and equal or below one part per thousand. The salinity downstream follows the salinity of the effluent and can reach levels as high as 14 ppt (Figure 23). Turbidity is constantly higher downstream compared to LRU and had a maximum level of 69 NTU in July 2004

(Figures 25-28). The dissolved oxygen levels measured by the refinery are not as high as those measured by ACAP. Levels upstream are usually higher than 5 mg/L except one period in late July 2004 where DO was as low as 2.6 mg/L

Dissolved Oxygen UP3 DWN5

12 Effluent 10 8 6 4

DO (mg/L) DO 2 0 July 3/03 July 9/03 June 4/03 July 16/03 Aug 13/03 Aug May 14/03 May 21/03 May 28/03 June 11/03 June 18/03 June 25/03 Date

Temperature UP3 DWN5

30 Effluent 25 20 15 10 5

Temperature (c) 0 July 3/03 July 9/03 Aug 6/03Aug June 4/03 July 16/03 July 23/03 July 30/03 July May 14/03 May 21/03 May 28/03 Aug 13/03 Aug June 11/03 June 18/03 June 25/03 Date

pH UP3 DWN5

7.8 Effluent 7.6 7.4 7.2 7 pH 6.8 6.6 6.4 6.2 July 3/03 July 9/03 Aug 6/03Aug June 4/03 July 16/03 July 23/03 July 30/03 July May 14/03 May 21/03 May 28/03 13/03Aug June 11/03 June 18/03 June 25/03 Date

Figure 25. Dissolved oxygen (DO), temperature (oC), and pH for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2003.

Fecal Coliforms UP3 DWN5

6000 Effluent 5000 4000 3000 2000 1000

Fecal (number/100mL) Fecal 0 July 9/03 July 23/03 Aug 6/03 Aug 13/03 Date

Tubidity UP3 DWN5 70 Effluent 60 50 40 30 20

Turbidity (NTU) Turbidity 10 0 July Aug 3/03 6/03 July July July Aug June 18/03 16/03 23/03 30/03 13/03 Date

Figure 26. Fecal coliforms (#/0.1 L) and turbidity (NTU) for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2003.

UP3 Dissolved Oxygen DWN5 Effluent 14 12 10 8 6 4 DO (mg/L) DO 2 0 Aug 4/04 July 08/04 July 14/04 July 21/04 July 28/04 Aug 11/04 June 02/04 June 10/04 June 16/04 June 23/04 June 30/04 Date

Temperature UP3 DWN5

30 Effluent 25 20 15 10 5

Temperature (c) Temperature 0 Aug 4/04Aug July 08/04 July 14/04 July 21/04 July 28/04 July Aug 11/04 Aug June 02/04 June 09/04 June 16/04 June 23/04 June 30/04 Date

UP3 pH DWN5 Effluent 9.00 8.50 8.00 7.50 7.00 pH 6.50 6.00 5.50 5.00 Aug 4/04 July 08/04 July 14/04 July 21/04 July 28/04 Aug 11/04 June 02/04 June 09/04 June 16/04 June 23/04 June 30/04 Date

Figure 27. Dissolved oxygen (DO), temperature (oC), and pH for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2004

Fecal Coliforms UP3 DWN5 Effluent 3000 2500 2000 1500 1000 500 0 Fecal (number/100mL) Fecal July 08/04 July 14/04 July 21/04 July 28/04 July June 02/04 June 09/04 June 16/04 June 23/04 June 30/04 Date

UP3 Tubidity DWN5 Effluent 120 100 80 60 40 20 Turbidity (NTU) Turbidity 0 Aug 4/04 July 08/04 July 14/04 July 21/04 July 28/04 Aug 11/04 June 02/04 June 09/04 June 16/04 June 23/04 June 30/04 Date

Figure 28. Fecal coliforms (#/0.1 L) and turbidity (NTU) for Little River Upstream (UP3), Downstream (DWN 5) and the final effluent. Irving Oil Little River summer monitoring, 2004

(Figures 25-28). The downstream site had relatively high DO levels in 2003, but, for 2004, many samples showed levels below 4 mg/L (Figures 25-28). Finally, faecal coliforms are usually low. The highest levels are found upstream for both years (Figures 25-28).

The effluent parameters usually follow the upstream and downstream patterns and tend to meet the general levels found in the river (Figures 23, 25-28). Some parameters such as pH and turbidity tend to have greater variation than the baseline measurement in the stream (upstream site) (Figures 25-28). Faecal coliforms were more important at the upstream site, especially in 2003.

When comparing the water quality data collected by the three different groups, we usually find a high similarity among the data sets (Figures 29-31). The temperature and salinity matched well, whereas dissolved oxygen had the biggest differences among data set particularly when low levels were recorded

(Figures 29-31).

28 24 20 16 Sonde 12 Temperature 8 IOL Temperature

Temperature (oC) 4 0 8/1 8/2 8/3 8/4 8/5 8/6 8/7 8/8 8/9 7/22 7/23 7/24 7/25 7/26 7/27 7/28 7/29 7/30 7/31 8/10 8/11 Date

14 Sonde Salinity 12 IOL Salinity 10 8 ACAP Salinity 6 4

Salinity (ppt) 2 0 8/1 8/2 8/3 8/4 8/5 8/6 8/7 8/8 8/9 7/22 7/23 7/24 7/25 7/26 7/27 7/28 7/29 7/30 7/31 8/10 8/11 Date

Sonde Dissolved Oxygen 1/10 ACAP DO 1/8

1/6

1/4

1/2

1/0 Dissolved Oxygen (mg/L) Oxygen Dissolved 8/1 8/2 8/3 8/4 8/5 8/6 8/7 8/8 8/9 7/22 7/23 7/24 7/25 7/26 7/27 7/28 7/29 7/30 7/31 8/10 8/11 Date

Figure 29. Combination of Irving Oil Little River summer monitoring (IOL), ACAP and sonde data for temperature (A), salinity (B), and dissolved oxygen (C), from July 22 to August 11, 2003.

28 24 20 16

12 Sonde 8 Temperature

Temperature (oC) IOL Temperature 4 0 6/1 6/2 6/3 6/4 6/5 6/6 6/7 6/8 6/9 6/10 6/11 6/12 6/13 6/14 6/15 6/16 6/17 6/18 6/19 6/20 6/21 Date

B) Sonde Salinity 16 14 IOL Salinity 12 10 8 6

Salinity (ppt) 4 2 0 6/1 6/2 6/3 6/4 6/5 6/6 6/7 6/8 6/9 6/10 6/11 6/12 6/13 6/14 6/15 6/16 6/17 6/18 6/19 6/20 6/21 Date

C) Sonde Dissolved Oxygen 12 10 IOL Dissolved Oxygen 8 6 4 2

Dissolved Oxygen (mg/L) 0 6/1 6/2 6/3 6/4 6/5 6/6 6/7 6/8 6/9 6/10 6/11 6/12 6/13 6/14 6/15 6/16 6/17 6/18 6/19 6/20 6/21 Date

Figure 30. Combination of IOL, ACAP and sonde data for temperature (A), salinity (B), and dissolved oxygen (C), from June 1 to 21, 2004.

27 24 21 18 15 12 Sonde Temperature 9 IOL Temperature Temperature (oC) 6 ACAP Temperature 3 0 7/1 7/2 7/3 7/4 7/5 7/6 7/7 7/8 Date B) Sonde Salinity 18 IOL Salinity 15

) ACAP Salinity 12

6 Salinity (ppt 3

0 7/1 7/2 7/3 7/4 7/5 7/6 7/7 7/8 Date C)

Sonde Dissolved Oxygen 12 IOL Dissolved Oxygen ACAP Dissolved Oxygen 9

Dissolved Oxygen (mg/L) 0

7/1 7/2 7/3 7/4 Date 7/5 7/6 7/7 7/8

Figure 31. Combination of IOL, ACAP and sonde data for temperature (A), salinity (B), and dissolved oxygen (C), from July 1 to 8, 2004.

3.4 Discussion

Effluent dilution in LRD is low with a maximum dilution of effluent to a

concentration of 15%. The majority of the refineries in Canada discharge in large

receiving environment such as the St. Lawrence River, the Great Lakes or the

Atlantic Ocean (St-Cyr 1995). The small size of the receiving system explains the

low dilution and confirms that the assimilative capacity of the stream may be

exceeded more easily.

The effect of tides on Little River and the plume dispersion is limited. Except for

October and November, the effluent plume usually does not move upstream

more than 120 m, which correspond to the first culvert (Figure 19). A beaver dam

constructed at the end of this culvert limits the water movement. In October and

November, water level is low in the streams and tides are the highest of the year, reaching as high as 8.4 m. During these extreme tides, salt water comes into

Little River and salinity increases when moving downstream; salinity in the downstream site is higher than effluent salinity. At extreme high tides, a mixture

of salt water and effluent can be found about 450 m upstream of the discharge

(Figure 19). Although salt water does not come in with every high tide, tides still

have an effect on the water level in Little River. Water level increases on a

regular basis up to 20-30 cm between low and high tide (personal observation).

During the highest tides of the year, water level can rise an extra meter. This type

of habitat would still be considered an estuary although it would more specifically be defined as tidal freshwater habitat (Elliott & Hemingway 2002).

The very low occurrence of salt water in the downstream section of Little River was unexpected. After the sampling season of 2003, it was decided to modify the design to better reflect the state of LRD. The 2004 design would focus more on

an upstream-downstream design rather than comparing LRD to other estuaries.

Although the other downstream sites were more affected by salt water, the

design was a good compromise as very few streams in the area are similar to

Little River.

Normally, the downstream salinity of LRD was mainly influenced by effluent

(Figure 23). Salinity upstream was stable and usually below 0.2 ppt. Salinity downstream varied greatly, and the change in salinity often occurred within few hours. The peaks in salinity were frequent and could be observed on a weekly basis (Appendix 1). These peaks correlate in timing to the addition of ballast water into the final effluent prior to its discharge into Little River (Richard Russell; pers. comm.). Irving Oil collects ballast water from tankers that transport refined petroleum. The ballast water is stored in tanks and mixed into the final effluent periodically (Richard Russell; pers. comm.).

The discharge of ballast water had two main consequences on fish: it periodically increases the salinity and it is associated with dramatic decreases in dissolved

oxygen levels in the stream. Downstream salinity increased during some periods

to levels similar to those found in the harbour, usually around 15 ppt during the

summer months (Tim Vicker, Executive Director, ACAP Saint John; pers.

comm.). Freshwater fishes such as the white sucker or the blacknose shiner

have been collected occasionally downstream of the discharge (Figures 4-5).

Freshwater fishes moving downstream could potentially be caught between the

effluent’s increased salinity and the harbour water.

The salinity fluctuation could also affect fish food availability and habitat quality.

Change of salinity can affect survival of aquatic organisms such as benthic

invertebrates and plants (CCME 1999). Salinity is a critical factor in distribution of

aquatic organisms and variation of salinity from fresh water to brackish water can limit the colonization of organisms requiring stable salinity (CCME 1999). A habitat with a large salinity range is more likely to have a reduced benthic community as well as a decreased habitat quality in terms of abundance of macrophytes that provide shelter and spawning grounds (Montague & Ley 1993).

The Canadian recommendations for salinity in marine and estuarine environments mention that human activity should not cause salinity fluctuations greater than 10% of natural levels (CCME 1999). The effect of the discharge of final effluent with ballast water actually caused a change in salinity of 140%.

The most important consequence of the ballast water input was the reduction of

stream levels of dissolved oxygen. During the sonde deployment, measurement

of dissolved oxygen in LRD revealed levels < 0.2 mg/L for up to three

consecutive days in 2003 and up to 10 consecutive days in 2004. Although, the increased salinity is not always a prerequisite to low oxygen level, it is often correlated. Ballast water contains oil, phenols, chlorides as well as organic and inorganic material (St-Cyr 1995). It has high levels of COD and, even if the ballast water is oxygenated prior to its discharge, it has a high potential to reduce dissolved oxygen level in the receiving environment (Arthur Niimi, Fish

Toxicology Scientist, Department of Fisheries and Oceans, pers. comm.). The

COD of the ballast water varies greatly depending on the type of ship and oil product. Water quality measurements on ballast water by the refinery showed level of COD averaging 1400 mg/L (Richard Russell, pers. comm.).

Extensive periods with low dissolved oxygen in LRD were measured during the summer of 2003 and 2004. During the same periods, dissolved oxygen levels were not as low when measured by ACAP or the refinery (Tables 8-9 and

Figures 25-28). The differences probably come from the methodology. ACAP and the refinery measure DO in the top portion of the water column, whereas the sonde recorded DO concentration in the lower portion of the water column. The oil refinery effluent is denser than the receiving water because of its higher concentration of organic and inorganic material, particularly when ballast water is added. The effluent consequently tends to occupy the lower portion of the water column before it is completely mixed. Effects from the effluent discharge were

more important in the lower portion of the water column and could not be detected by the monitoring programs from ACAP or the refinery.

In addition, changes in DO levels were constant and low dissolved oxygen periods would usually last for few hours to few days. Because the water was

monitored weekly by ACAP and IOL, the probability that the monitoring programs

measured the presence of low dissolved oxygen was reduced.

Moreover, ACAP measured DO at the end of the day while the refinery measures

it early in the day. DO levels tend to be lowest early in the morning as a result of

oxygen consumption coupled with absence of photosynthesis during the night.

However, the lowest DO level measured in LRD by ACAP was 1.17 mg/L in

August 2003 (Tables 8-9). The refinery recorded its lowest (2.1 mg/L) DO

concentration on July 28th, 2004 (Appendix 2).

The recommended minimum levels of dissolved oxygen for aquatic life are above

5.5 mg/L for warm freshwater habitats and above 8 mg/L for estuarine and

marine environments (CCME 1999). The DO level remained below that level as

long as 4 d in 2003 and 10 d in 2004. It is clear that prolonged periods of low levels of oxygen would be very stressful to fish, and would be expected to have a major effect on the fish community especially when levels are as low as 0.2 mg/L. Moreover, low DO would also have a negative effect on the benthic community, jeopardizing the food chain in this habitat.

Although fish are able to move upwards in the water column or out of the hypoxic region, the reduced concentration of DO represents an effect on fish habitat.

Furthermore, periods of hypoxia and anoxia may prevent fish establishment in

LRD. This could explain the reduced number of fishes found downstream of LRD, even outside of anoxic events.

LRD was not the only site were DO concentration was found to be lower than the recommended limits. The sonde deployment also recorded hypoxic periods in

LRU (Appendix 1). The decrease in DO is not as important as in LRD except for one extended period in May 2004 were DO level was near zero for more than 10 days (Appendix 1). This last situation is probably caused by the submersion of the sonde in the soft-bottom sediments (see below, problems related to the deployment of the sonde). Except for this incident, DO never went below 2 mg/L and are probably related to the organic composition of the substrate in LRU and would be a natural event in eutrophic habitats such as marshes during the summer months (Wetzel 1983).

Because of its eutrophic habitat, Little River Upstream is mainly inhabited by species tolerant to reduced DO level and increased turbidity. The killifish and stickleback species as well as the American eel and the white sucker are all considered tolerant to pollution and habitat degradation (Bergstedt & Bergersen

1997; Hughes et al. 1998; West et al. 1999; Belpaire et al. 2000; Schmidt &

Talmage 2001; Oberdorff et al. 2002). Only two species collected in LRU, the golden shiner and the blacknose shiner, were found to be moderately tolerant to intolerant to habitat perturbations (Simona et al. 2000; Schneider 2002).

The discharge of the oil refinery effluent also had an effect on water turbidity and temperature. Turbidity increased in LRD compared to its upstream site

sometimes with levels 60 times higher (Figures 25-28). Turbidity is a measure of

water clarity and is an indicator of the quantity of suspended matter (organic and

inorganic) in the water. Its impact on the fish populations is not known and very

little information is available on acceptable maximum levels for aquatic life

(CCME 1999).

Thermal effects from the addition of the effluent were visible in different ways

between upstream and downstream sites. On average, the temperature

downstream was two or three degrees higher with a maximum difference recorded of 6.6oC during the summer months. The highest bottom temperature recorded was 27 oC in LRD and 21.5 oC in LRU (Figure 24). Finally, water never

freezes downstream of the effluent during winter months as it does upstream

(Richard Russell; pers. comm.). The Canadian guidelines for freshwater habitat recommend that discharge should not affect the weekly average temperature, thermal stratification or turnover dates (CCME 1999). For estuaries, the guidelines recommend that activities should not exceed ± 1oC (CCME 1999).

Change in water temperature can affect physical water characteristics (solubility

of oxygen), physiological processes (respiration rate, spawning…) and behaviour of fishes as well as sensitivity to certain contaminants (CCME 1999). Exposure to very high temperatures can increase susceptibility to disease and toxic substances, and affect respiration and osmoregulation (CCME 1999). The presence of higher temperature combined with the periodic low dissolved oxygen level in LRD could increase stress on fish and their sensitivity to contaminants, as well as reduce their survival (Baer et al. 2002).

The deployment of the sondes for extended periods gave valuable information on level of dissolved oxygen, temperature and turbidity. However, it also led to some problems, first and foremost at the upstream site. Few periods in the water survey had equivalent upstream and downstream records. The main problems were related to:

1) submersion of the upstream sonde into the soft sediment (June to August

2003, June 2004);

2) loss of upstream sonde (July 2004); and

3) intermissions in data recording for downstream sonde (July 8th to August

3rd 2004).

The marshy nature of the upstream site substrate, with a top layer of soft sediment, contrasted with the hard substrate found in the downstream site. The changes in water level were frequent, and were dependent on both weather as well as dam effects. The upstream sondes did submerge in the soft substrate

100

during periods of low water. Consequently, some of the recorded periods at the

upstream site were suspected to be inaccurate especially for the dissolved

oxygen levels. However, the records from the downstream site were more likely

to reflect the characteristics of bottom water, as submersion in the sediments

was not possible. The absence of day-night changes in DO concentration,

coupled with the presence of very low levels at the upstream site, suggests a

cautious interpretation of the data.

Information provided by ACAP and the refinery permitted confirmation of some

trends in Little River (higher turbidity and temperature downstream) and

assessment of water quality at other sites. In general, sites in Marsh Creek and

Hazen Creek had similar water characteristics. The two creeks had warm water

temperature and relatively good dissolved oxygen levels upstream and

downstream. Few periods had DO records below the guidelines, all of them in the downstream sites.

MCD and LRD were the two sites receiving waste water. Marsh Creek received untreated sewage from the city of Saint John and Little River from the refinery.

The additional inputs were reflected in water quality parameters such as faecal coliforms and concentrations of ammonia and phosphate. MCD was the only site where faecal coliforms amounts were outstandingly high, with average amount above millions per 100 mL. Although such levels have human health and esthetic

101

implications, there is no evidence that faecal coliforms can have a deleterious effect on aquatic life (CCME 1999).

The highest average and maximum concentrations of free ammonia were found in LRD and MCD (Tables 8-9). Concentrations and averages would reach levels above the recommended level of 0.019 mg/L (CCME 1999). MCD and LRD also had the highest average and highest concentration of orthophosphate (Tables 8-

9). The Canadian water quality guidelines do not have a recommended limit for orthophosphate (CCME 1999). Nitrogen and phosphorus are generally the two limiting nutrients in aquatic systems. An increase in their concentrations in a system will stimulate plant and algae growth.

The water quality survey showed that Little River Downstream was impacted by the discharge of the oil refinery effluent. The most significant effect was the decrease in dissolved oxygen to stressful levels for extended periods of time.

The DO depletion was often correlated to increased salinity in the stream as a consequence of addition of ballast water in the final effluent. The addition of the effluent also increased water temperature and turbidity. The effect on temperature could increase fish susceptibility to diseases, chemical toxicity and low oxygen level. The water quality in LRD probably plays an important role in the reduced abundance of fish observed on site.

102

The discharge of waste both in LRD and MCD had an impact on amount of

macronutrient load. Both sites had high free ammonia and orthophosphate

levels. The levels found could cause eutrophication of the system. The level of

faecal coliforms in MCD was also extremely high and represents a risk for human

health although the effect on fish is unknown.

Given the confounding impacts associated with the discharge of ballast water in

the stream, it is not possible, under the present conditions, to evaluate the effect

on fish downstream of the oil refinery effluent. The company has planned a

switch to ships with double hulls by 2006, which will eliminate the need to

discharge ballast water through the effluent treatment system. Further studies

are required to determine what impacts this removal will have on water quality in

Little River. It was decided to evaluate the present-day impacts of the refinery discharge on fish performance of the dominant downstream species

(mummichog).

103

4 RESPONSE OF WILD MUMMICHOG TO THE EFFLUENT DISCHARGE

4.1 Introduction

The original intention of the study was to evaluate the potential impacts of the oil

refinery effluent using the adult fish survey component of an Environmental

Effects Monitoring approach. The estuarine portions of the small coastal streams

in the Saint John harbour do not appear to hold fish all year round (Chapter 2).

There is an increase in species and abundances during summer months, which

corresponds to an influx of marine species that spawn in fresh water, and an increase in freshwater species in the lower areas during summer periods of lower tidal influence. Designing an adult fish survey program for this situation is

challenging, and it is necessary to maximize the potential exposure period of

transient species to evaluate the potential consequences of effluent exposure.

The situation becomes even more complex in Little River. The fish community

downstream of the oil refinery effluent discharge was characterized by fewer

species, those species that are present are transient, and there is a low

abundance of those species of fish that are present (Chapter 2). The effects

seem to be related to water quality changes caused by the discharge of the oil

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refinery effluent, and particularly by the periodical addition of ballast water to the final effluent (Chapter 3).

There was still an interest in assessing the impacts of effluent from the oil refinery. This involved attempting to examine the most abundant species present, after a maximal period of residency, to determine whether there were consequences of the treated effluent. Of the species that were present, threespine and fourspine stickleback were only present as transient prespawning fish during May. The mummichog (Fundulus heteroclitus) moved in during May, and were present in smaller numbers throughout the summer. They were one of the most abundant species among sites and the most common one throughout the summer months.

The mummichog is a small bodied-fish that can live up to four years and reach a maximum length of 13 cm (Scott & Scott 1989). This schooling fish prefers marsh and brackish ponds with vegetation and can tolerate a wide range of salinity. It is an omnivorous species, feeding on small crustaceans and fish, invertebrates, vegetation and eggs (Scott & Crossman 1998). The mummichog, a multiple spawner, spawns from May to August in New Brunswick (Leblanc & Couillard

1995). It is a tolerant fish species that can be found in multiple habitats.

Mummichog move with the tidal cycle but have a low mobility during summer months and a high site fidelity at low tide (Butner & Battstrom 1960; Lotrich 1975;

Teo & Able 2003; Skinner 2005). Estimations of home range vary from 36 m for a

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linear home range to 15 ha for a home range area (Lotrich 1975; Teo & Able

2003). The differences in home range estimates are assumed to be mostly related to habitat characteristics and study design (Teo & Able 2003).

In the absence of data on background variability, it is recommended that surveys use 20 males and 20 females for each sentinel species to ensure sufficient power (Environment Canada 1998). This sample size was determined using a previous study showing that variability in organism characteristics stabilizes between 8 to16 individuals (Munkittrick et al. 2000). The parameters measured for the EEM fish survey include age, size at age, condition, gonad size, fecundity, and liver mass (Table 10).

Exposure to the effluent is also a major consideration for the design of the survey, and collections are recommended within an area where the effluent concentration is greater than 1% (Environment Canada 1998). The entire lower

Little River had an effluent concentration >15% during the sampling periods.

Chemical and biochemical (MFO) tracers can also be used to assess short-term exposure (days to weeks). However, they more often confirm that reference fish were not significantly exposed to effluent rather than confirming that exposed fish had spent most of their time in the effluent plume (Environment Canada 1998).

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Table 10. Definition of fish parameters used in the EEM Fish Survey.

Parameter Dependent Variable (Y) Covariate (X)

Age Age (log) None

Size-at-Age Body mass and length(log)* Age (log)

Condition Body mass (log) Length (log)

Gonad mass (GSI) Gonad mass (log) Body mass and length (log)*

Fecundity Number of eggs/female (log) Body mass and length

(log)*, and age

Liver mass (LSI) Liver mass (log) Body mass and length

(log)*

(Source: Environment Canada 1998)

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The oil refinery effluent contains PAHs, which are known to induce MFO activity

(Whyte et al. 2000). It was consequently possible to use MFO activity as a

biochemical tracer to assess the exposure of wild fish to the oil refinery effluent.

The characteristics of the mummichog and its availability at all sites make it a

good candidate to assess effects of the oil refinery effluent on Little River. It was

decided in July 2003 to evaluate this species as a potential sentinel.

4.2 Methodology

Minnow traps were set to collect 20 male and 20 female mummichog from each

site (MCD, LRD, LRU, HCD, and WC) from July 14th to 18th and from August 11th to 14th, 2003. Minnow traps were checked every day. Although traps were set for multiple days during the months of July and August 2003, it was not possible to collect 20 males and 20 females from the Little River or West Quako sites. Within two days, Marsh Creek and Hazen Creek collections were completed for both males and females. However, even when trapping ended in mid-August, all three other sites did not attain the required amount of fish. In order to reduce the variability caused by the change in the intensity of spawning activity between July

and August, it was decided to remove the data collected during August for the

analyses.

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After each site visit, fish were brought alive in coolers to the lab. They were measured for length (total length), then killed by spinal severance, and total mass, gonad mass and liver mass were recorded. Gonad and liver samples were placed in cryovials and placed in a –86OC freezer. Scales from the right side, below the pectoral fin, were taken and placed in a labelled envelope.

4.2.1 Statistics

The statistical software programs SYSTAT 9 (Levene’s test, analysis of variance

[ANOVA], Kruskal-Wallis, and analysis of covariance [ANCOVA]) was used. The data were analyzed per site for the following:

1) liversomatic index (LSI),

2) gonadosomatic index (GSI),

3) condition factor (K), and

4) mixed function oxygenase (MFO) level.

The calculation of indices are as follow:

LSI = liver mass (g) x 100 total body mass (g)

GSI = gonad mass (g) x 100 total body mass (g)

K = total body mass (g) x 100 total length (cm)3

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As for the community survey, type I error was set at ∝ = 0.05. When no

differences were found among sites, minimum detectable size was calculated

using α=β=0.05 and α=β=0.10. Data were assessed for homogeneity of residual

variance, using a Levene’s test. When data were homogeneous, ANOVA was

used for the analysis of variance. If data were not homogeneous, data were

transformed by log transformation to correct it. If transformation did not solve the

issue, the Kruskal-Wallis test for non parametric data was used. Minimum

detectable difference was calculated when no difference was found in order to

determine the capacity of the test to give a valid conclusion. MFO was analyzed

using analyse of variance whereas, GSI, LSI and K were analysed with analysis

of covariance.

4.2.2 Mixed Function Oxygenase Activity

Mixed function oxygenase level was measured using the activity of ethoxyresorufin-o-deethylase (EROD) in fish liver using a standardized protocol

(Whyte et al. 2000). For the preparation of positive and negative controls, mummichog from the Miramichi River were collected in November 2002. After anaesthesia with tricaine methane sulfonate (TMS) solution (Syndel International,

Vancouver, BC, Canada), positive controls were injected with 10 µg of β- naphthoflavone (Sigma Aldrich, Oakville, ON, Canada) per gram of body mass.

β-naphthoflavone was diluted in corn oil (5 mg/mL). Negative controls were injected with corn oil (20 µL/g). After 96 hours, fish were sacrificed by spinal severance and livers removed and frozen at -86oC.

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Livers were removed from the -86oC freezer and thawed on ice. For each

sample, a liver piece (total mass of 20 to 29 mg) was ground with 500 µl of

HEPES grinding buffer in a 1.5 mL microcentrifuge tube using a Kontes hand

tissue grinder. Dissecting tools and grinding tips were cleaned between each

sample. The samples were centrifuged for 20 minutes at 10 000 x g at 4oC. The

microsomal layer was resuspended and supernatant was pipetted and placed in

2.0 mL labelled cryovials. Liver homogenates were stored at –86oC while precipitated pellets were discarded (Whyte et al. 2000).

The protein analysis was done using the BIO-RAD® assay protocol (Whyte et al.

2000). Liver homogenates were diluted 1:10 ratio in water and positive and negative controls were diluted 1:20 ratio. Standards were made with bovine serum albumin (BSA) (Sigma Aldrich). Concentrations ranged from 0 to 0.5 mg/mL and were zeroed using distilled water. Diluted liver homogenates (10 µL) were added in triplicates to a 96-well plate, standards were added in duplicates

(Whyte et al. 2000). Two-hundred microliters of a diluted 1:4 ratio BIO-RAD solution was added to each well. After a 5 min incubation at room temperature, the plate was read using a LS 800 Lowry spectrophotometer at 595 nm. A linear standard curve for protein concentrations in relation to absorbance was produced from the BSA standards and used to calculate protein concentrations in the liver homogenates.

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For the determination of EROD activity, standards were made with resorufin

(Sigma Aldrich) in HEPES grinding buffer. Concentrations ranged from 0 to 0.5 mg/mL and were zeroed against distilled water. Standards (10 µL) were added to a 96-well plate in duplicates with 40 µL of grinding buffer. Liver homogenate (50

µL) was added in duplicates. Fifty microliters of 7-ethoxyresorufin (7ER)/HEPES buffer was added to each well and incubated in the dark for 10 min at room temperature (Appendix 3). The catalytic effect was started with the addition of 10

µL of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (ICN

Biochemicals, Aurora, OH, USA). Resorufin fluorescence was measured using a

FLX 800 microplate reader at 590 nm with an excitation wavelength of 530 nm.

Enzyme activity was adjusted by time and protein concentration (pmol/mg/min).

4.3 Results

Fish collected in July had bigger gonad size than fish in August (Table 11). Total mass, total length, and liver mass were similar between months. The female mummichog collected at the Little River downstream site had relative larger livers than the reference sites (ANCOVA p<0.005) (Figures 32-33). However, there were no differences in condition factor (K) nor relative gonad size between downstream fish and reference sites (ANCOVA p>0.334 and p>0.328, respectively) (Figures 34-35). Marsh Creek had higher LSI and K than only one reference site, Little River upstream (ANCOVA p=0.006 for female LSI and

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Table 11. Total mass (g), total length(cm), gonad mass(g) and liver mass(g) of male and female mummichog collected in each site in July and August 2003. Means ± standard errors (sample size). MCD LRU LRD HCD WC Male Mass July 2.12 ± 0.25 (20) 2.09 ± 0.25 (8) 1.92 ± 0.19 (8) 3.38 ± 0.30 (27) 1.96 ± 0.38 (7) (g) Aug. 2.91 ± 0.68 (4) 5.12 ± (1) 2.39 ± 0.29 (10) Length July 5.60 ± 0.17 (20) 5.7 ± 0.2 (8) 5.5 ± 0.1 (8) 6.4 ± 0.2 (28) 5.4 ± 0.3 (7) (cm) Aug. 5.9 ± 0.5 (4) 7.4 ± (1) 5.3 ± 0.6 (10) Gonad July 0.089 ± 0.010 (20) 0.024 ± 0.007 (8) 0.031 ± 0.006 (8) 0.055 ± 0.006 (28) 0.054 ± 0.008 (7) mass Aug. 0.013 ± 0.003 (4) 0.040 (1) 0.020 ± 0.003 (10) (g) Liver July 0.090 ± 0.037 (20) 0.058 ± 0.009 (7) 0.075 ± 0.010 (8) 0.091 ± 0.006 (28) 0.036 ± 0.008 (7) mass Aug. 0.110 ± 0.027 (4) 0.355 (1) 0.070 ± 0.011 (10) (g) Female Mass July 4.09 ± 0.51 (23) 2.19 ± 0.47 (8) 3.15 ± 0.45 (8) 5.27 ± 0.84 (18) 3.13 (1) (g) Aug. 2.30 ± 0.27 (5) 3.17 ± 0.81 (2) 3.35 ± 0.30 (5) Length July 6.6 ± 0.2 (23) 5.7 ± 0.4 (8) 6.2 ± 0.4 (8) 7.1 ± 0.3 (20) 5.7 (1) (cm) Aug. 5.6 ± 0.2 (5) 6.5 ± 0.6 (2) 6.5 ± 0.2 (5) Gonad July 0.912 ± 0.151 (22) 0.112 ± 0.047 (8) 0.286 ± 0.050 (8) 0.652 ± 0.105 (20) 0.771 (1) mass Aug. 0.031 ± 0.006 (5) 0.027 ± 0.014 (2) 0.068 ± 0.007 (5) (g) Liver July 0.210 ± 0.033 (23) 0.074 ± 0.017 (8) 0.193 ± 0.032 (8) 0.185 ± 0.023 (20) 0.102 (1) mass Aug. 0.102 ± 0.019 (5) 0.119 ± 0.048 (2) 0.142 ± 0.024 (5) (g)

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a 7

6 ac

bc 5 b

4 LSI %

0 MCD LRU LRD HCD WC MCD LRU LRD HCD WC FEMALE MALE SITE

Figure 32. Liversomatic index (LSI) for male and female mummichog (Fundulus heteroclitus) collected in July 2003 at each site. Values are means with standard errors (7≤n≤28). Different letters indicate statistical differences among sites (p<0.05). Statistical analysis could not be completed for males as interactions were found (ANCOVA p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

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Female

0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 -0.4

-0.8

-1.2 LOG LIVER

-1.6

-2 LOG WEIGHT

Male

0 MCD 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 LRU -0.5 LRD

-1 HCD WC

LOG LIVER -1.5 Linear (MCD)

-2 Linear (HCD) Linear (WC) -2.5 Linear (LRD) LOG WEIGHT Linear (LRU)

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17 b

b 13 b GSI % GSI

9 A

B B B B 5

1 MCD LRU LRD HCD WC MCD LRU LRD HCD WC

FEMALE MALE SITE

Figure 34. Gonadosomatic index (GSI) for male and female mummichog (Fundulus heteroclitus) collected in July 2003 at each site. Values are means with standard errors (7≤n≤28). Different letters indicate statistical differences among sites (p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

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1.5 a ab ab A A A b A A

1.2

0.9 K %

0.6

0.3

0 MCD LRULRDHCDWCMCDLRU LRD HCD WC FEMALE MALE SITE

Figure 35. Condition factor (K) for male and female mummichog (Fundulus heteroclitus) collected in July 2003 at each site. Values are means with standard errors (7≤n≤28). Different letters indicate statistical differences among sites (p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC).

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p=0.014 for female K). The gonad size of Marsh Creek females was statistically higher than all other sites including Little River downstream (ANCOVA p<0.001)

(Figure 34).

The statistical analysis for male liver size showed an interaction between sites

(ANCOVA p > 0.05). Analysis of the slopes and the intercept can give information on the relative size of the liver in relation to the total body size (Table 12). The slope for LRU and LRD was about 50% higher than the other sites suggesting that as fish increase in size, their liver get disproportionally larger in Little River compared to other sites (Table 12). Standard deviation for LRD slope is extremely high compared to other sites (Table 12).

No differences were found among sites for condition factor (K) (ANCOVA p=0.451)(Figure 35). The gonad size for males collected in Marsh Creek was higher than at other sites (Figure 34). Finally, no trend or correlation could be found among mixed-function oxygenase (MFO) activity in males and the presence or absence of waste input (Table 13). Some reference sites had statistical differences among them (LRU and HC were different than WC with p=0.008 and 0.013, respectively) whereas some reference sites had similar results to sites receiving wastes (LRU= LRD with p=0.600; MC = HC with p=0.107) (Table 13).

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Table 12. R2, slope with standard deviation, intercept with standard deviation, and sample size for liver mass in relation with total mass. Data for male mummichog collected in July 2003 at each site. Sample Site R2 Slope Intercept size

MCD 0.7327 0.0220 ± 0.0034 0.0072 ± 0.0082 20

LRU 0.8279 0.0372 ± 0.0074 -0.0255 ± 0.0170 8

LRD 0.8705 0.0485 ± 0.0423 0.0178 ± 0.0838 8

HCD 0.4806 0.0142 ± 0.0030 0.0423 ± 0.0112 27

WC 0.9604 0.0214 ± 0.0018 -0.0057 ± 0.0040 7

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Table 13. Mixed-function oxygenase (MFO) activity (pmol/min/mg) for mummichog collected in each site in July 2003. Values are mean ± standard error (sample size). Different letters represent statistical differences (p<0.05). Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Downstream (HCD), and West Quako Creek (WC). MCD LRU LRD HCD WC Female 132.5 ± 22.2 263.4 ± 59.8 361.4 ± 57.1 101.8 ± 23.0 20.3 ± (22) (8) (7) (20) (1) b a a b Male 235.4 ± 54.7 449.1 ± 105.6 369.3 ± 109.3 302.1 ± 37.8 114.9 ± 21.8 (20) (8) (8) (28) (7) bc a abc ab c

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The mixed-function oxygenase activity for female mummichog shows similar results for both sites in Little River (Kruskal-Wallis p=0.298). Both sites were significantly higher than Marsh Creek and Hazen Creek (Kruskal-Wallis p<0.032)

(Table 13).

For female data, HCD represented the baseline level, with standard deviation equals to 102.9. The MFO level recorded in female collected in Little River

Downstream stand outside of the two standard deviation range from the baseline level (Table 13).

4.4 Discussion

Relative liver sizes found in LRD were statistically higher than the reference sites, and presented increases greater than 25% of the pooled reference sites.

Such a difference in liver size is considered ecologically significant (Munkittrick et al. 2000).

The increased liver size found in both male and female mummichog collected in

LRD was not coupled with increases in GSI and/or K. This suggests that the increase is not related to higher food availability, but rather the consequence of higher exposure to chemicals (Munkittrick et al. 2000).

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The MFO activity found in Little River downstream is about 3.5 times higher than the baseline level found in HCD (Table 13). The induction of the mixed-function oxygenase may be related to polycyclic aromatic hydrocarbons (PAHs) found in the effluent (Whyte et al. 2000). Previous studies have shown that oil refinery effluent can induce MFO activity in fish (Khan 1998; Kiceniuk 1992; Ridlington et al. 1982).

Fish collected in LRD show common responses that differ from those at other sites (LSI for female, slope for male liver size, MFO activity for female for both LR sites). The MFO activity was considered to be induced when the level was at least two standard deviations above background levels, corresponding here to a three fold increase from the baseline level. The higher MFO activity in Little River sites corresponds to the higher LSI found in LRD. Increased MFO activity has been correlated in other studies to greater LSI as a result of higher exposure to

MFO-inducing compounds (Whyte et al. 2000). This suggests that fish are moving between sites, although they are spending sufficient time in Little River to show effects. It is not possible to clearly determine the source of MFO induction.

The induction could be caused by the exposure to the refinery effluent, historical contamination or an upstream source of contamination.

The community survey indicated that fish in LRD were actually transient fishes, either moving to spawning grounds or with the tides. The movement of fish between sites could be related to the poor habitat quality of LRD. The presence

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of hypoxic periods are probably causing or increasing the fish mobility between

sites. Data from June to August 2003 showed multiple extended periods with DO

concentrations below the required level to protect and maintain aquatic life

(Figure 20). Fish collected upstream of the discharge had increased MFO activity

suggesting a certain degree of exposure to the effluent plume or another source

of contamination. Consequently, the use of LRU as a reference site was

inappropriate.

Marsh Creek downstream has creosote sediment contamination, with measured

PAH concentrations over 6000 ppm in sediment (Astephen 2000). It was

expected that mummichog collected at this site would show increased MFO

activity. However, both males and females had MFO activity similar to reference

levels (Table 13). A previous study on the effects of Marsh Creek Downstream

sediment on fish health demonstrated MFO induction even when mummichog

were exposed to the lowest concentration of sediment (Astephen 2000). PAHs

have also been shown to accumulate in fish tissue (Astephen 2000). Because

no MFO induction was measured in fish collected in MCD, this supports the

hypothesis of mobile fish community, moving with tidal cycles. Mummichog

moving with tidal cycles would limit their exposure to MFO inducing compounds by spending the majority of their time in Saint John Harbour water. Although

Saint John Harbour contains PAHs, their concentrations might not be high enough to induce MFO. Vallis (2003) was not able to measure differences in

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EROD activity in fish exposed to Saint John harbour water compared to

reference sites.

Liver size and condition of fish from Marsh Creek Downstream were similar to

HCD and WC, but GSI was higher than at any other site for both sexes (Figures

30-35). The increase was approximately 400% compared to the relative gonad

size in pooled reference sites and 1000% when compared to LRU and WC only.

Increased GSI in fish collected in MCD does not seem to be related to sediments

or water exposure as it has been shown that fish residency in MCD was limited.

Moreover, previous studies showed negative effects on the reproductive system

from PAHs or no effects from the Saint John harbour water. The English sole

(Pleuronectes vetulus) threshold for impacts of PAHs on gonadal growth was 630

ppb PAH in sediment (Johnson et al. 2002). Other negative impacts on the reproductive system included inhibited spawning and infertile eggs (Johnson et al. 2002). Caged female mummichog in the Saint John harbour close to Little

River had no changes in testosterone and 17β-estradiol levels compared to the reference site (Leblanc 1998). More investigation will be necessary to determine the reason for higher relative gonad size of mummichog collected in the Marsh

Creek Downstream site.

MCD was not the only site to present an increased GSI. Hazen Creek

Downstream also had higher gonad size compared to the other reference sites.

GSI in HCD was about 350% higher for males and 750% higher for females

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(Figure 32). Both MCD and HCD are the most estuarine sites, receiving salt water at every tidal cycle. The mummichog is mainly a salt and brackish fish species and prefers higher salinity (Scott & Scott 1989). Mummichog prefer shallow waters with empty shells or aquatic vegetation for spawning habitat and move with tides, especially in macrotidal environments (Butner & Brattstrom

1960; Abraham 1985; Scott & Scott 1989; Sweeney et al. 1998). It is possible that the prevalence of very high GSI in MCD and HCD were related to the habitat characteristics, proximity of the harbour and the tidal influence rather than presence of contaminants and municipal sewage.

The mobility of mummichog in studied sites have been evident by looking at MFO activity in Little River sites as well as Marsh Creek Downstream. The mobility prevented assessing the effect of the oil refinery effluent of fish.

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5 FISH CAGING EXPERIMENT

5.1 Introduction

The sentinel species experiment was not capable of directly evaluating the

effects of the oil refinery effluent on fish health. The two major problems are the

same as those encountered for the EEM fish survey after the first two cycles:

lack of availability of proper sentinel species and difficulty in confirming fish

residency (Courtenay et al. 2002). EEM recommendations to overcome the

problems included caging bivalves and mesocosms (Courtenay et al. 2002).

However, development of new alternative approaches is still needed. One

potential methodology is that of caging finfish.

This chapter describes work done to assess the use of caged fish to monitor the

effects of oil refinery effluent on small coastal streams. Previous caging

experiments using finfish were used to assessing effects of point source

pollution. Species used include Atlantic cod (Gadus morhua), hulafish

(Trachinops taeniatus), pearl dace (Semotilus margarita) and mummichog

(Doebel et al. 2004; Goksoyr et al. 1994; LeBlanc, 1998; Smith & Suthers 1999).

Caging is used in various environments, particularly in marine habitat where fish

mobility can be problematic (Goksoyr et al. 1994; LeBlanc, 1998; Smith &

Suthers 1999).

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5.2 Methodology

Mummichog were collected using minnow traps in Saints Rest Marsh Creek (May

2004) and Hazen Creek (June and October 2004) a few days before the

experiments started. Fish were transported to the laboratory in local aerated

water. In the laboratory, total length and mass were taken and fish were sorted to

ensure uniformity among sites. Fish were held in coolers with 50% fresh water,

50% salt water, and aeration prior to caging. Each site was assigned a different

cooler and each cage had a different compartment in the cooler. Water was

changed daily and no food was provided during that period. When the

experiment was started, fish were transported to the sites in the coolers, placed

in the cages, and the cages were set up one at the time to minimize stress

related to air exposure.

At the end of a caging experiment, fish were brought back to the lab in aerated

coolers. They were measured for length (total length), then sacrificed by spinal

severance, and total mass, gonad mass and liver mass were recorded. Gonad

and liver samples were placed in cryovials and placed in a –86OC freezer.

The first caging experiment was based on Leblanc’s methodology (Leblanc

1998). The cages used were made of PVC (polyvinyl chloride) tubing with window openings covered with 2 mm mesh size (Figure 36). The dimensions of

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the cage were 30 cm long by 12 cm diameter. In each site, three cages were set on the stream bottom, attached to a buoy. In sites where current or tide could be strong, the cages were also attached to a cement block. All cages would be set or retrieved on the same day. No food was provided during the experiment.

The first caging experiment was based on a previous study done in the Saint

John Harbour (Leblanc 1998). The experiment took place in May using only females (Table 14). Because of high mortality at the end of the experiment, cages in the second experiment were set for only one week. Success was still limited and a third trial was done in October (Table 14). This time, a different cage design was used. The cage was a minnow trap with both openings blocked with fine mesh (Figure 36). The new cage design was tested for three days in

Saint Rests Marsh in August to ensure no fish would enter or escape from the modified trap.

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Table 14. Caging experiment designs. Starting date, duration of experiment, number of fish per cage, fish sex, sites used, and type of cages used. Experiment 1 2 3

Starting date May 07, 04 July 02, 04 October 6-7, 04

Duration of experiment 24 7 4-5 (days) Number of fish per 6 5 7 cage Fish gender Female Female Male

Sites All sites LRU, LRD, All sites

HCU, HCD

Type of cages PVC cages PVC cages Modified

minnow traps

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Figure 36. Cage dimensions for both cage types.

130

The last experiment was started on October 6th. However, sites in Little River were not accessible during the whole day for security reasons at the oil refinery.

Cages for LRU and LRD were set the next morning, October 7th. The caging

experiment was scheduled for one week, with a possibly of extending it to two

weeks if survival rate was high after six days. On the morning of October 11th, a storm, bringing 40-60 mm of rain, was predicted for the evening. The cages were consequently removed that day. Fish were held in coolers overnight, aeration was provided and no food was given to the fish. Each site had its cooler filled with local water. Sampling was conducted the next morning.

The first two caging experiments used mummichog larger than 50 mm, as this was determined in the sentinel species experiment to be the size where all fish are sexually mature. The last experiment used fish bigger than 60 mm as enough fish were collected to do such a selection. Table 15 shows average total mass and total length for each experiment.

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Table 15. Means and standard deviation for total mass and total length for each caging experiment. Experiment 1 2 3

Starting date May 07, 04 July 02, 04 October 6-7, 04

Ending date May 31, 04 July 08, 04 October 11, 04

Sample size 18 15 21

Total mass (g) 2.43 ± 19.57 5.48 ± 1.98 3.29 ± 1.05

Total length (cm) 6.1. ± 1.1 7.6 ± 0.7 6.6 ± 0.6

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5.2.1 Mixed Function Oxygenase Activity

Mixed function oxygenase level was measured as the activity of ethoxyresorufin- o-deethylase (EROD) in fish liver using a standardized protocol (Whyte et al.

2000). The protocol used is described in section 4.3.2. Data were assessed for homogeneity of residual variance, using a Levene’s test. When data were homogeneous, ANOVA was used for the analysis of variance. If data were not homogeneous, transformation such as log transformation, was applied to correct it. If transformation did not solve the issue, the Kruskal-Wallis test for non- parametric data was used (∝ = 0.05 and β= 0.20).

5.2.2 Testosterone Production

In vitro gonadal incubation was done following the protocol of MacLatchy et al.

(2004). Following dissection, testis tissue was placed in Medium 199 (Sigma

Aldrich) buffer solution and placed on ice (Appendix 3). The testes were very

small; therefore, for each sample, testis tissue was cut in pieces, weighed (total

mass varied from 1 to 28 mg) and placed in 1 mL of Medium 199 buffer solution

in polystyrene tissue culture plates (Fisher Scientific Co., Toronto, ON, Canada)

at 4ºC. Medium 199 in each well was removed and replaced with 1 mL of

Medium 199 with 3-isobutyl-1-methylxanthine (IBMX) solution (Appendix 3). Each

well received 5 µL of human Chorionic Gonadotropin (hCG) stock solution

(Sigma-Aldrich) to stimulate gonadal steroid production (MacLatchy et al. 2004).

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The plates were than incubated at 18ºC for 24 hours. After incubation, the media

was removed, placed in cryovials, and stored at –86ºC.

Testosterone production was measured using a radioimmunoassay (RIA)

protocol. In vitro media were removed from the -86oC freezer and thawed on ice.

Standards were made with stock testosterone (Sigma Aldrich) diluted in phosgel

buffer (Appendix 3). Concentrations ranged from 0 to 4000 pg/mL. Two hundred

microliters of each sample as well as interassay sample were placed in individual

tubes. Two hundred microliters of antibody (concentration necessary to have

approximately 50% binding of added radiolabelled steroid) was added to each

tube except the non-specific binding (NSB) tubes and 200 µL of tracer

(concentration to have 5000 disintegration per minute [DPM]) was added to each tube. Three total counts reference (TCR) tubes were made by placing 200 µL of tracer in each tube with 600 µL of phosgel. Tubes were incubated at 18 oC for 20 hours.

After incubation, the tubes were placed on ice for 10 min, 200 µL of charcoal solution was added to each tube except the TCR tubes (Appendix 3). Tubes were centrifuged at 4oC for 40 minutes at 3000 rpm. For each sample, supernatant was transferred to labelled 7 mL polyethylene scintillation tubes

(Fisher Scientific Co.) and 5 mL of scintillation cocktail (Fisher Scientific Co.,) were added. Tubes were vortexed and placed into a Beckman LS 6500 liquid scintillation counter. A non-linear standard curve was calculated using standard

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concentrations and NSB data from NSB and TCR tubes. The standard curve was used to calculate the amount of testosterone produced per mg of testis tissue.

As for MFO, testosterone data were assessed for homogeneity of residual variance. ANOVA or Kruskal-Wallis tests were used depending on homogeneity results.

5.3 Results

For the first caging experiment, fish survival was very low. Only the fish in MCD survived the exposure period. No analyses were conducted because of the small sample sizes. For the second experiment, fish caged at both sites in Little River died after the 7-day experiment, whereas fish in HCD survived. It was impossible to get the cages out of HCU as the setup drifted away from the shore and it was several days before the cages could be removed. At that point, all the fish were dead. Finally, after the caging experiment in October, all the fish survived except one fish in LRD.

Testosterone levels were very low at each site (Figure 37). Average gonadal steroid production was 0.3 pg/mg of tissue/hour. No statistical differences were found among sites (ANOVA df=20; p = 0.132). Minimum detectable size for n=21 and α=β=0.05 was 0.18 pg/mg of tissue/hour or 0.14 pg/mg of tissue/hour at

α=β=0.10.

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0.8

0.7

0.6

0.5

0.4

0.3 Testosterone (pg/mg/hr)

0.2

0.1

0 01234567 MCU MCD LRU LRD HCU HCD

Site

Figure 37. In vitro testosterone production in male mummichog (Fundulus heteroclitus) after caging, October 2004. Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), and Hazen Creek Downstream (HCD).

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MFO activity was similar in many sites except for fish caged in LRD and MCD

(Figure 38). Fish caged in MCD had the highest MFO activity (ANOVA df=20; p<0.001). LRD was statistically lower than the other sites (ANOVA df=20; p<0.031) (Figure 38). For both sites receiving waste, MFO activity was statistically different than their respective upstream sites (ANOVA df=20; p<0.001). Although statistical comparisons are not possible with the positive and negative controls, the levels measured even in the reference sites were higher than the negative control (Figure 38).

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1600 a 1400

1200

1000

800 b b b 600 bc

MFO (pmol/min/mg) MFO c 400

200

0 MCU MCD LRU LRD HCU HCD Cx BNF Site

Figure 38. Mixed-function oxygenase (MFO) activity in male mummichog (Fundulus heteroclitus) after caging experiment, October 2004. Different letters indicate statistical differences among sites (p<0.05).Marsh Creek Upstream (MCU), Marsh Creek Downstream (MCD), Little River Upstream (LRU), Little River Downstream (LRD), Hazen Creek Upstream (HCU), Hazen Creek Downstream (HCD), negative (Cx) and positive (BNF) controls.

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5.4 Discussion

The absence of effects on testosterone production was expected as fish were

regressed when collected. The actual testosterone level (0.3 pg/mg of

tissue/hour) was low compared to other studies. Testosterone levels are

generally higher early in the spawning period (May to July) and decrease rapidly

during the spawning season to reach levels close to zero at the end of August

(Leblanc & Couillard 1995). The low level of testosterone production during the

October experiment prevented the assessment of potential effects from the oil

refinery effluent on gonadal testosterone production.

The high MFO activity in MCD compared to its upstream site as well as the other downstream sites is probably related to the presence of creosote, which contains polycyclic aromatic hydrocarbons, in the sediment of MCD (Figure 38). MFO activity was also very high in mummichog exposed to MCD waters in a lab experiment (Astephen 2000). The concentration of PAHs in sediment collected was elevated, reaching a total concentration of 6367 ppm (Astephen 2000). The presence of PAHs in fish tissue in that study confirmed contaminant availability.

The generally high MFO levels found in studied sites is surprising (Figure 38).

The baseline level was found at HCD (320.7 pmol/min/mg). This was much higher than the negative control (159 pmol/min/mg) or the baseline level found in

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the previous year for males (WC = 114.9 pmol/min/mg). All fish were collected

from the same area, Hazen Creek Upstream and Downstream, which are sites with no known actual discharges. In chapter 4, the MFO activity found in male mummichog collected in HCD were of similar level (302.1 ± 37.8 pmol/min/mg).

Fish collected as sentinel species were pre spawning fish, as opposed to the

mummichog caged in sites, which were at a post spawning stage. MFO activity

can vary greatly with reproductive status, however differences are mainly visible

between reproductively active and inactive females (Whyte et al. 2000).

High MFO baseline level could be related to the quality of the Saint John Harbour

water. Previous studies done in the harbour showed presence of petroleum

residues, metals, PCBs, and PAHs, all chemicals that can induce MFO activity

(Godfrey Associated Ltd. 1993; Washburn & Gillis Associates Ltd 1993, Brillant

1999). Moreover, blue mussel (Mytilus edulis) and winter flounder

(Pseudopleuronectes americanus) collected in the harbour had increased MFO

activity (Washburn & Gillis 1993). However, Vallis (2003) could not show

induction in fish collected in the Saint John Harbour. Although MFO activity can

be induced within hours to a few days, it takes several days to weeks to return to

its normal level after exposure to inducing compounds (Whyte et al. 2000). Could

it be possible that mummichog collected in Hazen Creek have been exposed to

some level of contamination prior to their migration into the creek?

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The MFO activity found in fish exposed to LRD was also unexpectedly low. Not only the exposure to oil refinery effluent did not cause MFO induction, but the levels measured were statistically lower than the results for LRU and HCD.

Although the decrease in MFO activity is not ecologically relevant compared to the reference level, its decrease is surprising. The low EROD activity could not be explained by: 1) the inability of the oil refinery effluent to induce EROD activity; 2) the fact that mummichog cannot be induced; or 3) the absence of effluent where the cages were set. Oil refinery effluent has been shown to induce EROD activity in various studies (Ridlington et al. 1982; Kiceniuk 1992;

Parrott et al. 1996). The wild mummichog collected in Little River showed MFO induction (Table 13). Oil and grease are the main components present in oil refinery effluent capable of inducing EROD activity as they contain PAHs

(Ridlington et al. 1982; Parrott et al. 1996; Kirby et al 1999; Whyte et al. 2000).

Mummichog injected with β-naphthoflavone (positive control) showed a four-fold induction. Moreover, fish caged at other sites showed higher levels of MFO, including a six-fold increase between MCD and LRD. Finally, cages were set in an area determined to be constantly exposed to the effluent plume and consequently fish were exposed to PAHs contained in the effluent.

If exposure to the effluent and presence of MFO-inducing compounds in effluent are confirmed and cannot explain the reduced level of MFO, what could have caused the reduced level found in LRD? Whereas MFO induction is related to contaminant uptake rate, the depuration rate is related to the degradation rate,

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which can be related to seasonal factors (temperature) as well as physiological

factors (sex, age, reproductive cycle) (Whyte et al. 2000). Temperature

measured in LRD at the end of September of 2003 showed a difference of up to

5oC between the upstream and downstream site. The increased temperature

could have accelerated the depuration rate of fish caged downstream.

Nevertheless, intake of PAHs should have been greater in LRD than in LRU,

compensating for the faster depuration rate.

Food limitation has also been shown to affect MFO activity. Mummichog fed at

0.06% to 0.28% body mass/day showed increasing EROD activity after two and

18 days, respectively, compared to control fish fed at 2% body mass/day. EROD

activity increased until the end of the experiment on day 35 (Ferraro et al. 2003).

Similarly Arctic charr (Salvelinus alpinus) exposed to PCB and starvation showed

increasing EROD activity compared to fed fish exposed to the same contaminant

(Jørgensen et al. 1999). However, fish not exposed to PCB had similar EROD activity, whether they were fed or not (Jørgensen et al. 1999). Both experiments also showed decreased body mass for starved fish. However, fish in LRD were more likely to have suffered from starvation than fish from reference sites.

Consequently, EROD activity should have increased.

Other chemicals, such as metals and PCBs, have been shown to have the ability to inhibit EROD activity (Whyte et al. 2000). At this point it is unclear what caused a MFO activity reduction in mummichog exposed to the oil refinery effluent. More

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work will be needed to determine if this is a real effect and to better understand

its causes.

Even with the relative success of the caging experiment in October, many

problems where noticed. As cages were removed from sites with soft sediment

bottom, it appeared that cages were sinking into the sediment thus increasing the

risk of asphyxia for fish. The design of the cages has to be modified to ensure

that caging experiments reflect water characteristics. Effects on fish were visible

at some sites as fish were extremely pale and the presence of black colouring on

the snout and/or on the belly could be related to sediment exposure. The

moribund state of some mummichog suggested that the caging experiment could

not have lasted much longer without causing mortality. As mummichog had to

overnight in coolers before being sampled, it was possible to see recovery in some individuals the next morning (personal observations). It was also noted during sampling that the majority of fish had empty digestive tracks by the end of the five-day cage experiment, which could affect performance of the fish in a longer experiment. Suggestions for future caging design include: 1) use of bigger cages, especially in terms of height; and 2) suspension of cages in the water column using buoys (Figure 39).

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Figure 39. Suggestions for design of improved mummichog cages.

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In summary, the caging experiment failed to detect effects of the oil refinery effluent on testosterone production. However, it showed that MFO activity was affected downstream of the municipal discharges (MCD) as well as downstream of the oil refinery discharge (LRD). The increased MFO activity in MCD can be attributed to the presence of creosote deposition in the stream. The small MFO decrease in fish caged in LRD could not be explained.

The October caging experiment lasted for 4-5 days but observations showed that the actual design might not be suitable for an extended deployment. Survival could be compromised by food availability and by sediment accumulation in cages. Caging finfish was a challenging experience. Design will need improvement to accurately assess the effects of effluent discharge on fish health and increase its use as an alternative method in fish survey.

Caging experiments do not answer the question “Is the effluent affecting fish?” but rather “Might effluent affect fish?” (Courtenay et al. 2002). Even where effluent is discharged, fish can avoid extensive exposure to the effluent by moving away or by changing their behaviour (Westlake et al. 1983; Kierstead

1991). Nevertheless, caging experiments can provide information on potential effects especially in complex environments such as coastal streams where fish mobility and availability can be an issue.

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6 GENERAL CONCLUSION

This study had two main objectives: to assess the potential effects of the oil refinery effluent discharge on fish and fish habitat in Little River, and to evaluate the methodology used in the EEM program in a small coastal stream situation.

The fish community downstream of the outfall had lower numbers of fish species, lower abundances and a higher incidence of empty traps. The fish species found in LRD are presumed to be transient fish moving to spawning areas or simply following tidal movement.

The impacts on fish community and abundances were associated with periods of low dissolved oxygen and high conductivity in the stream caused by the presence of ballast water in the final effluent. The reduced abundance and species richness found in LRD could not ultimately be attributed to the discharge of the effluent as the occasional mix of ballast water with the final effluent had a confounding effect on the fish community.

In an effort to evaluate the impacts of the effluent from the refinery (as opposed to the ballast water), wild fish were examined. Mummichog were the most abundant species during the summer months, but they were still insufficient in number to collect enough to conduct a thorough evaluation of the potential

146

impacts of the effluent. Those fish that were collected downstream of the outfall

had increased liver size and MFO activity relative to reference sites in other

rivers. However, the MFO activity was not elevated relative to upstream

reference fish, and the Little River sites showed much higher variability than at

other sites. The similarity of liver detoxification enzymes upstream and

downstream of the effluent suggests that mummichog are either moving between

sites or are exposed to a common source of contamination. The increased liver size and EROD activity in Little River suggest that there is some exposure to

PAHs occurring, but whether this is from historical or existing discharges is

unclear.

It is apparent when looking at the community fish survey that the fish

downstream of the oil refinery effluent are not resident. The mobility hypothesis

is reinforced by the caging experiment. The increased mobility may be related to

the transient periods of poor water quality.

To reduce the problems with interpretation associated with high mobility in the

Little River system, several attempts were made to cage fish. High mortalities

suggest that the caging methodologies need modification to deal with some of

the habitat issues in these small coastal streams, including rapidly changing

flows associated with rainfall, soft bottom substrates, food availability and

appropriate dissolved oxygen level.

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As mentioned earlier, the refinery is moving towards double-hulled transport ships (2006) that will eliminate the potential impact of the ballast water dumping into Little River. This will potentially cause a substantial improvement in the quality of the Little River habitat for fish. As it was not possible under the current situation to evaluate the refinery effluent per se, the first objective (to evaluate the potential effects of the oil refinery effluent on the Little River fish populations) could not be met. Better caging methodologies (during periods of no ballast water discharge), stream side mesocosms or laboratory exposures will be required to evaluate the potential effects of the refinery effluent. The existing collections also were not able to evaluate the potential influence of instream historical depositions.

The second objective of the thesis was to evaluate the EEM methodologies for use in small coastal streams. The main problems encountered during the study were similar to the general difficulties found with the EEM program and were related to insufficient abundance of fish or fish species, and failure to confirm exposure to the effluent. The project explored different possibilities in the context of assessment in an estuarine environment. E.g., the studies involved a water quality survey instead of a benthos survey to assess effects on fish habitat. It also included the use of the monthly community survey to assess effects on fish community. Finally, the caging experiment was another attempt to assess effects on fish and cope with fish mobility.

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FUTURE DIRECTIONS

The elimination of ballast water from final effluent by 2006 will allow assessing the effect of the oil refinery effluent and historical contamination on fish. It will be possible to discriminate effects from present discharge and past contamination through mesocosm experiments or laboratory exposures. Mesocosms experiments can provide information of actual discharge effects on fish and its food web, while past contamination could be assessed through sediment laboratory exposures.

The development of caged fish methods will facilitate survival of fish and extended exposure duration. The development of such an alternative method to assess effects of discharge on fish are of great interest to the EEM program and will permit to resolve some of the problems encountered in this program including fish mobility and availability of sentinel species.

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7 REFERENCE

Abraham, B.J. 1985. Species profiles: Life histories and environmental requirements of coastal fishes and invertebrates (mid-Atlantic). Mummichog and striped killifish. Biological Report 82 (11.40). vi +23 pp.

Ambrose, R.F. & Meffert, D.J. 1999. Fish-assemblage dynamics in Malibu Lagoon, a small, hydrologically altered estuary in southern California. Wetlands 19: 327-340.

Astephen, S. 2000. Biological assessment of Marsh Creek, 1999. Atlantic Coastal Action Program (ACAP) Saint John, Saint John NB. 34 pp.

Baer, K.N., Olivier, K., & Pope, C.N. 2002. Influence of temperature and dissolved oxygen on the acute toxicity of profenofos to fathead minnows (Pimephales promelas). Drug and Chemical Toxicology: 25: 231-245.

Belpaire, C., Smolders, R., Vanden Auweele, I., Ercken, D., Breine, J., Van Thuyne, G., & Ollevier, F. 2000. An index of biotic integrity characterizing fish populations and the ecological quality of Flandrian water bodies. Hydrobiologia 434: 17–33.

Bergstedt, L.C. & Bergersen, E.P. 1997. Health and movements of fish in response to sediment sluicing in the Wind River, Wyoming. Canadian Journal of Fisheries and Aquatic Sciences 54: 312–319.

Bleckmann, C.A., Rabe, B., Edgmon, S.J., & Fillingame, D. 1995. Aquatic toxicity variability for fresh- and saltwater species in refinery wastewater effluent. Environmental Toxicology and Chemistry 14: 1219-1223.

Brillant, S., 1999. The marine food web in relation to the movement and accumulation of toxins in Saint John Harbour, New Brunswick, Canada. M.Sc. Thesis. University of New Brunswick. Saint John, New Brunswick. 145 pp.

Buikema, A.L. Jr., Niederlehner, B.R., Cairns, J. Jr., 1981. The effects of a simulated refinery effluent and its components on the estuarine crustacean, Mysidopsis bahia. Archives of Environmental Contamination and Toxicology 10: 231-240.

150

Butner, A. & Brattstrom, B.H. 1960. Local movement in Menidia and Fundulus. Copeia 2: 139-141.

Canadian Council of Ministers of the Environment (CCME). 1999. Canadian environmental quality guidelines. Chapter 4. Canadian water quality guidelines for protection of aquatic life. Canadian Council of Ministers of the Environment, Winnipeg.

Clarke, K.R. & Warwick, R.M. 2001. Change in marine communities: an approach to statistical analysis and interpretation. Second edition. PRIMER-E: Plymouth.

Cote, R.P., 1976. The effects of petroleum liquid wastes on aquatic life, with special emphasis on the Canadian environment. National Research Council Canada. Associate Committee on Scientific Criteria for Environmental Quality. 77 pp.

Courtenay, S.C., Munkittrick, K.R., Dupuis, H.M.C., Parkers, R., Boyd, J., 2002. Quantifying impacts of pulp mill effluent on fish in Canadian marine and estuarine environments: Problems and Progress. Water Quality Research Journal of Canada 37: 79-99.

Coyote Oil. (2004). The refining process. [online]. Available from: http://www.coyoteoil.com/process.html [Accessed March 30, 2004].

Culp, J.M. & Glozier, N.E. 1989. Experimental evaluation of a minnow trap for small lotic fish. Hydrobiologia 175: 83-87.

Culp, J.M., Podemski, C.L., Cash, K.J. 2000. Interactive effects of nutrients and contaminants from pulp mill effluents on riverine benthos. Journal of Aquatic Ecosystem Stress and Recovery 8: 67-75.

Doebel, C., Baron, C.L., Evans, R.E., Wautier, K.G., Werner, J., Palace, V.P. 2004. Caged pearl dace (Semotilus margarita) as sentinels for gold mining wastes in environmental effects monitoring. Bulletin of Environmental Contamination and Toxicology 72: 409-414.

Elliot, M. & Hemmingway, K.L. 2002. Fishes in estuaries. Blackwell Science, Great Britain. 627 pp.

Environment Canada. 1974. Petroleum refinery effluent regulations and guidelines regulations. Codes and Protocols Report EPS 1-WP-74-1. Water Pollution Control Directorate.

Environment Canada, 1995. 1992 Environmental status report on Canadian petroleum refinery effluents. Environmental Protection

151

Series. Report EPS 1/PN/4. 95 pp.

Environment Canada. 1998. Pulp and paper technical guidance for environmental effects monitoring. EEM/1998/8.

Environment Canada. (2003). About environmental effects monitoring [online]. The Green LaneTM, Environment Canada's World Wide Web site. Available from: http://www.ec.gc.ca/eem/english/default.cfm [Accessed April 23, 2003].

Environment Canada. (2004). Environmental acts and regulations. Acts Administered In Part by the Minister of the Environment. [online]. Available form: http://www.ec.gc.ca/EnviroRegs/Eng/SearchDetail.cfm?intAct=1017 [Accessed September 02, 2004].

Environment Canada. (2004)b. Environmental acts and regulations. Fisheries act. Petroleum refinery liquid effluent regulations. [online]. Available form: http://www.ec.gc.ca/EnviroRegs/Eng/SearchDetail.cfm?intReg=80 [Accessed March 29, 2004].

Ferraro, M., Curetsy, K., & Crivello, J. F. 2003. The effects of feeding restriction on metallothionein levels and ethoxyresorufin-o-deethylase activities in mummichogs. Journal of Aquatic Animal Health 15:31–38.

Glozier, N.E., Culp, J.M., Reynoldson, T.B., Bailey, R.C., Lowell, R.B., & Trudel, L. 2002. Assessing metal mine effects using benthic invertebrates for Canada’s environmental effects program. Water Quality Research Journal of Canada 37: 251-278.

Godfrey Associated Ltd. 1993. Saint John harbour wastewater strategy. Saint john, New Brunswick. 86 pp.

Goksøyr, A., Beyer, J., Husøy, A-M., Larsen, H.E., Westrheim, K., Wilhmsen, S., Klungsøyr, J. 1994. Accumulation and effects of aromatic and chlorinated hydrocarbons in juvenile Atlantic cod (Gadus morhua) caged in a polluted fjord (Sørfjorden, Norway). Aquatic Toxicology 29: 21-35.

Hall, J.A., Frid, C.L.J., & Gill, E. 1997. The response of estuarine fish and benthos to an increasing discharge of sewage effluent. Marine Pollution Bulletin 34:527-535.

Halpin, P.M. 1997. Habitat use patterns of the mummichog, Fundulus heteroclitus, in New England. 1. Intramarsh variation. Estuaries 20:

152

618-625.

Hughes, R.M., Kaufmann, P.R., Herlihy, A.T., Kincaid, T.M., Reynolds, L., & Larsen, D.P. 1998. A process for developing and evaluating indices of fish assemblage integrity. Canadian Journal of Fisheries and Aquatic Sciences 55: 1618-1631.

Irving Oil Limited. 2001. Deposits of combined liquid effluent streams. Saint John: Irving Oil Limited. Monthly reports from January to December 2001.

Irving Oil Limited. 2002. Deposits of combined liquid effluent streams. Saint John: Irving Oil Limited. Monthly reports from January to December 2002.

Irving Oil Limited. 2003. Deposits of combined liquid effluent streams. Saint John: Irving Oil Limited. Monthly reports from January to December 2003.

Irving Oil Limited. 2004. Deposits of combined liquid effluent streams. Saint John: Irving Oil Limited. Monthly reports from January to December 2004.

Irving Oil Limited. (2004b) A Backgrounder on the Irving Oil Refinery. Available from: http://www.irvingoilco.com/refining/isgref.htm [Accessed March 29, 2004]

Johnson, L.L., Collier, T.K., & Stein, J.E. 2002. An analysis in support of sediment quality thresholds for polycyclic aromatic hydrocarbons (PAHs) to protect estuarine. Aquatic Conservation: Marine & Freshwater Ecosystems 12: 517–538.

Jørgensen, E.H., Bye, B.E., & Jobling, M. 1999. Influence of nutritional status on biomarker responses to PCB in the Arctic charr (Salelinus alpinus). Aquatic Toxicology 44: 233–244.

Khan, R.A. 1998. Influence of petroleum at a refinery terminal on feral winter flounder, Pleuronectes americanus. Bulletin of Environmental Contamination and Toxicology 61: 770-777.

Kiceniuk, J.W. 1992. Aromatic hydrocarbons concentrations in sediments of Placentia Bay, Newfoundland. Canadian Technical Report of Fisheries and Aquatic Sciences 1888: iv + 12pp.

Kierstead, J.T. 1991. Refinery effluent biomonitoring. Proceedings of the Seventeenth Annual Aquatic Toxicity Workshop: Nov 5-7, 1990,

153

Vancouver, B.C. Vol. 1. Canadian Technical Report of Fisheries and Aquatic Sciences 1774.

King, M.M., Hite, R.L. 1993. A biological and water quality survey of Sugar Creek and tributaries, Crawford County, Illinois. August-September 1992. Monitoring and Assessment Unit, Illinois. Environmental Protection Agency, Division of Water Pollution Control. 49 pp.

Kirby, M.F., Neall, P., & Tylor, T. 1999. EROD activity measured in flatfish from the area of the Sea Empress oil spill. Chemosphere 38: 2929- 2949.

Kneib, R.T. & Craig, A.H. 2001. Efficacy of minnow traps for sampling mummichogs in tidal marshes. Estuaries 24: 884-893.

Knudsen, F.R., Schou, A.E., Wiborg, M.L., Mona, E., Tollefsen, K-E., Stenersen, J., Sumpter, J.P. 1997. Increase of plasma vitellogenin concentration in rainbow trout (Oncorhynchus mykiss) exposed to effluents from oil refinery treatment works and municipal sewage. Bulletin of Environmental Contamination and Toxicology 59: 802-806.

Krause, P.R. 1994. Effects of an oil production effluent on gametogenesis and gamete performance in the purple sea uchin (Strongylocentrotus purpuratus stimpson). Environmental Toxicology and Chemistry 13: 1153-1161.

Kuehn, R.L., Berlin, K.D., Hawkins, W.E., & Ostrander, G.K. 1995. Relationships among petroleum refining, water and sediment contamination, and fish health. Journal of Toxicology and Environmental Health 46:101-116.

Layman, C.A. & Smith, D.E. 2001. Sampling bias of minnow traps in shallow aquatic habitats on Eastern shore of Virginia. Wetlands 21: 145-154.

Leblanc, J. & Couillard, C.M. 1995. Description de la période de reproduction d’un poisson sentinelle : le choquemort (Fundulus heteroclitus) de l’estuaire de la Miramichi. Rapport Technique Canadien des Sciences Halieutiques et Aquatiques 2057: vii + 32 pp.

Leblanc, K. 1998. Effects on the reproductive endocrine function of the mummichog (Fundulus heteroclitus) and the goldfish (Carassius auratus) exposed to Saint John. New Brunswick, harbour waters. Thesis. University of New Brunswick. 118 pp.

Lotrich, V.A. 1975. Summer home range and movements of Fundulus

154

heteroclitus (Pisces : Cyprinodontidae) in a tidal creek. Ecology 56: 191-198.

Lowell, R.B., Ribey, S.C., Ellis, I.K., Porter, E.L., Culp, J.M., Grapentine, L.C., McMaster, M.E., Munkittrick, K.R., Scroggins, R.P. 2003. National assessment of the pulp and paper environmental effects monitoring data. Environment Canada. National Water Research Institute Contribution No. 03-521.

MacLatchy, D.L., Gormley, K.L., Ibey, R.E.M., Sharpe, R.L., Shaughnessy, K.S., Courtenay, S.C., Dubé, M.G., and Van Der Kraak, G.J. 2004. A short-term mummichog (Fundulus heteroclitus) bioassay to assess endocrine responses to hormone-active compounds and mixtures. In G. Ostrander (ed.) Techniques in Aquatic Toxicology, Volume 2, CRC Press, Boca Raton, FL, USA. In press.

Montague, C.L. & Ley, J.A. 1993. A possible effect of salinity fluctuation on abundance of benthic vegetation and associated fauna in Northeastern Florida Bay. Estuaies 16: 703-717.

Munkittrick, K.R., Megraw, S.R., Colodey, A., Luce, S., Courtenay, S., Paine, M., Servos, M., Spafford, M., Langlois, C., Martel, P., and Levings, C. 1997. Fish survey expert working group final report. Recommendations from cycle 1 review. Environment Canada, EEM/1997/6.

Munkittrick, K.R., McMaster, M.E., Van Der Kraak, G.J., Portt, C., Gibbons, W.N., Farwell, A., Gray, M. 2000. Development of methods for effects-driven cumulative effects assessment using fish populations: Moose River project. Society of Environmental Toxicology and Chemistry (SETAC). SETAC Technical Publication Series. 236 pp.

Oberdorff, T., Pont, D., Hugueny, B., & Porcher, J-P. 2002. Development and validation of a fish-based index for the assessment of river health in France. Freshwater Biology 47: 1720–1734

Ostrander, G.K., Kuehn, R.L., Berlin, K.D., & Hawkins, W.E. 1995. Anthropogenic contaminants and fish health along an urban waterway. Environmental Toxicology and Water Quality : An International Journal 10: 207-215.

Parrott, J.L., Backus, S.M., Swyripa, M. 1996. MFO inducers from refinery effluent, Mackenzie River, Norman Wells. Proceedings of the 23rd annual aquatic toxicity workshop: October 7-9, 1996, Calgary, Alberta. Canadian Technical Report of Fisheries and Aquatic Sciences #2144.

155

Putman, D.L. 1989. Developments on legislation related to treatment of petroleum refinery effluents: A Canadian overview. Water Pollution Research Journal of Canada 24: 345-353.

Ridlington, J.W., Chapman, D.E., Boese, B.L., Johnson, V.G., Randall, R., 1982. Petroleum refinery wastewater induction of the hepatic mixed- function oxidase system in Pacific staghorn sculpin. Archives of Environmental Contamination and Toxicology, 11: 123-127.

Rowe, D.W., Sprague, J.B., Heming, T.A., Brown, I.T., 1983 a). Sublethal effects of treated liquid effluent from a petroleum refinery. 2. Growth of rainbow trout. Aquatic Toxicology, 2: 161-169.

Rowe, D.W., Sprague, J.B., Heming, T.A., 1983 b). Sublethal effects of treated liquid effluent from a petroleum refinery. 1. Chronic Toxicity to Flagfish. Aquatic Toxicology 3: 149-159.

Rozas, L.P. & Minello, T.J. 1997. Estimating densities of small fishes and decapod crustaceans in shallow estuarine habitats: A review of sampling design with focus on gear selection. Estuaries 20: 199-213.

Saha, M.K., Konar, S.K., 1984. Sublethal effects of effluent of petroleum refinery on fish. Environment and Ecology, 2: 262-265.

Schmidt, K. & Talmage, P. 2001. Fish community surveys of Twin Cities metropolitan area streams. Minnesota Department of Natural Resources Special Publication 156, October 2001. 33 pp.

Schneider, J.C. 2002. Fish as indicators of lake habitat quality and a proposed application. Michigan Department of Natural Resources fisheries Division, Fisheries Research Report Number 2061. 40 pp.

Scott, W.B. & Corssman, E.J. 1998. Freshwater fishes of Canada. Fisheries Research Board of Canada - Bulletin 184.

Scott, W.B. & Scott, M.G. 1988. Atlantic fishes of Canada. Canadian Bulletin of Fisheries and Aquatic Sciences 219: 731 pp.

Sergy, G.A. & Scroggins, R.P. 1991. Biological testing development in Environment Canada. Canadian Technical Report of Fisheries and Aquatic Sciences. Proceedings of the 17th Annual Aquatic Toxiciy Sciences 1774: 101-105.

Sherry, J.P., Scott, B.F., Nagy, E. & Dukta, B.J. 1994. Investigation of the sublethal effects of some petroleum refinery effluents. Journal of Aquatic Ecosystem Health 3: 129-137.

156

Simona, T.P., Jankowskib, R., & Morrisb C. 2000. Modification of an index of biotic integrity for assessing vernal ponds and small palustrine wetlands using fish, crayfish, and amphibian assemblages along southern Lake Michigan. Aquatic Ecosystem Health and Management 3: 407-418.

Skinner, M. 2005. The site fidelity of mummichogs (Fundulus heteroclitus) in an Atlantic Canadian estuary: Implications for use as a sentinel species in environmental monitoring. M.Sc. Thesis. University of New Brunswick, Saint John, New Brunswick, Canada. In press.

Smith, A.K. & Suthers, I.M. 1999. Effects of sewage effluent discharge on the abundance, condition and mortality of hulafish, Trachinops taeniatus (Plesiopidae). Environmental Pollution 106: 97-106.

St-Cyr, M. 1995. 1992 Environmental status report on Canadian petroleum refinery effluents, Report EPS 1/PN/4 October 1995, Environment Canada. Environmental Protection Series.

Sweeney, J., Deegan, L., & Garritt, R. 1998. Population size and site fidelity of Fundulus heteroclitus in a macrotidal saltmarsh creek. Biological Bulletin 195: 238-239.

Teo, S.L.H. & Able, K.W. 2003. Habitat use and movement of the mummichog (Fundulus heteroclitus) in a restored salt marsh. Estuaries 26: 720-730.

Tourism Saint John. (2004). Tide tables (Reversing Falls). [online]. Available form: http://www.tourismsaintjohn.com/files/fuse.cfm?section=54&lang= [Accessed November 12, 2004].

Vallis, L.E. 2003. The potential use of rock gunnel (Pholis gunnellus) as a bioindicator in Saint John Harbour, New Brunswick, Canada. M.Sc. Thesis. University of New Brunswick, Saint John, New Brunswick, Canada. 97 pp.

Walker, S.L., Hedley, K, & Porter E. 2002. Pulp and paper environmental monitoring in Canada: An overview. Water Quality Research Journal in Canada 37: 7-19.

Walker, S.L., Dixit, S.S., Andersen, D., Caux, P.Y., Chambers, P.A., Charlton, M.C., Howes, L.A., & Kingsley, L. 2003. Scoping science assessment of the impacts of freshwater aquaculture on the Canadian environment. National Water Research Institute, Environment Canada.

157

NWRI Contribution 03-522. 87 pp.

Washburn & Gillis Associates Ltd. 1993. Saint John Harbour environmental quality study. Fredericton, New Brunswick. xiii + 215 pp + appendices.

West, J.L., Bailey, J.R., Almeida-Val, V.M.F., Val, A.L., Sidell, B.D.,& Driedzic, W.R. 1999. Activity levels of enzymes of energy metabolism in heart and red muscle are higher in north-temperate-zone than in Amazonian teleosts. Canadian Journal of Zoology 77: 690–696.

Westlake, G.F., Sprague, J.B., Hines, R.J., Brown, I.T. 1983. Sublethal effects of treated liquid effluent from a petroleum refinery. III. Avoidance and other locomotor responses of rainbow trout. Aquatic Toxicology 4: 235-245.

Wetzel, R.G. 1983. Limnology 2nd Edition. Saunders College Publishing.

753 pp.

Whyte, J.J., Jung, R.E., Schmitt, C.J., & Tillitt, D.E. 2000. Ethoxyresorufin- O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Critical Reviews in Toxicology 30: 347-570.

Zar, J.H. 1984. Biostatistical Analysis. Second Edition Prentice Hall Press. 718 pp.

158

8 APPENDICES

APPENDIX 1. Sonde deployment in Little River upstream and downstream, 2003 and 2004.

14 Salinity 30 DO Temperature 12 25

10 20

8 15 6

10 (oC) Temperature 4 DO (mg/L) and SalinityDO (mg/L) and (ppt) 5 2

0 0 07/22/03 07/23/03 07/24/03 07/25/03 07/26/03 07/27/03 07/28/03 07/29/03 07/30/03 07/31/03 08/01/03 08/02/03 08/03/03 08/04/03 08/05/03 08/06/03 08/07/03 08/08/03 08/09/03 08/10/03 08/11/03 Date Figure 38. Salinity, dissolved oxygen and temperature, Little River Downstream site, July 22 to August 4, 2003.

159

LRU DO LRD DO 10 LRU Salinity 6 LRD Salinity 9

5 8

7 4

5 3 DO (mg/L) 4 Salinity (ppt)

2 3

2 1

0 0 9/24/03 9/25/03 9/26/03 9/27/03 9/28/03 9/29/03 9/30/03

Date Figure 41. Salinity and dissolved oxygen data, Little River upstream and downstream sites, September 24 to October 1, 2003.

160

14 LRU DO 9 LRD DO LRU Salinity 8 12 LRD Salinity 7

10 6

8 5

4 DO (mg/L) 6 Salinity (ppt)

3 4

2 1

0 0 5/10/04 5/11/04 5/12/04 5/13/04 5/14/04 5/15/04 5/16/04 5/17/04 5/18/04 5/19/04 5/20/04 5/21/04 5/22/04 5/23/04 5/24/04 5/25/04 5/26/04 5/27/04 5/28/04 5/29/04 5/30/04 5/31/04

Date

Figure 42. Salinity and dissolved oxygen data, Little River upstream and downstream sites, May 10 to 31, 2004.

161

14 14 LRU DO LRD DO

12 LRU Salinity 12 LRD Salinity

10 10

8 8 DO (mg/L)

6 6 Salinity (ppt)

4 4

2 2

0 0 6/1/04 6/2/04 6/3/04 6/4/04 6/5/04 6/6/04 6/7/04 6/8/04 6/9/04 6/10/04 6/11/04 6/12/04 6/13/04 6/14/04 6/15/04 6/16/04 6/17/04 6/18/04 6/19/04 6/20/04 6/21/04 Date

Figure 43. Dissolved oxygen and salinityin Little River upstream and downstream sites, June 1 - 21, 2004.

162

Salinity

Dissolved Oxygen 18 30 Temperature

15 25

12 20

9 15 Temperature (oC) 6 10 DO (mg/L) and Salinity (ppt) Salinity DO (mg/L) and

3 5

0 0 7/1/04 7/2/04 7/3/04 7/4/04 7/5/04 7/6/04 7/7/04 7/8/04 Date

Figure 44. Salinity, dissolved oxygen and temperature, Little River downstream site, July 1-6 2004.

163

12 30 Salinity Dissolved Oxygen Temperature 25

6 15 Temperature (oC) 10 DO (mg/L) and Salinity (ppt) Salinity DO (mg/L) and 3

0 0 08/18/04 08/19/04 08/20/04 08/21/04 08/22/04 08/23/04 08/24/04 08/25/04 08/26/04 08/27/04 08/28/04 08/29/04 08/30/04 08/31/04 Date

Figure 45. Salinity, dissolved oxygen and temperature, Little River downstream site, August 16-31 2004.

164

9 Salinity 25 Dissolved Oxygen Temperature

6 15

3 (oC) Temperature DO (mg/L) and Salinity (ppt) Salinity DO (mg/L) and 5

0 0 9/1/04 9/2/04 9/3/04 9/4/04 9/5/04 9/6/04 9/7/04 9/8/04 9/9/04 9/10/04 9/11/04 9/12/04 9/13/04 9/14/04 9/15/04 9/16/04 9/17/04 Date

Figure 46. Salinity, dissolved oxygen and temperature, Little River downstream site, September 1-17 2004.

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APPENDIX 2. Data collection from ACAP.

Marsh Creek 2003. Fecal Free Salinity Turbidity colifiorm Total Ammonia Ammonia Total PO4- Date (ppt) pH (NTU) DO (mg/L) (#/100ml) (mg/L) (mg/L) P (mg/L) 18-Jun 0.05 7.05 1.0 11.03 >240 0.03 0.00 0.02 25-Jun 0.39 7.08 0.0 7.51 >870 0.69 0.00 0.01 2-Jul 0.06 6.87 0.0 6.05 311 0.05 0.00 0.01

stream 9-Jul 0.07 7.29 0.2 7.92 673 0.09 0.00 0.01 p

u 16-Jul 0.09 7.16 0.0 6.80 425 0.04 0.01 0.02 30-Jul 0.04 7.42 0.5 8.30 40 0.03 0.00 0.01 13-Aug 0.04 7.28 0.0 8.20 210 0.00 0.00 0.01 20-Aug 0.03 7.38 0.5 9.52 27 0.02 0.00 Means 0.10 7.19 0.3 8.17 >349 0.12 0.00 0.01 Fecal Free Salinity Turbidity colifiorm Total Ammonia Ammonia Total PO4- Date (ppt) pH (NTU) DO (mg/L) (#/100ml) (mg/L) (mg/L) P (mg/L) 18-Jun 3.35 7.05 1.0 6.03 TMTC* 0.10 0.00 0.03 25-Jun 1.25 8.46 2.0 12.73 3600000 0.22 0.03 0.10 2-Jul 16.93 7.76 0.0 9.73 1550000 0.05 0.00 0.09 9-Jul 5.61 8.49 0.8 15.20 2033333 0.34 0.05 0.11 16-Jul 11.01 7.10 0.5 4.30 1050000 0.43 0.01 0.11

downstream 30-Jul 9.64 7.42 0.5 9.77 0** 0.27 0.01 0.10 13-Aug 2.30 7.22 0.0 7.97 2900000 0.22 0.00 0.07 20-Aug 0.27 7.80 0.5 11.32 3600000 0.01 0.01 0.07 Means 7.63 7.63 0.4 9.72 2455555.50 0.22 0.01 0.09 * TMCT: Too many to count ** Data not included in mean calculation

166

Marsh Creek 2004. Water Fecal Total Total Temp Salinity Turbidity DO colifiorm Ammonia Free Ammonia PO4-P Date (oC) (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) (mg/L) (mg/L)

7-Jul 16.8 0.06 7.35 1.5 9.70 >240 0.03 0.00 0.03 14-Jul 21.0 0.08 7.24 0.0 8.91 290 0.01 0.00 0.08 21-Jul 16.7 0.06 7.22 1.8 9.72 212 0.04 0.00 0.01 28-Jul 17.5 0.10 6.96 1.6 9.56 0.04 0.00 0.00 Upstream 4-Aug 18.1 0.08 7.14 3.4 9.44 >>800 0.67 0.01 0.03 11-Aug 18.2 0.09 7.33 2.7 9.42 460 0.14 0.00 0.01 18-Aug 18.7 0.06 7.16 0.4 9.33 300 0.02 0.00 0.01 Means 18.1 0.07 7.20 1.6 9.44 315.50 0.13 0.00 0.02 Water Fecal Total Total Temp Salinity Turbidity DO colifiorm Ammonia Free Ammonia PO4-P Date (oC) (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) (mg/L) (mg/L) 7-Jul 17.6 0.22 7.48 1.5 9.53 5800000 0.55 0.01 0.17 14-Jul 23.5 0.22 7.55 1.4 8.48 5000000 1.04 0.03 0.18 21-Jul 17.3 0.21 7.33 0.5 9.59 1166667 0.83 0.01 0.15 28-Jul 19.0 0.21 7.07 4.5 9.26 1.03 0.01 0.12

Downstream 4-Aug 18.1 0.22 7.29 6.0 9.43 7800000 1.29 0.02 0.26 11-Aug 20.3 0.21 7.31 6.4 9.02 3200000 1.29 0.02 0.22 18-Aug 21.4 0.19 7.37 3.0 8.83 3310000 0.38 0.01 0.10 Means 19.6 0.21 7.34 3.3 9.16 4379444.44 0.92 0.02 0.17

167

Little River 2003. Fecal Free Salinity Turbidity colifiorm Total Ammonia Ammonia Total PO4- Date (ppt) pH (NTU) DO (mg/L) (#/100ml) (mg/L) (mg/L) P (mg/L) 22-Jun 0.00 7.78 0.00 10.84 47 0.01 0.00 0.01 29-Jun 0.06 7.75 1.00 8.72 >700 0.02 0.00 0.01 6-Jul 0.06 7.32 0.00 7.30 >116 0.06 0.00 0.01 13-Jul 0.04 7.82 0.50 10.20 72 0.00 0.00 0.00 stream

p 20-Jul 0.43 7.33 0.50 6.33 108 0.04 0.00 0.00 u 27-Jul 0.05 7.21 2.50 6.02 79 0.04 0.00 0.01 3-Aug 0.04 7.30 0.50 7.00 176 0.04 0.00 0.00 17-Aug 0.04 6.94 1.00 9.38 222 0.07 0.00 0.00 24-Aug 6.01 7.50 0.50 14.43 156 0.00 0.00 0.00 Means 0.84 7.40 0.81 8.67 >194,3 0.03 0.00 0.00 Fecal Free Salinity Turbidity colifiorm Total Ammonia Ammonia Total PO4- Date (ppt) pH (NTU) DO (mg/L) (#/100ml) (mg/L) (mg/L) P (mg/L) 22-Jun 0.00 7.72 0.00 120 1.70 0.05 0.05 29-Jun 1.27 8.24 1.50 10.60 90 1.88 0.19 0.38 6-Jul 1.12 7.23 0.50 11.53 >40 1.70 0.03 0.07 13-Jul 0.85 7.33 1.00 8.52 >240 1.78 0.03 0.04 20-Jul 1.50 6.84 2.00 4.10 70 2.29 0.03 0.02 27-Jul 0.74 6.84 5.30 3.76 93 1.70 0.02 0.06

downstream 3-Aug 2.41 6.78 3.00 1.30 24 0.93 0.01 0.02 10-Aug 244 17-Aug 5.93 6.58 1.00 1.17 36 1.73 0.02 0.02 24-Aug 5.81 6.71 3.00 7.90 270 1.73 0.02 0.05 Means 2.62 6.90 2.26 5.47 >122,7 1.69 0.02 0.04

168

Little River 2004. Water Fecal Total Total Temp Salinity Turbidity DO colifiorm Ammonia Free Ammonia PO4-P Date (oC) (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) (mg/L) (mg/L)

4-Jul 20.6 0.06 7.59 0.00 8.98 61 0.03 0.00 0.01 14-Jul 21.3 0.05 7.65 2.00 8.86 58 0.00 0.00 0.01 18-Jul 22.0 0.06 7.65 0.00 8.51 56 0.02 0.00 0.01 -0.05 14.62 Upstream -0.05 14.62 -0.05 14.62 15-Aug 21.5 0.02 7.35 8.13 8.82 60 0.01 0.00 0.02 Means 21.4 0.01 7.56 2.53 11.29 58.67 0.01 0.00 0.01 Water Fecal Total Total Temp Salinity Turbidity DO colifiorm Ammonia Free Ammonia PO4-P Date (oC) (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) (mg/L) (mg/L) 4-Jul 22.5 1.54 7.03 20.00 8.58 50 2.29 0.03 0.07 14-Jul 23.6 4.33 6.73 19.00 8.27 76 1.73 0.02 0.05 18-Jul 24.0 2.02 6.87 2.70 8.74 5 1.88 0.03 0.18 -0.05 14.62

Downstream -0.05 14.62 -0.05 14.62 15-Aug 21.5 0.24 7.15 1.03 8.81 190 0.96 0.02 0.06 Means 22.9 1.14 6.95 10.68 11.18 80.33 1.72 0.02 0.09

169

Hazen Creek 2003. Fecal Free Salinity Turbidity colifiorm Total Ammonia Ammonia Total PO4- Date (ppt) pH (NTU) DO (mg/L) (#/100ml) (mg/L) (mg/L) P (mg/L) 6-Jul 0.79 7.61 0.00 7.16 27 0.04 0.00 0.01 13-Jul 0.26 7.77 0.00 9.82 56 0.05 0.00 0.00 20-Jul 0.35 7.94 0.50 9.28 15 0.05 0.00 0.00

stream p

u 3-Aug 0.16 7.65 0.50 9.55 34 0.04 0.01 0.00 10-Aug 0.07 7.35 1.00 8.87 176 0.01 0.00 0.02 17-Aug 0.15 7.64 0.50 9.28 28 0.04 0.00 0.00 24-Aug 0.24 7.79 2.00 9.68 110 0.02 0.00 0.00 Means 0.29 7.68 0.64 9.09 63.71 0.03 0.00 0.0049 Fecal Free Salinity Turbidity colifiorm Total Ammonia Ammonia Total PO4- Date (ppt) pH (NTU) DO (mg/L) (#/100ml) (mg/L) (mg/L) P (mg/L) 6-Jul 0.26 7.58 0.00 9.23 >240 0.03 0.00 0.01 13-Jul 0.56 8.66 0.50 12.18 92 0.03 0.00 0.00 20-Jul 0.40 8.05 1.00 8.19 23 0.02 0.00 0.00

3-Aug 0.29 7.46 0.50 6.33 11 0.08 0.00 0.00

downstream 10-Aug 0.12 7.12 0.50 5.00 50 0.01 0.00 0.00 17-Aug 0.16 7.17 0.50 4.78 17 0.03 0.00 0.01 24-Aug 0.35 8.39 1.00 11.03 32 0.00 0.00 0.01 Means 0.31 7.78 0.57 8.11 >66,43 0.03 0.00 0.00

170

Hazen Creek 2004. Water Fecal Total Total Temp Salinity Turbidity DO colifiorm Ammonia Free Ammonia PO4-P Date (oC) (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) (mg/L) (mg/L)

4-Jul 13.5 0.17 7.76 0.00 10.41 >200 0.04 0.00 0.00 11-Jul 15.3 0.17 7.64 0.00 10.01 36 0.02 0.00 0.00 18-Jul 15.6 0.41 7.95 1.10 9.93 43 0.02 0.00 0.01 0.00 14.62 Upstream 1-Aug 14.4 0.24 7.88 0.40 10.20 20 0.02 0.00 0.00 8-Aug 13.2 0.27 7.80 0.00 10.47 356 0.01 0.00 0.00 0.00 14.62 Means 14.40 0.18 7.81 0.30 11.46 113.75 0.02 0.00 0.00 Water Fecal Total Total Temp Salinity Turbidity DO colifiorm Ammonia Free Ammonia PO4-P Date (oC) (ppt) pH (NTU) (mg/L) (#/100ml) (mg/L) (mg/L) (mg/L) 4-Jul 21.7 0.70 7.74 1.00 8.76 34 0.04 0.00 0.00 11-Jul 24.4 0.93 9.23 0.85 8.31 2 0.05 0.05 0.01 18-Jul 24.1 0.90 8.77 0.00 8.36 12 0.08 0.03 0.01 0.00 14.62

Downstream 1-Aug 20.7 11.22 7.84 4.50 8.39 53 0.03 0.00 0.00 8-Aug 17.7 4.27 8.37 1.60 9.28 1 0.00 0.00 0.00 -0.05 14.62 Means 21.72 2.57 8.39 1.59 10.33 20.40 0.04 0.02 0.00

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APPENDIX 3. Instruction to prepare solutions needed to measure EROD activity and testosterone level.

All products come from Sigma-Aldrich except when otherwise mentioned.

All solutions should be kept at 4oC otherwise mentioned.

• BSA standard

Mix 25 mg of bovine serum albumin (BSA) in 50.0 ml of distilled

water.

• Resorufin standard

Mix 5.0 mg of resorufin in 10 ml dimethyl sulfoxide (DMSO).

• HEPES GRINDING buffer

Mix 11.184 g KCl and 5.206 g HEPES in 1 L of water. Refrigerate

until cold, than adjust pH to 7.5 with 1mol/l HCl.

• HEPES buffer solution

Mix 26.03g HEPES in 1 L of water. Refrigerate until cold, than

adjust pH to 7.8 with 1 mol/l HCl.

• 7ER solution

172

Mix 0.022 mg of 7-ethoxyresorufin (7ER) per ml of DMSO. Measure

absorbance at 461.5 nm and dilute until reading is between 1.60

and 1.70 absorbance units. Store in dark at room temperature.

• 7ER/HEPES buffer

Mix 550 µl 7ER with 4550 µl of HEPES buffer solution. Store in dark

at room temperature.

• NADPH solution

Mix 20 mg of reduced nicotinamide adenine dinucleotide

phosphate (NADPH) (ICN Biochemicals, Aurora, OH, USA) in 1.0

ml of double distilled water

• TMS solution

Add 0.005 to 0.1 g of tricaine methane sulphonate (TMS) (Syndel

International Inc., Vancouver, BC, Canada) per liter of water.

Keep at room temperature.

• Medium 199 solution

Mix one bottle (11.0 g) of medium 199 containing Hank’s salts

without bicarbonate with 6.0 g HEPES 0.35g sodium bicarbonate,

0.1 g streptomycin sulfonate, and 1.0 g BSA in 1 L of double

173

distilled water. Refrigerate until cold, than adjust pH to 7.4 with 1

mol/l HCl or NaOH.

• Medium 199 with IBMX solution

Mix 0.222 g of 3-isobutlyl-1-methylxanthine (IBMX) with 1 L

medium 199 solution.

• Phosgel RIA buffer

Mix 2.875 g of Na2HPO4 with 0.64 g NaH2PO4-H2O, 0.5 gelatin, and

0.05 g thimerosal in 400 ml double distilled water. Heat at 45-50oC

for about 15 minutes. Adjust to 500 ml with double distilled water.

Adjust pH to 7.6.

• Charcoal solution

Mix 0.25 g activated charcoal with 0.025 g of dextran T70 in 50 ml

phosgel. Keep at room temperature.

174

VITA

Candidate’s full name: Geneviève Vallières

University attended: Université de Sherbrooke

Sherbrooke, Québec

From September 1995 to May 1998

Bachelor in Biology, concentration in ecology

Conference Presentations:

Society of Environmental Toxicology and Chemistry – World congress,

November 2004.

Platform presentation

Title: Challenges with monitoring multiple stressors in nearshore and

estuarine areas using sentinel species

Aquatic Toxicity Workshop, October 2004.

Platform presentation

Title: Assessing effects on fish from a stream receiving oil refinery

effluent

Environmental Effects Monitoring Conference, February 2004.

Poster presentation

Title: Assessing the health of fish in a stream receiving large industry

effluent

175