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Throughout North America populations of white sturgeon (Acipenser transmontanus) are threatened, in part due to poor annual recruitment. Definitive causes for this are not yet known, but the effects of contaminants are suspected to contribute. White sturgeon are exposed to a range of contaminants as they tend to inhabit industrialized river systems such as the Columbia and Fraser. White sturgeon are not commonly studied in ecotoxicology and their vulnerability as a species to contaminants of environmental concern is not well defined. To date, few exposure studies have been conducted with larvae, fry, and/or juveniles of this species; life stages often considered most susceptible to pollutants. Specifically, little work has been conducted to characterize effects of metals on white sturgeon. In the Upper Columbia River (UCR) a population of white sturgeon has been experiencing poor annual recruitment for over thirty years, and the effects of metal pollution have been hypothesized as a potential contributing factor. In particular, Teck Metals Ltd. (Teck) operates a metallurgical facility in Trail, BC, Canada that currently discharges processed effluent into the river and historically released other metal containing tailings such as slag. There are concerns that concentrations of trace-elements, such as copper, lead, cadmium, and zinc, associated with the effluent and/or slag, might have detrimental impacts on the surrounding ecosystem, including the local white sturgeon population. In 2006, a remedial investigation and feasibility study (RI/FS) was initiated in the UCR, under the oversight of the US EPA, and this project is contributing to the portion dedicated to the risk assessment of the exposure of white sturgeon to metals. The goals of this project were to develop information on toxicity of water, sediments and associated slag to help characterize sensitivity of white sturgeon to metals, and assess associated risks of metals on the population of white sturgeon in the UCR. Previous work conducted as part of a MSc degree, examined the effects of liquid effluent released by Teck on early life stages of white sturgeon. In addition, baseline information of toxicity due to sub-chronic exposure of early life stage sturgeon to copper, cadmium, and zinc, were developed. The thesis presented herein builds upon this previous work and has three major components to further characterize sensitivity of white sturgeon to metals and risk of exposure in the UCR. Specifically, a series of acute dose-response experiments were conducted with various early life stages of white sturgeon and resulting threshold values compared to water quality standards to assess protectiveness. Sensitivity of white sturgeon to metals was characterized by conducting parallel experiments with standard test species, such as rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas), as well as parallel field exposures in UCR water to develop water effect ratios (WERs) and assess relative bioavailability. A second set of experiments investigated whether exposure to water downstream of the metal smelter in Trail, BC affected survival or growth of white sturgeon. Mobile laboratories were situated riverside upstream and downstream of the smelter and the effects of potential contaminants within UCR water to early life stage white sturgeon were investigated under chronic exposure conditions. The third set of experiments involved characterizing UCR sediment toxicity and potential effects to sturgeon. Results from this research indicate that early life stage white sturgeon are relatively sensitive to copper, cadmium, lead, and zinc in comparison to other fishes. Sturgeon were particularly sensitive to copper, especially during early life stage development when larvae are transitioning to exogenous food. Thresholds for effects of copper on early life stage white sturgeon (LC50’s ranged between 9 and 22 μg/L) bracket water quality criteria for the protection of aquatic life (7.9 μg/L ± 1.5). This result indicated that white sturgeon in the UCR might not be adequately protected. Environmentally relevant concentrations of metals, such as copper, found in water, sediment, or waters associated with sediment of the UCR, including pore water and overlying water, may approach or exceed water quality criteria and lethal concentration (LC) values for sturgeon. Results from the risk assessment portion of this project, however, indicated that contaminants in the water column downstream of the metal smelter at Trail did not likely affect survival of white sturgeon. Dilution of Teck effluent in the river is such that, at the major spawning site where early life stages of sturgeon are likely to be present and where the riverside experiments from the present project were conducted, there would be no toxicity expected. Contaminants associated with sediments in the UCR and their impact on survival of sturgeon is also of concern as early life stages inhabit benthic habitats, on surface sediments, or in interstitial space between stones. Analytical results from this project did indicate that UCR sediment downstream of the smelter facility were significantly greater (p < 0.01) in concentrations of trace-elements, such as copper, lead, cadmium, and zinc, relative to reference sites. However, survival of white sturgeon was not adversely affected following exposure to UCR sediments. This project provided valuable information to help assess potential causes for poor recruitment of white sturgeon in the Columbia River. Advancements were made in characterizing the effects of metals to white sturgeon. In particular, life stage-specific sensitivities were identified that could have a significant impact on current risk assessment approaches and the derivation of protective water quality standards. There are several hypotheses as to why the number of white sturgeon have been decreasing in the UCR over the last few decades, but as of yet, no definitive cause for poor recruitment has been identified. As more research is conducted, possible causes for recruitment failure can be eliminated. Based on results from this project, metals in the UCR do not appear to be contributing directly to decreased survival of early life stage sturgeon.

Most environmental studies concerning the environmental health of the Swan- Canning River system have focussed on nutrient inputs from both rural and urban catchments that are the cause of algal blooms. On occasions these algal blooms have resulted in fish deaths attributed to oxygen starvation. Relatively few studies have examined whether non-nutrient contamination is affecting the health of the riverine environment. Those studies that have, have concentrated on measuring the levels of heavy metals, organochlorines, organophosphates, and hydrocarbons in the sediments and water of the river system, and in the flesh of the biota. However, chemical analysis often fails to detect chemicals of concern due to high laboratory detection limits. In addition, analysis of the body burden of contaminants within biota does not necessarily convey if exposure is inducing adverse effects at the individual or ecosystem levels. The use of biochemical markers as a tool for the assessment of the health of the Swan-Canning River system was examined under a collaborative research project with the Waters and Rivers Commission, established in response to the recognition of the paucity of information from chemical analyses. The present study focussed on the estuarine portion of the Swan-Canning River system, using the black bream (Acanthopagrus butcheri), an estuarine dependent fish species, as a biomonitoring tool. Prior to the commencement of this study it had been determined that the black bream was a suitable fish species for use as a biomonitoring tool when using mixed function oxygenase (MFO) activity induction under laboratory conditions. / Biopsies taken from feral black bream collected from eight sites during the period 2000 to 2002 from the estuary confirmed that the use of MFO induction in this fish species as a biomarker of exposure to organic contaminants is a reliable biomarker. Fish gender was a confounding factor in the interpretation of MFO induction when using the enzyme ethoxyresorufin-O-deethylase (EROD) as EROD activity was suppressed in both pre- and post-spawning female black bream. No such suppression was identified when using the MFO enzyme ethoxycoumarin-O-deethylase (ECOD). However, due to differences in the pattern and intensity of the induction of EROD and ECOD activities it was concluded that ECOD activity was not a substitute for EROD activity to detect certain chemical as ECOD activity represents a different cytochrome P450 pattern to EROD activity. No spatial, seasonal or interannual differences in the level of the enzyme sorbitol dehydrogenase (SDH) in the blood of the black bream were measured indicating that the interpretation of MFO activity induction was not compromised by hepatocellular damage. This study has shown that the black bream in the Swan-Canning Estuary are exposed to, and are metabolising polycyclic aromatic hydrocarbons (PAHs), notwithstanding that the chemical analysis of the contaminant load of these substances in the estuarine waters is consistently below laboratory detection limits. In addition, biomarker responses such as ECOD activity indicate that various other organic pollutants are present and are being metabolised by the black bream. / The measurement of biliary metabolites clearly show that, under winter conditions, the comprehensive drainage system of the Swan Coastal Plain contributes PAHs from pyrogenic sources such as burnt fuels into the estuary although the onset and intensity of rainfall events notably impacts on the volume of stormwater inflow. During the summer months, when freshwater flow is minimal, petrogenic sources of PAHs are dominant. Metabolic enzyme analysis points to the black bream being challenged in their aerobic capacities during summer, and that gill tissue was the most suitable tissue to evaluate the aerobic and anaerobic capacity of this fish species. Furthermore, there was a significant negative correlation between stress protein (hsp70) expression and DNA integrity in field-collected fish suggesting that the black bream within the estuary are highly stressed. No gradient of response in biomarker levels was identified in the Swan-Canning Estuary under either winter or summer conditions indicating there are multiple sources of inputs of potential pollutants along the length of the estuary. Stormwater and road runoff are the primary source of pollutant input into the estuary in the winter months, while summer biomarker levels, particularly PAH, appear to reflect the high usage of the estuary for recreational purposes and runoff from poorly irrigated parks and gardens. Significant rainfall events at any time of the year have the potential to adversely impact the biota of the estuary, particularly when these events result in a flush of water from the drains following long dry periods. / The study shows that the black bream is a suitable fish species to use under field conditions to detect the presence of bioavailable non-nutrient contamination within the Swan-Canning Estuary. A suite of biomarkers in black bream have been tested seasonally and annually but only a small number of biomarkers have proven suitable for routine monitoring of the health of the Swan-Canning Estuary. This treatise concludes with several recommendations for further investigations into biomarkers of fish health for the purpose of increasing our understanding on the sources and type of contamination entering the estuary, and potential effects on the aquatic biota of the Swan-Canning River system. These recommendations include, but are not limited to: (1) the need to determine baseline levels for the different biomarkers investigated in this study, (2) the examination of the Moore River or the Warren River estuaries as potential reference sites for biomarker studies in the Swan- Canning Estuary, (3) the advantage of identifying a second estuarine-dependent indigenous fish as a biomonitoring tool, (4) the requirement for a targeted study aimed at clarifying the relationship between major drain discharges, biomarker levels and impacts on river biota, and (5) a study of estuarine waters utilising SPMDs be undertaken in tandem with biomarker analysis of field captured fish would be beneficial.

Pharmaceutical drugs are used extensively by humans and domestic animals. The detection of compounds such as non-steroidal anti-inflammatory drugs (NSAIDs) in effluents of sewage treatment plants and surface waters has raised concerns about whether these drugs have the potential to impact aquatic organisms. However, little is known about either the mechanism of action of NSAIDs or their impact on aquatic organisms. A key indicator of animal stress performance is the elevation in plasma cortisol levels, the primary circulating corticosteroid in teleosts, and the associated metabolic changes in response to stressor exposure. The secretion of cortisol is under the control of the hypothalamus-pituitary-interrenal (HPI) axis with the terminal step involving the activation of interrenal steroidogenesis by adrenocorticotropic hormone (ACTH) from the pituitary gland. Cortisol, predominantly via glucocorticoid receptor (GR) activation, is involved in a wide array of animal functions, including growth and metabolism, osmo- and iono-regulation, stress and immune function and reproduction, all of which play a role in regaining homeostasis after stressor insult. The overall objective of this thesis was to investigate the role of NSAIDs in impacting the evolutionarily-conserved adaptive stress response in a model teleost fish, the rainbow trout (Oncorhynchus mykiss). Specifically, the impact of NSAIDs on stress coping mechanisms was investigated by examining: i) interrenal steroid biosynthetic capacity and cortisol production, ii) target tissue GR function, iii) cellular heat shock protein response, iv) tissue-specific metabolic response to stressors, and iv) ionoregulatory performance in seawater. The experimental approach involved a series of whole animal in vivo and in vitro studies, using rainbow trout interrenal cell preparations, with two NSAIDs, salicylate and ibuprofen, commonly detected in our surface waters. Fish were subjected to stressors of varying intensity and duration, including handling disturbance, heat shock and salinity exposures, to identify targets impacted by NSAIDs in fish. NSAIDs did not affect resting plasma cortisol levels but disrupted the acute ACTH-stimulated corticosteroidogenesis in vitro and stressor-induced plasma cortisol response in vivo. The mode of action of NSAIDs in disrupting cortisol production involves inhibition of the key rate-limiting step, the steroidogenic acute regulatory protein (StAR), in steroidogenesis. Also, tissue (brain, liver and gill) GR protein content is a target for endocrine disruption by NSAIDs leading to abnormal negative feedback regulation of plasma cortisol levels and reduced target tissue responsiveness to cortisol after stressor exposure. The drugs also clearly affected the cellular stress response in rainbow trout by perturbing the expression of heat shock protein 70 (hsp70), a highly conserved stress coping mechanism. This impaired heat shock response with NSAIDs corresponded with an altered tissue metabolic capacity suggesting disturbances in biochemical adjustments to stressor. Specifically, the dynamics of glucose, the primary fuel to cope with the enhanced tissue metabolic demand, was disrupted in a drug-specific manner in rainbow trout. Exposure to NSAIDs also disrupted the ionoregulatory mechanisms critical for seawater acclimation in rainbow trout. The targets for ionoregulatory disturbance in seawater by NSAIDs include the major ion transporter gill Na+/K+-ATPase as well as gill GR, a key signaling protein for Na+/K+-ATPase upregulation in fish. Altogether, NSAIDs disrupt the adaptive endocrine and metabolic stress coping mechanisms in rainbow trout. The targets for endocrine disruption by NSAIDs include multiple sites along the HPI axis as well as target tissue response to cortisol action in fish. Specifically, the mode of action of NSAIDs involves disruption of StAR and GR, two key proteins critical for cortisol production and target tissue responsiveness to this steroid, respectively. While the work presented here identified the mechanism(s) of action of NSAIDs, the environmental relevance of this finding, specifically the impact of concentrations of NSAIDs present in our waterways on fish stress performance, remains to be explored.

Pharmaceutical drugs are used extensively by humans and domestic animals. The detection of compounds such as non-steroidal anti-inflammatory drugs (NSAIDs) in effluents of sewage treatment plants and surface waters has raised concerns about whether these drugs have the potential to impact aquatic organisms. However, little is known about either the mechanism of action of NSAIDs or their impact on aquatic organisms. A key indicator of animal stress performance is the elevation in plasma cortisol levels, the primary circulating corticosteroid in teleosts, and the associated metabolic changes in response to stressor exposure. The secretion of cortisol is under the control of the hypothalamus-pituitary-interrenal (HPI) axis with the terminal step involving the activation of interrenal steroidogenesis by adrenocorticotropic hormone (ACTH) from the pituitary gland. Cortisol, predominantly via glucocorticoid receptor (GR) activation, is involved in a wide array of animal functions, including growth and metabolism, osmo- and iono-regulation, stress and immune function and reproduction, all of which play a role in regaining homeostasis after stressor insult. The overall objective of this thesis was to investigate the role of NSAIDs in impacting the evolutionarily-conserved adaptive stress response in a model teleost fish, the rainbow trout (Oncorhynchus mykiss). Specifically, the impact of NSAIDs on stress coping mechanisms was investigated by examining: i) interrenal steroid biosynthetic capacity and cortisol production, ii) target tissue GR function, iii) cellular heat shock protein response, iv) tissue-specific metabolic response to stressors, and iv) ionoregulatory performance in seawater. The experimental approach involved a series of whole animal in vivo and in vitro studies, using rainbow trout interrenal cell preparations, with two NSAIDs, salicylate and ibuprofen, commonly detected in our surface waters. Fish were subjected to stressors of varying intensity and duration, including handling disturbance, heat shock and salinity exposures, to identify targets impacted by NSAIDs in fish. NSAIDs did not affect resting plasma cortisol levels but disrupted the acute ACTH-stimulated corticosteroidogenesis in vitro and stressor-induced plasma cortisol response in vivo. The mode of action of NSAIDs in disrupting cortisol production involves inhibition of the key rate-limiting step, the steroidogenic acute regulatory protein (StAR), in steroidogenesis. Also, tissue (brain, liver and gill) GR protein content is a target for endocrine disruption by NSAIDs leading to abnormal negative feedback regulation of plasma cortisol levels and reduced target tissue responsiveness to cortisol after stressor exposure. The drugs also clearly affected the cellular stress response in rainbow trout by perturbing the expression of heat shock protein 70 (hsp70), a highly conserved stress coping mechanism. This impaired heat shock response with NSAIDs corresponded with an altered tissue metabolic capacity suggesting disturbances in biochemical adjustments to stressor. Specifically, the dynamics of glucose, the primary fuel to cope with the enhanced tissue metabolic demand, was disrupted in a drug-specific manner in rainbow trout. Exposure to NSAIDs also disrupted the ionoregulatory mechanisms critical for seawater acclimation in rainbow trout. The targets for ionoregulatory disturbance in seawater by NSAIDs include the major ion transporter gill Na+/K+-ATPase as well as gill GR, a key signaling protein for Na+/K+-ATPase upregulation in fish. Altogether, NSAIDs disrupt the adaptive endocrine and metabolic stress coping mechanisms in rainbow trout. The targets for endocrine disruption by NSAIDs include multiple sites along the HPI axis as well as target tissue response to cortisol action in fish. Specifically, the mode of action of NSAIDs involves disruption of StAR and GR, two key proteins critical for cortisol production and target tissue responsiveness to this steroid, respectively. While the work presented here identified the mechanism(s) of action of NSAIDs, the environmental relevance of this finding, specifically the impact of concentrations of NSAIDs present in our waterways on fish stress performance, remains to be explored.

Runoff and effluent discharge from mining activities has resulted in elevated concentrations of selenium in aquatic ecosystems. Bioavailability is dependent on chemical speciation. Although dissolved inorganic Se species are not directly toxic to organisms, uptake by primary producers and subsequent biotransformation to organo-selenium species substantially increase risk and bioaccumulation potential, potentially impairing reproduction in high-order organisms. The effects of dietary selenium exposure were assessed in two aquatic invertebrates: Ceriodaphnia dubia and Chironomus dilutus. Two generations of these organisms were exposed to seleniferous algae grown in 0-40 μg L-1 selenate. Dissolved selenate was readily absorbed and concentrated by algal species Pseudokirchneriella subcapitata and Chlorella kesslerii. Se had no effect on survival at treated concentrations. Dietary selenium exposures had little effect on reproductive endpoints in either invertebrate species, suggesting that invertebrates have the ability to regulate chronic Se exposures from dietary sources. These results provide valuable information concerning the effects of dietary selenium in aquatic invertebrates.

Runoff and effluent discharge from mining activities has resulted in elevated concentrations of selenium in aquatic ecosystems. Bioavailability is dependent on chemical speciation. Although dissolved inorganic Se species are not directly toxic to organisms, uptake by primary producers and subsequent biotransformation to organo-selenium species substantially increase risk and bioaccumulation potential, potentially impairing reproduction in high-order organisms. The effects of dietary selenium exposure were assessed in two aquatic invertebrates: Ceriodaphnia dubia and Chironomus dilutus. Two generations of these organisms were exposed to seleniferous algae grown in 0-40 μg L-1 selenate. Dissolved selenate was readily absorbed and concentrated by algal species Pseudokirchneriella subcapitata and Chlorella kesslerii. Se had no effect on survival at treated concentrations. Dietary selenium exposures had little effect on reproductive endpoints in either invertebrate species, suggesting that invertebrates have the ability to regulate chronic Se exposures from dietary sources. These results provide valuable information concerning the effects of dietary selenium in aquatic invertebrates.

Neuroendocrine processes coordinate the behavioural, physiological, and seasonal aspects of reproduction. Some chemicals can disrupt the hypothalamus-pituitary-gonadal axis, impacting reproductive health. Naphthenic acids (NAs), the carboxylic acids in petroleum, are of emerging concern as they contaminate coastlines after oil spills and aquatic ecosystems of the Athabasca oil sands area. They are acutely toxic in fish and tadpoles and possibly endocrine disrupting at sublethal levels. I characterized courtship behaviours and disruption by NAs in the Western clawed frog, Silurana tropicalis. Courtship primarily consists of males producing low trills and achieving amplexus, a mating position where a male clasps a female. Adult frogs were exposed for five days to 20 mg/L NA, a dose low enough to not affect physical activity. In males, absolute calling activity was reduced. Other acoustic parameters such as dominant frequency, click rate, and trill length were not affected. Injecting human chorionic gonadotropin had a slight rescue effect. Vocalization and amplexus were both inhibited after exposure and restored after 2 weeks of recovery. However, calling behaviour did not predict competitive ability or mating success. In females, NA exposure reduced mating success, possibly through decreased attractiveness or receptivity. Receptivity can be indicated by attraction towards the sound of mating calls (phonotaxis), which is cryptic and subjective. I created an apparatus that measures phonotaxis by placing speakers inside traps with infrared lights to detect the time of entry. This novel method is widely applicable for low-visibility observations and studies of choice and preference. This work shows that an aquatic contaminant can reduce mating success in otherwise healthy frogs, and provides a detailed foundation for further investigation.

Metformin is the most common first-line oral therapeutic agent used in the treatment of type-2 diabetes. Because of its widespread use, metformin has been increasingly detected in wastewater effluent. It is partially bio-transformed into guanylurea is subsequently released into aquatic environments. Since the literature concerning the effect of metformin and guanyl urea on early life stage of fish is scant, the aim of this research was to understand the potential influence of metformin and guanylurea on developmental, cardiometabolic and behavioral responses in zebrafish embryos, from the 4 cell stage (3 hours post fertilization, hpf) to first feed (120 hpf). To this end, embryos were exposed to environmentally relevant (0.4, 4, 40 μg·L−1) and supra-environmental (400 and 4000 μg·L−1) concentrations of the two chemicals. Metformin caused an increased mortality and spinal abnormalities in all concentrations compared to controls. and increased pericardial and yolk sac edema at the highest tested concentration. Metformin did not cause alterations in hatch or heart rate over the examined developmental stages. In addition, metformin did not cause alterations in general swimming, light-dark movement, startle response or thigmotaxis, irrespective of exposure concentration. Exposure to guanylurea over the same developmental stages caused a significant difference in mortality at 40 μg·L−1 only. Guanylurea did not cause alterations to any of the other tested endpoints. Our data suggests that metformin and guanylurea caused modest impacts to embryonic development of zebrafish at these concentrations. / Thesis / Master of Science (MSc) / Pharmaceuticals have been detected at the ng to µg L-1 range in aqueous environments for decades. These compounds are designed to be biologically active at low concentrations and can cause elicit adverse effects in non-target species. Among the more recently detected compounds are the antihyperglycemic drug metformin and its biotransformation product (guanylurea), which have been the focus of few studies in fish. This thesis addresses multiple knowledge gaps by examining the potential impacts of metformin and guanylurea during the embryonic and early larval zebrafish period (3-120 hours post-fertilization). Exposure to metformin resulted in increased mortality and abnormalities. Guanylurea exposure increased mortality at one dose. We suggest that metformin and guanylurea cause modest effects in developing larval zebrafish.