Pyrethroid-resistant Hyalella azteca with genetically-confirmed voltage gated sodium channel mutations were identified at three geographically isolated locations in Central California. In December 2013, H. azteca were collected from Mosher Slough in Stockton, CA, a site with reported pyrethroid concentrations at approximately twice the LC50 for laboratory-cultured H. azteca and shipped to Southern Illinois University Carbondale. These H. azteca have been maintained in pyrethroid-free culture since December, 2013 with one supplement of organisms from the same site in March, 2014. Abundant research exists on fitness costs of insecticide resistance to pest species including reduced fecundity, fertility, reduced overwintering success, and reduced survival to adulthood. The current study showed that after 22 months in culture, resistant animals had approximately 53 times higher tolerance to permethrin than non-resistant H. azteca. After 16 months in culture, the resistant animals maintained complete loss of the wild-type allele at the L925 locus and had non-synonymous substitutions that resulted in either a leucine-isoleucine or leucine-valine substitution. Finally, the resistant animals showed lower reproduction, lower upper thermal tolerance, and were more sensitive to the common contaminants DDT, copper (II) sulfate, and NaCl. As shredders that consume epiphytes, animal and plant detritus, and filamentous algae, H. azteca aid in nutrient cycling through the breakdown and fragmentation of detritus and can serve as an important food source for larger invertebrates, fish, amphibians, and waterfowl. The results of the current study also hold implications for biomonitoring programs, toxicity testing, and laboratory culture procedures, as cryptic species complexes with varied sensitivities could dramatically alter results of these programs.

Chronic exposure to pyrethroid insecticides can result in sublethal impacts to non-target species in aquatic systems, driving population-level changes. Characterizing the underlying mechanisms of resistance is essential to better understanding the role and potential consequences of contaminant-driven microevolution. The current study found that multiple mechanisms enhance the overall phenotypic expression of resistance characteristics in Hyalella azteca. In VGSC mutated H. azteca, both adaptation and acclimation traits appear to play a role in the attenuation of the adverse effects to pyrethroid exposures. Pyrethroid resistance is primarily attributed to the heritable mutation at various loci of the voltage-gated sodium channel, resulting in reduced target-site sensitivity. However, some additional reduced pyrethroid sensitivity was also conferred through reversible physiological responses to environmental conditions, such as enhanced enzyme-mediated detoxification. Cytochrome p450 monooxygenases (CYP450) and general esterases (GE) were the biotransformation pathways that significantly contributed to the detoxification of permethrin in H. azteca. Over time, VGSC mutated H. azteca retained most of their pyrethroid resistance, though there was some increased sensitivity from parent to offspring when reared in the absence of pyrethroid exposure. The permethrin 96 h LC50 declined from 1809 ng/L in P0 individuals to 1123 ng/L in the F1 generation, though still remained well above the 20.4 ng/L of wild-type individuals. This reduction in tolerance was likely related to alterations in acclimation mechanisms conferring resistance traits, rather than changes to target-site sensitivity. Enzyme bioassays indicated decreased CYP450 and GE enzyme activity from P0 to F1, whereas the VGSC mutation was retained. The permethrin LC50 values in resistant H. azteca were still two orders-of-magnitude higher than non-resistant populations indicating that the largest proportion of resistance was maintained through the inherited VGSC mutation. Thus, some variation in phenotypic expression of resistance characteristics in H. azteca over time is likely associated with uninheritable genetic factors or non-constitutively expressed traits controlling enzyme pathways which overlie a strong heritable component of resistance. A better understanding of the mechanistic and genomic basis of variable acclimation will be necessary for better predicting the ecological and evolutionarily consequences of contaminant-driven change in H. azteca.

The food of a common freshwater deposit-feeding amphipod Hyalella ctzteca (Saussure) and the effect of its feeding on energy transfer within the sediments of Marion Lake were examined. By use of C¹⁴ labelling techniques and chemical analyses of food and faeces, Hyalella was shown to digest algal and bacterial cells from ingested sediment particles at a rate sufficient to supply energy necessary for observed growth, respiration and egg production. Non-living sediment organic substances (cellulose and lignin-like materials) were not assimilated. Changes in oxygen concentration over undisturbed sediment cores incubated under light and dark conditions and with antibiotic additions were used to provide an in situ measure of epibenthic algal production and community and bacterial respiration. Seasonal and spatial differences in sediment microflora production potentially available for consumption by Hyalella were positively correlated with amphipod growth, distribution and the onset of egg production. In laboratory experiments amphipods selected sediments with the highest concentrations of microflora and growth was proportional to the abundance of this food source. Numbers of Hyalella were varied in undisturbed sediment cores to evaluate the effect on microflora production. After 43 hr incubation, algal production stimulated at natural amphipod densities and declined, with higher numbers. Lacterial respiration was stimulated at densities four times those observed in Marion Lake and this also decreased with crowding. Total microflora production and amphipod energy requirements were compared to calculate that at natural densities during the summer Hyalella would consume less than 10% of the daily production. The remaining microflora production may be consumed by numerous other deposit-feeding species in the benthic community. Physical leaching and heterotrophic organisms lowered the organic content of faeces in the dark, while autotrophic organisms and dissolved organic compounds released by amphipods added organic matter to faecal pellets. Thus, while Hyalella's browsing activities may reduce the biomass of sediment microflora, it concurrently aided recolonization of faecal material and sediment particles and thus appears to stimulate production of its own food supply. Additional data are presented which compare the respiration of different benthic communities. The similarity of the rates of oxygen exchange, after correction for temperature difference, suggests that processes affecting oxygen availability are important in determining the overall rate of energy transfer within many benthic communities. / Science, Faculty of / Zoology, Department of / Graduate

Polycyclic aromatic hydrocarbons (PAH) are a class of common environmental contaminants known to be phototoxic. PAH Photoinduced toxicity is caused by two mechanisms: photomodification and photosensitization. The photomodification process results in modified PAHs, usually via oxygenation, forming new compounds (oxyPAHs), which are often more soluble than their parent PAHs. The process of photosensitization usually leads to the production of singlet oxygen, a reactive oxygen species (ROS), which in turn is extremely damaging to organic molecules. Both of these processes occur at environmentally relevant levels of actinic radiation. Metals are ubiquitous environmental contaminants found extensively in many aquatic systems. Many metals are toxic at very low levels, and exhibit toxicity via ROS production or via direct binding to a ligand in an organism (Biotic Ligand Model). PAHs and metals often occur as co-contaminants in the environment, and there combined effects have only been examined in a few organisms. The goal of this thesis was to examine the toxicity of PAHs, oxyPAHs, metals and their mixtures to Hyalella azteca in 96 h acute toxicity tests. All of the tests were performed under varying spectra of light; photosynthetically active radiation (PAR), PAR + UVA or simulated solar radiation (SSR) (PAR + UVA radiation + UVB radiation). In addition, chemical exposures in the Dark were performed to assess toxicity in the absence of light. The PAHs chosen represent 3 of the most common PAHs anthracene (ANT), phenanthrene (PHE), benzo(a)anthracene (BAA). The 12 oxyPAHs studied were quinolated analogues of the 3 parent compounds as well as anthraquinone derivatives that are hydroxylated at various positions. The toxicity of the parent PAHs increased in the presence of increasing amounts of actinic radiation. The toxicity of the oxyPAHs also increased as PAR, UVA and UVB was added. Furthermore, most PAHs and oxyPAHs were found to be more toxic than the parent PAHs in the absence of actinic radiation. The metals cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) were used in toxicity tests. These metals were selected based on their high prevalence in aquatic environments and the large amount of data in the published work. The order of metal toxicity in the Dark was Cd > Cu > Zn > Ni. The order of metal toxicity for the PAR and PAR/UV-A regime was Cd = Cu > Ni > Zn. The order of metal toxicity for the SSR treatment was Cu > Cd > Ni > Zn. The toxicity of several metal/PAH mixtures was determined using one of the four metals and ANT, ATQ and 1-hATQ. The mixtures generally had additive toxicity under Dark and PAR lighting regimes. Under SSR lighting most mixtures showed a strictly additive toxicity, however synergistic toxicity was observed for the redox active metals (Cu, Ni) mixed with ANT. In the aquatic environment complex mixtures of PAHs and metals occur. The results of this study illustrate the effects that these mixtures may have on benthic invertebrates.

Polycyclic aromatic hydrocarbons (PAH) are a class of common environmental contaminants known to be phototoxic. PAH Photoinduced toxicity is caused by two mechanisms: photomodification and photosensitization. The photomodification process results in modified PAHs, usually via oxygenation, forming new compounds (oxyPAHs), which are often more soluble than their parent PAHs. The process of photosensitization usually leads to the production of singlet oxygen, a reactive oxygen species (ROS), which in turn is extremely damaging to organic molecules. Both of these processes occur at environmentally relevant levels of actinic radiation. Metals are ubiquitous environmental contaminants found extensively in many aquatic systems. Many metals are toxic at very low levels, and exhibit toxicity via ROS production or via direct binding to a ligand in an organism (Biotic Ligand Model). PAHs and metals often occur as co-contaminants in the environment, and there combined effects have only been examined in a few organisms. The goal of this thesis was to examine the toxicity of PAHs, oxyPAHs, metals and their mixtures to Hyalella azteca in 96 h acute toxicity tests. All of the tests were performed under varying spectra of light; photosynthetically active radiation (PAR), PAR + UVA or simulated solar radiation (SSR) (PAR + UVA radiation + UVB radiation). In addition, chemical exposures in the Dark were performed to assess toxicity in the absence of light. The PAHs chosen represent 3 of the most common PAHs anthracene (ANT), phenanthrene (PHE), benzo(a)anthracene (BAA). The 12 oxyPAHs studied were quinolated analogues of the 3 parent compounds as well as anthraquinone derivatives that are hydroxylated at various positions. The toxicity of the parent PAHs increased in the presence of increasing amounts of actinic radiation. The toxicity of the oxyPAHs also increased as PAR, UVA and UVB was added. Furthermore, most PAHs and oxyPAHs were found to be more toxic than the parent PAHs in the absence of actinic radiation. The metals cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) were used in toxicity tests. These metals were selected based on their high prevalence in aquatic environments and the large amount of data in the published work. The order of metal toxicity in the Dark was Cd > Cu > Zn > Ni. The order of metal toxicity for the PAR and PAR/UV-A regime was Cd = Cu > Ni > Zn. The order of metal toxicity for the SSR treatment was Cu > Cd > Ni > Zn. The toxicity of several metal/PAH mixtures was determined using one of the four metals and ANT, ATQ and 1-hATQ. The mixtures generally had additive toxicity under Dark and PAR lighting regimes. Under SSR lighting most mixtures showed a strictly additive toxicity, however synergistic toxicity was observed for the redox active metals (Cu, Ni) mixed with ANT. In the aquatic environment complex mixtures of PAHs and metals occur. The results of this study illustrate the effects that these mixtures may have on benthic invertebrates.

Past monitoring has noted benthic macroinvertebrate community impairment downstream of both the Key Lake and Rabbit Lake uranium operations in northern Saskatchewan, Canada. The objective of this research was to try to identify the cause(s) of these impacts using a weight-of-evidence approach. Given that sediments generally accumulate contaminants that are related to metal mining activities (such as metals and radionuclides), the initial hypothesis for this research was that contaminated sediments were the primary cause of benthic community impairment at both operations.<p>In 2003 and 2004 a Sediment Quality Triad (SQT) approach confirmed the presence of an effect on benthic community structure, in addition to significant differences in surface-water, pore-water and whole-sediment chemistry at the immediate down-stream exposure sites at both uranium operations. However, no significant adverse effects were noted in 10-d whole-sediment bioassays with <i>Hyalella azteca</i>, although this lack of response could be partially due to sediment pore-water dilution resulting from the automated clean overlying water renewal process employed. Potential causes of benthic community impairment identified through the 2003 and 2004 SQTs for Key Lake include physical sediment composition, surface water pH and total ammonia, in addition to pore-water total ammonia and arsenic. Potential stressors identified at Rabbit Lake included high surface water manganese and uranium concentrations, and increases in pore-water total ammonia, manganese, iron, arsenic, and uranium levels.<p>In the summer of 2004, 4-d in-situ bioassays using <i>H. azteca</i> were conducted along with the SQTs to investigate the role both contaminated surface water and sediment played in benthic community impairment in-situ. Results from the Key Lake in-situ bioassay demonstrated that surface-water was the primary cause of acute toxicity to <i>H. azteca</i>. Results from the Rabbit Lake in-situ study also demonstrated that surface water as the primary cause of acute toxicity to <i>H. azteca</i>, although the relationship was not as strong. The cause of in-situ toxicity at Key Lake could not be correlated with any of the variables measured within the in-situ study, including trace metals, total ammonia, and pH. Of the measured constituents at Rabbit Lake, only concentrations of uranium in both surface water and pore-water were suspected of causing the observed in-situ mortality. Two data sets from two methods of surface water and pore-water collection supported these conclusions.<p>Due to time constraints and stronger cause-effect relationships, efforts were focused on the in-situ toxicity observed at Key Lake. Surface water collected in 2004 at the time of the related in-situ study was also found to be acutely toxic to <i>H. azteca</i> in separate laboratory surface water bioassays, thus verifying that contaminated surface water, not sediment, was the primary cause of the observed in-situ <i>H. azteca</i> mortality. Further information revealed that organic mill-process chemicals, which have been previously linked with sporadic effluent toxicity, were released at the Key Lake operation during the time of the in-situ experiment and associated surface water collection. Additional surface water samples collected in June and August, 2005, were not acutely toxic to <i>H. azteca</i>. Furthermore, a second bioassay with archived surface waters from the initial 2004 collection demonstrated that the water was no longer acutely toxic (i.e., acute toxicity disappeared after one-year storage). Chemistry comparisons of the toxic and non-toxic surface water samples, verified that trace metals, ammonia, pH, and major ions, including sulphate, were not the cause of toxicity, leaving only organic mill-process chemicals as a possible cause. Subsequent 4-d laboratory toxicity tests demonstrated that these process chemicals (kerosene, amine, and isodecanol) are toxic to H. azteca at the levels released in 2004, and are therefore believed to be the cause of the <i>H. azteca</i> mortality seen in the earlier in-situ experiment.<p>In short, this weight-of-evidence research provided new information on the possible causes of benthic macroinvertebrate community impairment downstream of both the Key Lake and Rabbit Lake uranium operations.

There is increasing global recognition of the effects of large scale land disturbance by wildfire on a wide range of water and ecosystem services. In 2003, the Lost Creek wildfire burned a contiguous area of 21,000 ha on the eastern slopes of the Rocky Mountains in southern Alberta. This disturbance had a significant and prolonged impact on the water quantity and quality of downstream river reaches and reservoirs in the Oldman watershed. Previous research in this watershed demonstrates that dissolved metal concentrations in rivers draining burned landscapes were 2 to 15 times greater than in unburned reference streams (Silins et al. 2009a). Currently there is no information on the effects of wildfire on the bioaccumulation and toxicity of sediment-associated metals in fire-impacted streams in Alberta. This study was designed to evaluate the linear downstream disturbance effects of wildfire in the Crowsnest River located in southern Alberta. The toxicity and bioaccumulation of particulate-associated metals from wildfire impacted tributaries to the Crowsnest River on freshwater amphipod Hyalella azteca were evaluated. Phillips samplers were deployed to collect suspended solids in streams draining burned zone impacted by the Lost Creek wildfire and reference (unburned) zones within the area. Metal toxicity and bioaccumulation were determined in the laboratory by exposing the epi-benthic freshwater amphipod Hyalella azteca to particulates collected from the Crowsnest River. A metal effects addition model (MEAM) was used to assess the impact of metal mixtures and to predict chronic mortality (Norwood et al. 2013). Increased concentrations of Al, Ba, Co, Cr, Mn and Zn were found in the tissues of H. azteca exposed to particulates from burned watersheds in comparison to the unburned watersheds. H. azteca mean survival was similar when exposed to the particulates samples from both burned and unburned sites indicating that 9 years after this landscape disturbance, there was little impact due to the wildfire. However, at burned site (B1), the observed survival was lower than survival predicted by MEAM. The data suggests that factors other than the metals examined in this study were influencing the survival of H. azteca. The concentrations of sediment-associated metals have decreased in the nine years since the wildfire, and minimal metal toxicity was observed in H. azteca. Although metal toxicity in H. azteca was minimal 9 years after the Lost Creek Fire, the short term effects of wildfire on metal toxicity remain largely unknown. In addition, other factors such as burn severity, stream size and hydroclimatic conditions can influence the effects of wildfire on abundance and diversity of aquatic invertebrates (Minshall et al. 2001). Therefore, the influences of those factors on metal toxicity as a result of wildfire should be rigorously assessed in future studies.

Past monitoring has noted benthic macroinvertebrate community impairment downstream of both the Key Lake and Rabbit Lake uranium operations in northern Saskatchewan, Canada. The objective of this research was to try to identify the cause(s) of these impacts using a weight-of-evidence approach. Given that sediments generally accumulate contaminants that are related to metal mining activities (such as metals and radionuclides), the initial hypothesis for this research was that contaminated sediments were the primary cause of benthic community impairment at both operations.<p>In 2003 and 2004 a Sediment Quality Triad (SQT) approach confirmed the presence of an effect on benthic community structure, in addition to significant differences in surface-water, pore-water and whole-sediment chemistry at the immediate down-stream exposure sites at both uranium operations. However, no significant adverse effects were noted in 10-d whole-sediment bioassays with <i>Hyalella azteca</i>, although this lack of response could be partially due to sediment pore-water dilution resulting from the automated clean overlying water renewal process employed. Potential causes of benthic community impairment identified through the 2003 and 2004 SQTs for Key Lake include physical sediment composition, surface water pH and total ammonia, in addition to pore-water total ammonia and arsenic. Potential stressors identified at Rabbit Lake included high surface water manganese and uranium concentrations, and increases in pore-water total ammonia, manganese, iron, arsenic, and uranium levels.<p>In the summer of 2004, 4-d in-situ bioassays using <i>H. azteca</i> were conducted along with the SQTs to investigate the role both contaminated surface water and sediment played in benthic community impairment in-situ. Results from the Key Lake in-situ bioassay demonstrated that surface-water was the primary cause of acute toxicity to <i>H. azteca</i>. Results from the Rabbit Lake in-situ study also demonstrated that surface water as the primary cause of acute toxicity to <i>H. azteca</i>, although the relationship was not as strong. The cause of in-situ toxicity at Key Lake could not be correlated with any of the variables measured within the in-situ study, including trace metals, total ammonia, and pH. Of the measured constituents at Rabbit Lake, only concentrations of uranium in both surface water and pore-water were suspected of causing the observed in-situ mortality. Two data sets from two methods of surface water and pore-water collection supported these conclusions.<p>Due to time constraints and stronger cause-effect relationships, efforts were focused on the in-situ toxicity observed at Key Lake. Surface water collected in 2004 at the time of the related in-situ study was also found to be acutely toxic to <i>H. azteca</i> in separate laboratory surface water bioassays, thus verifying that contaminated surface water, not sediment, was the primary cause of the observed in-situ <i>H. azteca</i> mortality. Further information revealed that organic mill-process chemicals, which have been previously linked with sporadic effluent toxicity, were released at the Key Lake operation during the time of the in-situ experiment and associated surface water collection. Additional surface water samples collected in June and August, 2005, were not acutely toxic to <i>H. azteca</i>. Furthermore, a second bioassay with archived surface waters from the initial 2004 collection demonstrated that the water was no longer acutely toxic (i.e., acute toxicity disappeared after one-year storage). Chemistry comparisons of the toxic and non-toxic surface water samples, verified that trace metals, ammonia, pH, and major ions, including sulphate, were not the cause of toxicity, leaving only organic mill-process chemicals as a possible cause. Subsequent 4-d laboratory toxicity tests demonstrated that these process chemicals (kerosene, amine, and isodecanol) are toxic to H. azteca at the levels released in 2004, and are therefore believed to be the cause of the <i>H. azteca</i> mortality seen in the earlier in-situ experiment.<p>In short, this weight-of-evidence research provided new information on the possible causes of benthic macroinvertebrate community impairment downstream of both the Key Lake and Rabbit Lake uranium operations.

There is increasing global recognition of the effects of large scale land disturbance by wildfire on a wide range of water and ecosystem services. In 2003, the Lost Creek wildfire burned a contiguous area of 21,000 ha on the eastern slopes of the Rocky Mountains in southern Alberta. This disturbance had a significant and prolonged impact on the water quantity and quality of downstream river reaches and reservoirs in the Oldman watershed. Previous research in this watershed demonstrates that dissolved metal concentrations in rivers draining burned landscapes were 2 to 15 times greater than in unburned reference streams (Silins et al. 2009a). Currently there is no information on the effects of wildfire on the bioaccumulation and toxicity of sediment-associated metals in fire-impacted streams in Alberta. This study was designed to evaluate the linear downstream disturbance effects of wildfire in the Crowsnest River located in southern Alberta. The toxicity and bioaccumulation of particulate-associated metals from wildfire impacted tributaries to the Crowsnest River on freshwater amphipod Hyalella azteca were evaluated. Phillips samplers were deployed to collect suspended solids in streams draining burned zone impacted by the Lost Creek wildfire and reference (unburned) zones within the area. Metal toxicity and bioaccumulation were determined in the laboratory by exposing the epi-benthic freshwater amphipod Hyalella azteca to particulates collected from the Crowsnest River. A metal effects addition model (MEAM) was used to assess the impact of metal mixtures and to predict chronic mortality (Norwood et al. 2013). Increased concentrations of Al, Ba, Co, Cr, Mn and Zn were found in the tissues of H. azteca exposed to particulates from burned watersheds in comparison to the unburned watersheds. H. azteca mean survival was similar when exposed to the particulates samples from both burned and unburned sites indicating that 9 years after this landscape disturbance, there was little impact due to the wildfire. However, at burned site (B1), the observed survival was lower than survival predicted by MEAM. The data suggests that factors other than the metals examined in this study were influencing the survival of H. azteca. The concentrations of sediment-associated metals have decreased in the nine years since the wildfire, and minimal metal toxicity was observed in H. azteca. Although metal toxicity in H. azteca was minimal 9 years after the Lost Creek Fire, the short term effects of wildfire on metal toxicity remain largely unknown. In addition, other factors such as burn severity, stream size and hydroclimatic conditions can influence the effects of wildfire on abundance and diversity of aquatic invertebrates (Minshall et al. 2001). Therefore, the influences of those factors on metal toxicity as a result of wildfire should be rigorously assessed in future studies.

Pyrethroid insecticides applied on crops and in urban areas are being found in aquatic ecosystems due to natural processes, such as run-off. Although highly toxic to invertebrates and fish, populations of Hyalella azteca have become resistant to some of these compounds, which pose risks to not only their populations, but higher trophic level populations via bioaccumulation (in this thesis, the Inland silverside (Menidia beryllina)). Concurrently, the impact of global climate change (GCC) is impacting environmental water parameters, such as temperature and salinity. The objective of this thesis was to analyze the relationship between varying water parameters due to GCC on the fate of permethrin (a type of pyrethroid) in resistant H. azteca (by measuring toxicokinetic rates) and in M. beryllina (by measuring bioaccumulation after consuming permethrin-dosed resistant H. azteca). Permethrin bioconcentration testing used two distinct populations (Mosher Slough and Escondido Creek) of pyrethroid-resistant H. azteca and showed that temperature and salinity affected toxicokinetic rates. Statistical differences in metabolite formation rates (km) across temperatures were found between and within populations. Salinity also exhibited statistical differences in the elimination of parent compound (kep). No statistically significant differences in uptake rates (ku) were found for either population. In the M. beryllina testing, the ability for the fish to bioaccumulate permethrin via a dietary route of exposure was confirmed, contradicting previous findings. Statistically significant bioaccumulation was found across salinities, whereas no statistically significant temperature effects were observed. With the predicted increased use of pyrethroids over the course of the next century, the emergence of resistant populations of H. azteca may increase, simultaneously increasing the risk for bioaccumulation by higher trophic species. With rates of biotransformation in H. azteca affected by changing water parameters due to GCC change, the ratio of parent and metabolite compound transferred to fish will also be altered. Pyrethroid metabolites in fish act as endocrine disruptors rather than inhibit nerve function like the parent compound, which can significantly affect fish development. Overall, this thesis demonstrates important potential effects of GCC on the rates and biological transfer of pyrethroids by aquatic species, and the potential combined effects of these multiple stressors on two trophic levels of aquatic organisms.