Fish skin is a metabolically active tissue that responds quickly to stressors and is the first line of defence against physical damage. Club cells, characteristic components of Ostariophysian fish skin, release their contents into the surrounding water upon rupture (e.g. during predation). These chemical cues act as public information of predation risk. Despite the assumption that club cells evolved under the selective force of predation, studies demonstrated that predation has no effect on club cell investment. Rather, club cell production is stimulated by skin penetrating pathogens and parasites. The experiments in this thesis investigate the responses of fish skin to manipulated pathogen risk. In the first experiment, fathead minnows (Pimephales promelas) were exposed to varying infective risk from two pathogen species that differ in pathogenicity, Saprolegnia ferax and S. parasitica. Although there was no difference in club cell density between fish exposed to the two Saprolegnia species, fish exposed to high concentrations of the pathogens had smaller club cells than those exposed to low concentrations. These results are the first to demonstrate a pathogen effect on the size of club cells. The second experiment investigated whether the physical presence of the pathogen was necessary for an alteration in epidermal parameters or whether Saprolegnia parastitica conditioned water was the only stimulus necessary to evoke a change. Results indicated a lack of treatment effect on club cell density, club cell size or epidermal thickness. The third experiment investigated the timing of club cell changes following a pathogen challenge. Although fish exposed to the Saprolegnia ferax treatment had higher club cell density than fish exposed to the control, there was no difference in club cell density between fish sacrificed on day 3, 6, 9 or 12. A portion of the test population for the third experiment was infected with black spot disease. When analyzed separately, trematode infected fish had smaller club cells than those that were uninfected. In light of inconsistent epidermal responses to pathogen challenges, and comparison with other studies, assessment of environmental stressors and population differences that may affect experimental outcomes and potentially interact with infectious agents is advised.

Cadmium (Cd) is a priority pollutant in ecosystems worldwide. It is highly toxic to aquatic organisms including fish at fairly low concentrations. Numerous studies have investigated the influence of Cd exposure on fish, but few of them have considered how environmentally relevant levels of Cd affect reproduction, particularly reproductive behaviour. To assess the toxicity of Cd on fish reproduction, breeding fathead minnows (Pimephales promelas) were exposed to water-borne Cd for 21 days at four different concentrations (0, 1, 2.5 and 5 µg/L, respectively) based on a standard short-term reproductive assay and reproductive performance as well as behaviour were examined during or at the end of the exposure period. The results showed that Cd accumulated in a dose-dependent manner in the livers and ovaries of female fish. Brood size and mean egg production were significantly reduced in Cd-exposure treatment groups. When fertilized eggs were incubated in the water containing 2.5 µg/L or higher Cd, there was delayed hatching, but at the same time there was greater synchronous hatching after hatching started. Hatching success of Cd-exposed eggs also declined compared to the control. No significant difference was observed among treatments in adult fish survival, the number of breeding attempts, fertilization success, egg size, plasma β-estradiol levels of female, larval deformities, reproductive behaviour, gonadosomatic index or liver somatic index. The results of this study demonstrate that Cd is able to impair reproduction of fathead minnow at the concentration as low as 0.64 µg/L. It is harmful to both breeding fish and their offspring. The traditional endpoints used in standard reproduction assay (e.g. egg production and brood size) are probably more sensitive than behavioural endpoints, but the traditional method of interpreting reproductive impairment may underestimate toxic effects. The findings of this study have important implications for understanding the effects of chronic Cd exposure in metal-impacted feral fish populations. It can be applied to the protection or restoration of fish populations in Cd contaminated aquatic systems.

Fish skin is a metabolically active tissue that responds quickly to stressors and is the first line of defence against physical damage. Club cells, characteristic components of Ostariophysian fish skin, release their contents into the surrounding water upon rupture (e.g. during predation). These chemical cues act as public information of predation risk. Despite the assumption that club cells evolved under the selective force of predation, studies demonstrated that predation has no effect on club cell investment. Rather, club cell production is stimulated by skin penetrating pathogens and parasites. The experiments in this thesis investigate the responses of fish skin to manipulated pathogen risk. In the first experiment, fathead minnows (Pimephales promelas) were exposed to varying infective risk from two pathogen species that differ in pathogenicity, Saprolegnia ferax and S. parasitica. Although there was no difference in club cell density between fish exposed to the two Saprolegnia species, fish exposed to high concentrations of the pathogens had smaller club cells than those exposed to low concentrations. These results are the first to demonstrate a pathogen effect on the size of club cells. The second experiment investigated whether the physical presence of the pathogen was necessary for an alteration in epidermal parameters or whether Saprolegnia parastitica conditioned water was the only stimulus necessary to evoke a change. Results indicated a lack of treatment effect on club cell density, club cell size or epidermal thickness. The third experiment investigated the timing of club cell changes following a pathogen challenge. Although fish exposed to the Saprolegnia ferax treatment had higher club cell density than fish exposed to the control, there was no difference in club cell density between fish sacrificed on day 3, 6, 9 or 12. A portion of the test population for the third experiment was infected with black spot disease. When analyzed separately, trematode infected fish had smaller club cells than those that were uninfected. In light of inconsistent epidermal responses to pathogen challenges, and comparison with other studies, assessment of environmental stressors and population differences that may affect experimental outcomes and potentially interact with infectious agents is advised.

Endocrine disrupting compounds and pesticides have been detected in rivers and irrigation canals of Southern Alberta, a semiarid region with irrigation-dependent crop production, intensive livestock operations, and a growing human population. However, little is known about the effects of agricultural runoff or wastewater treatment plant (WWTP) effluent in Southern Alberta on fish. Reproductive effects of WWTP effluents from the cities of Lethbridge and Medicine Hat, as well as agricultural runoff in the Lethbridge Northern Irrigation District canals, were investigated in a field study with wild fathead minnows (FHMN) in the Oldman and the South Saskatchewan rivers, in Alberta, Canada, and in a laboratory study with laboratory reared FHMN exposed in vivo to the city of Lethbridge WWTP effluent for 21 days. Biochemical and morphological endpoints were measured to characterize reproductive status. Liver vitellogenin, a biomarker of exposure to estrogen mimics, was analyzed using quantitative RT-PCR, and gonadal histology was used to determine sex, gonadal maturity, and intersex. Adverse reproductive effects were detected in FHMN exposed for 21 days to 10 and 25% of Lethbridge WWTP effluent. In the field, effluents from both Lethbridge and Medicine Hat had an effect on the reproductive systems of FHMN. In canals, reproductive effects were detected in wild fathead minnows in years when water quality in irrigation drain canals decreased. Exposure to pesticides was estimated using acetylcholinesterase (AChE) inhibition. Exposure to Lethbridge WWTP effluent did not inhibit AChE, whereas results from the field study were inconclusive. In conclusion, reproductive systems of fathead minnows in Southern Alberta were impacted by anthropogenic chemicals. / xi, 104 leaves : ill. (some col.), maps ; 29 cm

The oil sands, located in north-eastern Alberta, are one of the largest deposits of oil worldwide. Because the Alberta Environmental Protection and Enhancement Act prohibits the release of oil sands process-affected material into the environment, industry is storing vast quantities of tailings on mine lease sites. The oil sands industry is currently accumulating tailings waste at a rate of >105 m3/day, for which reclamation strategies are being investigated. Naphthenic acids (NAs) have been identified as the most toxic component of oil sands tailings as they are considered acutely toxic to a variety of biota, and are therefore a target contaminant for tailings pond reclamation strategies. Current literature based on Microtox® assays (marine bacteria Vibrio fischeri) suggests that lower molecular weight NAs are more toxic than higher molecular weight NAs. The following thesis involves the utilization of NA fractions and their relative toxicities to determine if NA toxicity is related to NA molecular weight. A previous study generated an oil sands-derived naphthenic acid extract (NAE), which was fractionated by distillation at stepped temperatures, yielding five fractions with increasing median molecular weights (Daltons). In the present study, the same extract and five fractions were utilized. To expand on the earlier characterization which involved a low resolution electrospray ionization mass spectrometry (ESI-MS), the whole extract and five fractions were analysed using electrospray ionization high-resolution mass spectrometry (ESI-HRMS) and synchronous fluorescence spectroscopy (SFS). Mean molecular weights were generated for each fraction, and an increase in molecular weight with increasing fraction number was confirmed. Respective mean Daltons and relative proportions for each fraction are as follows: 237 and 11.9 % (fraction 1), 240 and 32.3% (fraction 2), 257 and 33.4% (fraction 3), 308 and 16.8% (fraction 4), and 355 and 5.6% (fraction 5). When chemical analyses of fractions were compared, it was determined that structures contributing to increased molecular weight included increased cyclic structures (up to 7-ring structures), aromaticity (mono- and diaromatics), nitrogen, sulfur, and oxygen heteroatoms, and dihydroxy/dicarboxy compounds. In addition, characterization data suggested the presence of NAs exhibiting estrogenic structures. Following chemical characterization, NA fractions were subject to embryo/larval bioassays using two fish species: Oryzias latipes (Japanese medaka) and Pimephales promelas (fathead minnow). Endpoints evaluated were mortality, time to hatch, hatch length, and abnormalities. Results suggest that relative NA fraction toxicity is not related to molecular weight, as no trend relating mean Dalton weight to toxicity was observed for any endpoint in both species. Acute toxicity data indicated differences between fractions as high as 2-fold, although results were species-dependent. Fraction 1 displayed the lowest potency (highest LC50) for both Japanese medaka (0.291 mM) and fathead minnow (0.159 mM). Fractions 3 and 2 for Japanese medaka (0.149 and 0.157 mM, respectively), and fractions 5 and 2 for fathead minnow (0.061 and 0.080 mM, respectively) displayed the greatest potencies for mortality (lowest LC50). When fraction LC50s for Japanese medaka were compared to the whole NAE (0.143 mM), the mid molecular weight fractions (fractions 2 and 3) appeared most similar to the whole NA. . In terms of relative toxicity and proportion, constituents in the mid molecular range fractions (2 and 3) likely represent greater risk compared to other fractions, and further chemical and toxicological characterization of constituents within these fractions is warranted particularly for long-chained, monocarboxylic acids, with low aromaticity. Japanese medaka and fathead minnow varied in their sensitivity and their relative response to different fractions. In general, fathead minnow were more sensitive than Japanese medaka based on lower estimates of LC50 and threshold (growth) values in addition to the presence of developmental abnormalities (predominately yolk sac edema) associated with a few of the fractions. Compared to differences in toxicity between fractions for a given species (>2-fold for fathead minnow), there was more variability between species for a given fraction (> 3-fold for fraction 5). Also, the relative toxicity of fractions as indicated in the present study is contrary to the results generated using Vibrio fischeri for the same fractions. Thus, there is a need for multi- endpoint and species toxicity evaluations to assess the efficacy of remediation and reclamation options for reducing toxicity of oil sands tailings.

A number of pharmaceuticals have been shown to have adverse effects on key biological processes of aquatic organisms at low concentrations (ng/l range). Key questions for chemical risk assessment are whether such pharmaceuticals can produce adverse effects on organisms when present in the environment in combination and at low concentrations, whether these can be classified as additive, and under what circumstances can they be predicted. The main purpose of this study was to assess the potential for combination effects of a multicomponent steroid pharmaceutical mixture of dissimilarly acting compounds on an ecologically relevant end point – reproduction, using the existing predictive toxicity models Concentration Addition (CA) and Independent Action (IA). Concentrations of steroids close to those reported in the environment were shown to produce adverse effects on reproduction when present in combination with other steroids. Clear combination effects significantly larger than the effects of the individual compounds were observed when each compound was present at a concentration below the detection limit of the assay, demonstrating a ‘something from nothing’ mixture effect. Furthermore, IA predicted more pronounced effects on egg production that CA, an observation previously unreported from the literature. Actual observed effects were closer to the IA prediction. Additional biomarker and molecular endpoints were examined in subsequent studies to establish the mechanisms of disrupted reproduction in pair-breeding FHMs exposed to the steroid mixture. Results from this study indicate that reproductive impairment of fish exposed to the steroid mixture is likely due to the masculinisation of female fish due to the androgenic activity of the mixture. These results have implications for chemical risk assessment, and in particular, highlight the need for caution when using CA as a worst-case approximation of mixture effects.

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Single species toxicity tests are widely used to assess the potential effects of a toxicant on aquatic life. Increasingly, it is necessary to understand how the results of these tests relate to toxicant effects in natural communities. This dissertation presents the methodology and validation for a community level toxicity test that bridges the gap between single species tests and natural community responses. The research involved control of environmental parameters, improvement of feeding regimes and testing of the final community. The results are presented as four separate papers. The first paper addresses the development and validation of a standardized reconstituted water for culturing and toxicity testing of algae, cladocerans, a rotifer and two fish species. The next two papers address the substitution of the food source currently used in the fathead minnow survival-growth impairment test (<i>Artemia</i>) with a freshwater food source (the rotiter, <i>Brachionus calyciflorus</i>). Along with the alga <i>Chlorella vulgaris</i> (producer), B. calyciflorus (primary consumer) and the fathead minnow larvae (secondary consumer) comprise a three level food chain that was used to address trophic level interactions (feeding reduction and growth impairment) in the final phase of this research. The end result is an experimental procedure in which environmental parameters (water quality, temperature, etc.) and trophic structure parameters (Le. producer and primary consumer density) can be controlled well enough to insure that any shifts in community structure can be attributed to toxicant related effects. / Ph. D.

Toxic effects of the pesticide Lindane on the eggs, yolksac fry and larvae of the fathead minnow (Pimephales promelas) were investigated over 14 days at 25 and 18°C. The general objective was to understand the effects of the pesticide on the early life stages of the fish, and to evaluate the suitability of these stages as indicators of contaminated environments. Egg, yolk-sac and larval stages that had been exposed to 300, 120, 90, 60, 30, 10, 5, and 0 μg/L of Lindane at 25°C exhibited a 14d-LC₅₀ of 44 μg/L. Similarly, the same stages exposed to 300, 120, 90, 60, 30, 10, and 0 μg/L of Lindane at 18°C exhibited a 14d-LC₅₀ of 37 μg/L. Lower temperature thus seemed to increase the susceptibility of the fish to the pesticide. Mortality data were also used to determine the sensitivity of the early developmental stages of fish to the pesticide. At 25°C, the larval stage was the most sensitive stage. At 18°C, on the other hand, the most sensitive stage was the yolk-sac. Results obtained from experiments in which Lindane doses were applied at different intervals within the 14d-period supported these observations. Low temperatures had a marked effect on the sensitivity of the fathead minnow to Lindane. Embryos exposed to Lindane at 18°C showed different temporal distribution of mortality than those at 25°C. A delay in hatching and emergence of smaller larvae were also noted at 18°C. Lordoscoliosis, edemas and hemorrhages were the most common morphological changes induced by the pesticide during the larval stage. The frequency of these abnormalities was linearly related to doses of Lindane. Impaired swimming behavior was also noted, but it was not related to dosages. / Master of Science