The primary objective of this work was to determine the tissue-specific bioconcentration factors (BCFs) of the selected pharmaceuticals: norethindrone (NET), ibuprofen (IBU), verapamil (VER), clozapine (CLZ) and fenofibrate (FFB) in two freshwater fishes: fathead minnow and channel catfish. BCF tests on fathead followed the standard OECD 42-day test while a 14-day abridged test design was used in catfish exposures. Additional objectives included a) comparing the measured BCFs to the US EPA's BCFWIN model predicted values, b) comparing the BCF results from the standard and reduced tests, and c) prediction of chronic risk of the pharmaceuticals in fish using the human therapeutic plasma concentrations. Each test included uptake and depuration phases to measure tissue-specific kinetic BCFs. The results indicated that all the pharmaceuticals, except IBU, have the potential for accumulation in fish. Estimated BCFs for NET, VER and FFB may not be significant in view of the current regulatory trigger level (BCF ≥ 2000); however, CLZ's BCF in the liver had approached the criterion level. Significant differences were noticed in the tissue-specific uptake levels of the pharmaceuticals with the following general trend: (liver/kidney) > (gill/brain) > (heart/muscle) > plasma. IBU uptake was highest in the plasma. When compared to the measured BCFs, predicted values for NET, IBU, VER and FFB were slightly overestimated but did not differ largely. However, the measured BCF of CLZ in the liver was approximately two-orders of magnitude higher than the predicted level. The tissue-BCFs for the two species were not widely different indicating the potential usefulness of the reduced BCF test. Comparison of fish and human plasma levels indicated that NET, CLZ and VER have the potential to cause chronic effects in fish.

Three suspended solids types containing a range of physicochemical characteristics were used to determine the effect of suspended solids on the bioavailability of acenaphthene, 1,2,4,5-tetrachlorobenzene, zinc, and chlordane to Daphnia magna and Pimephales promelas. Generally, the bioavailability of zinc and chlordane decreased due to interactions with all suspended solids types while bioavailability of acenaphthene and 1,2,4,5-tetrachlorobenzene were not clearly reduced. Partition coefficients and slope of dose-response curves related chemical characteristics and organism sensitivity, respectively, to experimentally determined results. It is believed that the biologically available form of these chemicals to Daphnia magna and Pimephales promelas resides in the aqueous phase.

Due to a policy of no release, oil sands process-affected water (OSPW) produced by the surface-mining oil sands industry in North Eastern Alberta, Canada, is stored on-site in tailings ponds. There is concern regarding the toxic effects of OSPW on aquatic organisms. Knowledge of the chemical composition and toxicity of OSPW is limited. Research is necessary for potential remediation and release of OSPW back into the environment. Due to the large volume and persistency of OSPW, active efforts are necessary for the remediation of OSPW before release and habitat reclamation. Currently, ozonation is considered one possible method for remediation of OSPW by reducing the concentrations of dissolved organic compounds, including naphthenic acids (NAs), which are considered among the primary toxic constituents. However, further work is needed to evaluate the effectiveness of ozonation in reducing the toxicity of OSPW and to ensure that ozonation does not increase the toxicity of OSPW. The overall objective of this work was to determine the toxic effects of OSPW on endocrine disruption and embryo development, using both in vitro and in vivo models, and the effectiveness of ozone treatment for reducing the toxicity of OSPW. In the first study, untreated and ozone-treated OSPW were examined for effects on sex steroid production using the H295R cell line steroidogenesis Assay. The results indicate that exposure to untreated OSPW can significantly decrease synthesis of testosterone (T) and increase synthesis of 17β-estradiol (E2) by 0.55±0.06 and 2.0±0.13-fold, respectively, compared to that of control groups (ρ < 0.05). These effects were due to increased aromatase enzyme activity and decreased E2 metabolism. The results also suggest that ozonation is an effective treatment to reduce concentrations of NAs in OSPW without altering steroidogenesis. In the second study, the T47D-kbluc (estrogen responsive) and MDA-kb2 (androgen responsive) cell assays were used to determine whether OSPW might act as either agonists or antagonists of the estrogen receptor (ER) or androgen receptor (AR), respectively. The estrogenic responses to untreated OSPW were significantly greater by 2.6±0.22-fold compared to control group (ρ < 0.05). Exposure to untreated OSPW produced significant antiandrogenic response in the presence of 0.01, 0.05 and 0.1 nM T by 16±6.5%, 47±7.6% and 75±9.7%, respectively, of that of the corresponding concentrations of T alone (ρ < 0.05). The results suggest that compounds in the dissolved organic fraction of OSPW have estrogenic and anti-androgenic properties, acting as ER agonists and/or AR antagonists. Ozonation of the OSPW partially mitigated the antiandrogenicity but had no effect on the estrogenicity of OSPW. In the third study, the endocrine-disrupting effects of OSPW and ozone-treated OSPW were determined by quantifying relative changes in the abundances of transcripts of genes along the brain-gonad-liver (BGL) axis in male and female fathead minnows (Pimephales promelas). The results indicate that OSPW has endocrine-disrupting effects at all levels of BGL axis and these effects of impaired expression of genes along the BGL axis are sex specific. For example, exposure to OSPW resulted in significantly greater abundances of transcripts of vtg (Vitellogenin), chg-l (Choriogenin L) and chg-h (Choriogenin H minor) by 4.9±1.2, 5.4±1.5 and 3.4±0.78-fold, respectively, compared to those of control groups (ρ < 0.05) in livers from male fathead minnow. However, in livers from female fathead minnows, exposure to OSPW resulted in significantly lesser abundances of transcripts of vtg, chg-l and chg-h by 0.002±0.0011, 0.022±0.007 and 0.036±0.024-fold, respectively, compared to those of control fish (ρ < 0.05). Ozonation of OSPW attenuated the effects on abundances of transcripts of some genes, and the attenuation was more prominent in males than in females. However, impact of ozonation on endocrine-disrupting effects of OSPW was less evident than in the in vitro studies described in Chapter 2 and 3. The results also provide a mechanistic basis for the endocrine-disrupting effects of OSPW from other studies, including impaired reproduction of fathead minnows exposed to OSPW. In the final study the effects of untreated, ozone-treated, and activated charcoal-treated OSPW (OSPW, O3-OSPW, and AC-OSPW) on the survival, growth, and development of embryos of fathead minnows were determined. Compared to the control group, which had an embryo survival rate of 98±2.1%, survival was significantly less when exposed to OSPW (44±7.1%; ρ < 0.05). Eggs exposed to untreated OSPW exhibited a significantly greater rate of premature hatching, and embryos exhibited more frequent spontaneous movements. Incidences of hemorrhage (50±3.4%), pericardial edema (56±7.1%), and malformation of the spine (38±5.4%) were significantly greater in embryos exposed to OSPW compared to control group (ρ < 0.05). Significantly greater concentrations of ROS (1.7±0.11-fold), and greater abundances of transcripts cyp3a, gst, sod, casp9, and apopen (2.4±0.34, 2.2±0.26, 3.1±0.74, 3.3±0.57 and 2.4±0.25-fold, respectively) compared to control groups (ρ < 0.05), indicated that exposure to OSPW caused oxidative stress, which can result in damage to mitochondria and promote activation of caspase enzymes and apoptotic cell death. Removal of dissolved organic constituents in OSPW by ozone treatment, or by activated charcoal, significantly attenuated all of the adverse effects associated with untreated OSPW. The results suggest that the organic fraction of OSPW can negatively impact the development of fathead minnow embryos through oxidative stress and apoptosis, and that ozonation attenuates this developmental toxicity. Overall, the findings from the research described in this thesis provide novel and important insights into the toxicity and mechanisms of the toxicity of OSPW with respect to endocrine disruption and development of embryos of fish. In addition, the research provides compelling evidence that ozonation might be an effective method for accelerating the remediation of OSPW. The results of the research might help regulators develop effective strategies for reclamation, remediation and potential release of OSPW back to the environment.

The overall objective of the work described in this thesis was to examine the effects of both waterborne and dietary routes of exposure to fathead minnow (Pimephales promelas) when exposed to complex metal mining mixtures. This was conducted using a 21-day, multi-trophic, short-term fathead minnow (FHM) reproductive bioassay. The endpoints that were measured were used to assess the effects on multiple levels of biological organization (sub-organismal to population endpoints). The first phase of this research was conducted in situ using environmentally realistic concentrations of 3 separate metal mining effluents [20% surface water effluent (SWE), 30% mine water effluent (MWE), 45% process water effluent (PWE)] from Sudbury, Ontario, Canada. Metals were analyzed in several media (water, sediments) and tissues (biofilm, Chironomus dilutus, female fathead minnow carcass, ovaries, liver and gills). The incorporation of the biofilm (primary producers) into the bioassay also added another level of organization that was novel to this study. Significant increases in metal concentrations were observed in the water and biofilm tissues in all treatments [SWE, MWE, PWE], compared to reference. Cobalt and nickel increased significantly in C. dilutus tissues in SWE (1.4-fold and 1.5-fold respectively), and copper and selenium in PWE (5.2-fold and 3.3-fold respectively), however no significant increases occurred in MWE compared to reference. There were no significant increases in metal concentrations in female FHM tissues (carcass, liver, gonads, gills) in any of the treatments, suggesting that metal bioavailability was reduced. Cumulative number of eggs per female per day increased significantly (+127%) after exposure to SWE and decreased significantly (-33%) after exposure to PWE when compared to the reference fish. Mean total number of days to hatch was also reduced in PWE compared to reference. In order to gain a better understanding of the routes of exposure causing toxicity in FHM, the second phase of this research examined the effects of exposure through diet, through water or through both using a fully factorial food exposure design in a laboratory setting. In this experiment we pre-exposed C. dilutus to both 45% PWE and laboratory control water until they reached the 3rd-4th instar stage of development (approximately 21 days) where they were collected and frozen until the start of the FHM reproductive bioassay. We further examined the role of food quality on fish toxicity by assessing differences between multi trophic (where fish were fed both a live and frozen diet of C. dilutus) in the laboratory. This research was conducted at the Toxicology Centre in Saskatoon, Saskatchewan, Canada. The results showed that significant effects were observed when fish were fed a live diet versus a frozen diet. Condition factor and body weight increased, although inconsistent effects were observed for liver somatic index (LSI) in fathead minnows in both experiments when exposed to one or both routes of exposure. Cumulative total egg production and cumulative spawning events were both significantly affected by both waterborne and dietborne exposures with the greatest effects seen in the multi-trophic streams and particularly when fish were fed a live diet. This significance of this research has demonstrated the importance of including both routes of exposure when assessing effects of mine effluent. This research also shows that the artificial stream technology is a useful tool in isolating the effects of a particular point source input (metal mining mixtures) when a system is highly confounded. The results suggest that under environmentally relevant exposure conditions, trophic transfer and live diet may lead to greater reproductive effects and increased fish toxicity. This also suggests that trophic transfer is an important route of exposure that is virtually impossible to attain using typical laboratory bioassay techniques (food-borne study using artificial diets or waterborne exposures only).

Throughout their lives, prey organisms must balance the tradeoff between fitness-related activities and the risk of predation. To successfully mediate such tradeoffs, prey must have an accurate method to gauge current predation risk. For many aquatic organisms, the use of chemosensory information has been shown to be a ubiquitous and useful tool in mediating predation risk. The chemical cues to which aquatic organisms respond include the odour of known predators and the odour of a damaged conspecific or known or closely related heterospecific. In fishes, the response to damage-released cues from conspecifics or closely related heterospecifics has been shown to be innate, while the response to distantly related unknown heterospecific cues are likely learned. In a series of laboratory and field studies I examined the role of learning in the ability of fathead minnows to respond to damage-released cues of brook stickleback as an indication of predation risk. My results indicate that minnows from a population without stickleback do not recognize stickleback cues as dangerous. However, following the introduction of stickleback, minnows learn to recognize stickleback cues as dangerous. Further study indicated a low ratio of stickleback to minnows in a given population will decrease the likelihood of learning when compared with a similar sized population containing a higher ratio of stickleback to minnows. I also demonstrated that an increase in habitat complexity decreases the ability of minnows to learn to recognize stickleback cues. Studies have further demonstrated that in the face of predation (as indicated by chemical cues from minnows and stickleback) minnows will decrease their antipredator response when in the presence of a fish shoal, especially a shoal of conspecifics. Finally, an examination of the effects of a minnows length, body condition and breeding status indicate that morphological parameters can play a significant role in the intensity of response to heterospecific and conspecific damage-released cues. Previous works have demonstrated that behavioural responses to heterospecific damage-released cues increase the probability of the receiver surviving a predation event. This has important implications for predator/prey interactions and consequently factors affecting the learning and use of such cues deserve increasing attention from ecologists.

Previous studies at the Key Lake uranium mill (Saskatchewan, Canada) suggested the complex effluent discharged alters energetic stores of resident fish species. A second study at the same site demonstrated certain fish from lakes downstream of the mill produce larvae with elevated incidence of developmental deformities. The mechanisms by which energy homeostasis is affected in fish downstream of the Key Lake uranium mill are unknown, and the effects of deformities and altered metabolism on swimming ability have not been explored. Therefore, the overall objective of this thesis was to investigate whether effluent exposed fish exhibited differences in swimming performance and energy homeostasis. To achieve this objective two experiments were conducted. In the first experiment juvenile spottail shiner (Notropis hudsonius) were collected from a lake downstream of the Key Lake uranium mill, and compared to fish collected from a nearby reference lake. In the second experiment larvae were collected from laboratory raised fathead minnow (Pimephales promelas) exposed to 5% diluted uranium mill effluent or control (dechlorinated municipal) water, and reared in the same treatments to 60 days post hatch (dph). No gross deformities were observed in any fish, and only shiner collected from the exposure lake in the field experiment had enlarged heart ventricles relative to body size compared to fish from the reference lake. Swimming performance was similar between shiner from the exposure and reference lakes in the field study, but effluent exposure impaired swimming ability in 60 dph fathead minnow in the laboratory experiment compared to fish from the control water treatment. After swimming performance tests fish were considered fatigued and metabolic endpoints were compared to non-fatigued fish. In both non-fatigued and fatigued shiner, liver glycogen was significantly greater in fish collected from the exposure lake compared to the reference lake. There was no difference in liver triglycerides in non-fatigued shiner between lakes, but liver triglycerides decreased after swimming in the field study reference fish. Muscle energy stores were unaffected by site or swimming in the field experiment. Conversely, whole body triglycerides and glycogen were similar between treatments in non-fatigued fathead minnow in the laboratory experiment. Swimming significantly decreased whole body triglycerides in fathead minnow from both treatments, but whole body glycogen was unaffected. In the field experiment blood endpoints (hematocrit, plasma glucose, lactate) in fatigued and non-fatigued shiner from both lakes further supported the possibility of altered intermediary metabolism or blunted stress response in fish downstream of the Key Lake uranium mill. In the field study, shiner muscle citrate synthase activity (an indicator of tissue aerobic capacity) was similar between lakes, but muscle âhydroxyacyl coenzyme A dehydrogenase activity (an indicator of tissue lipolytic capacity) was elevated. In contrast, laboratory fathead minnow whole body âhydroxyacyl coenzyme A dehydrogenase activity was similar between treatments, but citrate synthase activity was significantly lower in fathead minnow from the 5% effluent treatment. In summary, shiner from the exposure lake in the field experiment had similar swimming endurance and greater energy stores compared to fish from the reference lake, despite metabolic alterations. Fathead minnow from the 5% effluent treatment in the laboratory experiment had reduced swimming endurance that was matched by reduced whole body citrate synthase activity, but no other metabolic alterations were observed. Therefore, effluent exposure caused metabolic alterations in both fathead minnow and shiner, but specific effects between experiments were inconsistent. Overall, the physiological significance of the metabolic and swimming effects of effluent exposure is unclear, but suggests discharged effluent has the potential to negatively affect wild fish survivability.

The overall objective of the research conducted and described in this thesis was to develop an environmentally relevant bioassay to assess the effects of complex effluents on a sentinel fish species. A short-term fathead minnow (FHM) reproductive bioassay was utilized to assess the effects of industrial effluents on multiple levels of biological organization (sub-organismal to population endpoints). The FHM bioassay was tested in both lab and on-site investigations using an artificial stream system. The incorporation of trophic-transfer into the bioassay was also developed to quantify the importance of contaminated food as a source of exposure. This work was conducted in two key phases. Phase I focused on testing and developing the FHM bioassay, in the lab and on-site with pulp mill effluent (PME), to firstly document response patterns and, secondly, to conduct an investigation of cause study. Phase II focused on developing the trophic-transfer system to document responses to metal mine effluent (MME) in the lab and on-site in an artificial stream system. Development of the trophic-transfer system was also conducted during this phase to compare responses to standard water-only exposures. <p>In Phase I, exposure to PME in both the lab and field studies resulted in disruptions in egg production and spawning events. By focusing on identifying response patterns I was able to determine that the effects observed were indicative of an estrogenic response. I was also able to identify a process stream that was the potential cause of responses observed after exposure to final effluent. Isolation of this process stream will assist the mill in developing approaches for future mitigation. The results from this research will also provide additional data for the environmental effects monitoring (EEM) program for pulp and paper and investigation of cause studies on a national basis. <p> In Phase II, in both the field and laboratory investigations, significant decreases in reproductive output (egg production and spawning events) were observed in the water-only system exposures. Significant decreases in hatching success and increases in deformities were observed in the trophic-transfer system only, suggesting that the combination of both food and water was important in assessing the effects on the F1 generation. Overall, the responses in the trophic-transfer system were not comparable between the lab and field studies. In the lab study, significant decreases in reproductive output occurred, compared to the field study where significant increases in egg production and spawning events occurred. In addition, the effects on the F1 generation in the field study were not as severe as those observed in the lab investigation. It was concluded that the presence of reference water and the environment within the trophic-transfer system were responsible for this reduction in toxicity. <p> Phases I and II of this research have made significant contributions to artificial stream development within Canada for the assessment of industrial effluents and their effects on aquatic biota. The results from these studies have also demonstrated that environmentally relevant testing is essential if we are to accurately assess effects on aquatic biota. Future development and application of this bioassay should be towards developing a standardized approach for not only assessing the effects of industrial effluents in a comparative manner, but also in investigation of cause studies.

Metal mines release treated effluents that contain a variety of metals, metalloids, and organics into the aquatic environment. A number of metal mine effluents (MMEs) have been found to contribute to adverse effects in fish and benthic invertebrates, such as decreased diversity and density, however the specific causal factors of toxic responses during chronic exposures to the MMEs are often unknown. Therefore, the overall objective of this dissertation was to explore causative and modifying factors of MME toxicity to a resident fish species, the fathead minnow (Pimephales promelas), during chronic, multi-trophic exposures. The representative MME used in this study was the process water effluent (PWE) of a Canadian metal mine, which is released into Junction Creek in Sudbury, Ontario, Canada. Chronic exposure to the MME has been a source of decreased reproductive output in fathead minnows in several previous studies, however, these same studies were not able to determine the potential causal factors of the reproductive impairment. In order to address the overall objective, several laboratory mesocosm studies were conducted, which consisted of three separate components. The first component included exploring several metals (Cu, Ni, and Se; alone and in mixture) that are consistently present in the MME and are known to cause toxicity at fairly low concentrations as potential causes for decreased egg production in fathead minnows. The second component included evaluating the role of decreased food availability (a possible indirect effect of MME in the receiving environment) as a potential cause of decreased egg production in fathead minnows. The third and final component included examining the role of water chemistry [(increased alkalinity and dissolved organic carbon (DOC)] as potential modifying factors of chronic MME toxicity to fathead minnows. In general, my results suggest that the metals present in the MME likely do not contribute directly to decreased reproductive performance in fathead minnows during chronic exposures, under the conditions examined. Instead, the MME appears to decrease food availability, therefore indirectly influence fathead minnow egg production. Furthermore, water chemistry modifications tested in this thesis were not able to entirely mitigate the reproductive effects in fish induced by the MME, although they did improve egg production relative to unmodified MME. Metal concentrations in fish tissues were not influenced by increases to alkalinity or DOC level in the exposure water, suggesting that bioavailability of metals during chronic exposure to metal-mixtures cannot be fully explained based on our understanding of metal complexation with abiotic ligands (inorganic and organic) during single metal or acute exposures. From a regulatory perspective, water chemistry modifications may somewhat improve fathead minnow reproductive performance during chronic exposure to the MME, however the MME would still not be entirely free of effects relative to the uncontaminated water. Future studies should focus on understanding the factors responsible for decreased food availability in MME-impacted aquatic ecosystems, and further explore potential approaches for ameliorating effluent quality.

Throughout their lives, prey organisms must balance the tradeoff between fitness-related activities and the risk of predation. To successfully mediate such tradeoffs, prey must have an accurate method to gauge current predation risk. For many aquatic organisms, the use of chemosensory information has been shown to be a ubiquitous and useful tool in mediating predation risk. The chemical cues to which aquatic organisms respond include the odour of known predators and the odour of a damaged conspecific or known or closely related heterospecific. In fishes, the response to damage-released cues from conspecifics or closely related heterospecifics has been shown to be innate, while the response to distantly related unknown heterospecific cues are likely learned. In a series of laboratory and field studies I examined the role of learning in the ability of fathead minnows to respond to damage-released cues of brook stickleback as an indication of predation risk. My results indicate that minnows from a population without stickleback do not recognize stickleback cues as dangerous. However, following the introduction of stickleback, minnows learn to recognize stickleback cues as dangerous. Further study indicated a low ratio of stickleback to minnows in a given population will decrease the likelihood of learning when compared with a similar sized population containing a higher ratio of stickleback to minnows. I also demonstrated that an increase in habitat complexity decreases the ability of minnows to learn to recognize stickleback cues. Studies have further demonstrated that in the face of predation (as indicated by chemical cues from minnows and stickleback) minnows will decrease their antipredator response when in the presence of a fish shoal, especially a shoal of conspecifics. Finally, an examination of the effects of a minnows length, body condition and breeding status indicate that morphological parameters can play a significant role in the intensity of response to heterospecific and conspecific damage-released cues. Previous works have demonstrated that behavioural responses to heterospecific damage-released cues increase the probability of the receiver surviving a predation event. This has important implications for predator/prey interactions and consequently factors affecting the learning and use of such cues deserve increasing attention from ecologists.

The overall objective of the research conducted and described in this thesis was to develop an environmentally relevant bioassay to assess the effects of complex effluents on a sentinel fish species. A short-term fathead minnow (FHM) reproductive bioassay was utilized to assess the effects of industrial effluents on multiple levels of biological organization (sub-organismal to population endpoints). The FHM bioassay was tested in both lab and on-site investigations using an artificial stream system. The incorporation of trophic-transfer into the bioassay was also developed to quantify the importance of contaminated food as a source of exposure. This work was conducted in two key phases. Phase I focused on testing and developing the FHM bioassay, in the lab and on-site with pulp mill effluent (PME), to firstly document response patterns and, secondly, to conduct an investigation of cause study. Phase II focused on developing the trophic-transfer system to document responses to metal mine effluent (MME) in the lab and on-site in an artificial stream system. Development of the trophic-transfer system was also conducted during this phase to compare responses to standard water-only exposures. <p>In Phase I, exposure to PME in both the lab and field studies resulted in disruptions in egg production and spawning events. By focusing on identifying response patterns I was able to determine that the effects observed were indicative of an estrogenic response. I was also able to identify a process stream that was the potential cause of responses observed after exposure to final effluent. Isolation of this process stream will assist the mill in developing approaches for future mitigation. The results from this research will also provide additional data for the environmental effects monitoring (EEM) program for pulp and paper and investigation of cause studies on a national basis. <p> In Phase II, in both the field and laboratory investigations, significant decreases in reproductive output (egg production and spawning events) were observed in the water-only system exposures. Significant decreases in hatching success and increases in deformities were observed in the trophic-transfer system only, suggesting that the combination of both food and water was important in assessing the effects on the F1 generation. Overall, the responses in the trophic-transfer system were not comparable between the lab and field studies. In the lab study, significant decreases in reproductive output occurred, compared to the field study where significant increases in egg production and spawning events occurred. In addition, the effects on the F1 generation in the field study were not as severe as those observed in the lab investigation. It was concluded that the presence of reference water and the environment within the trophic-transfer system were responsible for this reduction in toxicity. <p> Phases I and II of this research have made significant contributions to artificial stream development within Canada for the assessment of industrial effluents and their effects on aquatic biota. The results from these studies have also demonstrated that environmentally relevant testing is essential if we are to accurately assess effects on aquatic biota. Future development and application of this bioassay should be towards developing a standardized approach for not only assessing the effects of industrial effluents in a comparative manner, but also in investigation of cause studies.