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.

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 &#8805; 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.

Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in significant alteration of concentrations of PGE2 at environmentally relevant concentrations.

The thick shelled river mussel belongs to a highly endangered group of organisms worldwide. A parasitic larval stage is included in the mussel's life cycle where its glochidia larvae attach to the gills of a host fish. The glochidia infection can affect the ability of fish populations to form schools, a behavior that is fundamental to the survival of many fish species. As the mussel is involved in conservation projects where the reintroduction of the glochidia larva is included, one should know more about how the parasite affects its host to be able to make well-founded decisions regarding possible reintroduction. In this study, a group of glochidia-infected minnows is compared to a control group where it was analyzed if the infected fish obtained a higher nearest neighbor distance (NND), a larger average school area (ASA) and a lower movement of the schools (MOS), two and four weeks after being infected. The minnows were infected in vitro where their schooling behavior was recorded with a video camera of which the footage material where analyzed. Collected data were analyzed with 2-way ANOVAS, where the results showed that there is a relationship between infection and changed schooling behavior with respect to the density and area of the fishes in general. NND and ASA increased in the infected group, but not in the non-infected group, at two and four weeks. If the goal through conservation measures is to restore the original balance of the parasite-host interactions of the thick shelled river mussel and the minnow, more research on how mussel parasites affect its fish hosts should be done. / Den tjockskaliga målarmusslan tillhör en starkt hotad organismgrupp över hela världen. Ett parasitiskt larvstadium ingår i musslans livscykel där dess glochidielarver fäster vid gälarna hos en värdfisk. Denna glochidieinfektion kan påverka fiskpopulationens förmåga att bilda stim, ett beteende som är grundläggande för många fiskarters överlevnad. Då musslan är med i bevarandeprojekt där återintroduktion av glochidielarven ingår bör kunskapen fördjupas gällande hur parasiten påverkar sin värd för att kunna ta välgrundade beslut inför eventuella åtgärder. I den här studien jämfördes glochidieinfekterade elritsor med en kontrollgrupp där det undersöks om de infekterade fiskarna får en högre nearest neighbour distance (NND), en större average school area (ASA) och en lägre movement of the schools (MOS) två och fyra veckor efter att de infekterats. Elritsorna infekteras in vitro där deras stimbeteende spelades in med en videokamera för att sedan bildanalyseras. Insamlade data analyserades med 2-vägs-ANOVOR, där resultaten visade att det finns samband mellan infektion och förändrat stimbeteende med avseende på fiskarnas täthet och area generellt. NND och ASA ökade hos de infekterade, men inte för de icke-infekterade under två och fyra veckor. Om målet genom bevarandeåtgärder är att återställa den ursprungliga balansen i parasit-värdinteraktionen för den tjockskaliga målarmusslan och elritsan, bör det undersökas vidare hur musselparasiter påverkar sina värdfiskar.

Since many estuarine zones are impacted by copper contamination, there is an on-going effort to develop Biotic Ligand Models (BLMs) predicting copper toxicity in transitional environments. In the first stage of this project, a critical analysis of the BLM framework identified some aspects of the model that required further investigation. In particular, a BLM for estuaries needed (a) a better characterization of the dissolved organic matter (DOC) and its effect on copper availability, and (b) the inclusion in the model’s equation of a salinity-correction factor modulating the relationship between copper accumulation on the biotic ligand and toxicity. The first issue was addressed by modelling the data produced using a Chelex resin method to determine the labile fraction of copper in samples of mixed riverine and estuarine waters. A refined and simplified BLM equation was then presented, accounting for both the DOC characteristics and the relevance of the osmotic gradient in modulating the relationship between copper accumulation and toxicity. A critical analysis of the literature on copper toxicity and salinity led to the hypothesis that copper-exposed fish are more sensitive to osmotic stresses, as copper interferes with their osmoregulatory pathways. In particular, the cytosolic isoform-2 of the enzyme carbonic anhydrase (CA2) was identified as an osmotic effector protein targeted by copper and involved in osmotic stress response pathways, hence representing a mechanistic link between the combined effects of copper exposure and osmotic stress. To test this hypothesis, two in vivo studies were performed, using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) and applying different rates of salinity changes as a way of dosing osmotic stress. The results showed a disturbance in plasma ion homeostasis after the salinity transitions, but notably the magnitude of the disturbance was greater in the copper-exposed individuals, suggesting a sensitizing effect of copper on the responses of fish to osmotic stress. Gene expression data demonstrated that CA2 is targeted by copper and confirmed the role of the enzyme in osmoregulatory pathways, as further supported by a promoter analysis of the gene coding for zebrafish CA2, which revealed the presence of osmotic-stress related elements. Overall, these results suggest that CA2 is an osmotic effector protein whose response can be activated by a medium level of osmotic stress through a combination of transcriptional and post-translational control circuits.

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.

All of New Zealand's five endemic species of mudfish (Neochanna) are threatened, and translocation has been recommended as an option for conservation. This research undertakes a translocation of black mudfish (Neochanna diversus) into wetland margins of Lake Kaituna, in the Waikato region, and addresses research questions applicable to improving translocation success. Results from this research are intended to aid possible future translocations of the more threatened Northland mudfish (Neochanna heleios) and other genetically distinct populations of black mudfish. Captive rearing of juveniles collected from the wild is currently the most feasible option for sourcing translocation stock. Mudfish juveniles (greater than 25 mm T.L.) had greater survival rates, compared to mudfish fry less than 25 mm T.L. Mudfish growth was far greater when fed on a combined diet of brine shrimp (Artemia salina) and white worms (Enchytraeus albidus) than when fed exclusively on brine shrimp. Temperature was found to have a small effect on mudfish growth, with a slightly greater growth in fish at 15 C than those at 10 C. The introduced species Gambusia affinis has been the subject of concern for mudfish conservation and commonly found at wetland sites suitable for mudfish translocation. Found to prey on mudfish fry and eggs in aquaria, it was important to determine the effects of Gambusia density prior to undertaking a translocation to a location where Gambusia were present. Investigations were made into the effect of Gambusia density on black mudfish juveniles in 9 outdoor mesocosms. Increasing Gambusia density was found to have an inhibitory effect on black mudfish growth. This may be due to increased competition for food, a theory supported by analysis of zooplankton communities, where, in the presence of Gambusia, large zooplankton had been removed and smaller rotifers flourished. Monitoring programmes are required to assess any impacts or improvements of mudfish populations, including those created by translocation. A Gee minnow trapping programme in outdoor mesocosms was conducted to test the reliability of traps, finding that water depth, mudfish density, mudfish memory and trap shyness had no effect on the trapability of mudfish. Trap position was found to have the most significant effect, with a greater number of mudfish caught when traps were set overnight at the surface than when set on the bottom of mesocosms. Black mudfish adults and juveniles were translocated into 18 pools (~1 m diameter) on the wetland margins of Lake Kaituna in September 2006, followed by monthly monitoring. Water quality monitoring and an assessment of hydrology and vegetation was undertaken. Habitat characterisation was found to be a key factor, with correlations between water quality data and trapping results finding fewer fish remaining in pools with less suitable characteristics for mudfish (e.g. high turbidity and conductivity). Other species were found to have a large impact, with predation by shortfinned eels (Anguilla australis) thought to have eliminated mudfish from some pools. In addition fewer mudfish were caught in pools with Gambusia, possibly due to increased competition.

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.