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No description available.

Lead was shown to inhibit both callus initiation and callus growth in cultures of Daucus carota L. subsp. sativus (Hoff. Thell.) cv. Nantes ''Tiptop'' and ''Nanthya''. Taproot explants of Daucus carota were stressed with lead. The callus cell lines which initiated under this stress were shown to exhibit resistance to the effects of lead ions. The growth of the selected and nonselected cell lines on non-lead containing media was comparable and the resistance possessed by the selected cell lines did not result in reduced growth rates in the presence of lead. The resistance characteristic was shown to be stable and to be successfully transmitted over mitotic and meiotic barriers. Plants were regenerated from the selected cell lines and ion uptake studies were conducted on isolated cortical tissue from mature taproots. The uptake of lead into the cortical cell tissue from the selected lines was shown to be reduced and a greater proportion of the lead that did enter the tissue was present in the Apparent Free Space and did not enter the cells. The regenerated plants were self-pollenated to produce an F1 generation. F1 plantlets were grown in hydroponic culture containing various concentrations of lead. The selected plants were seen to be resistant to the lead stress. The sites of lead accumulation in these roots were determined using x-ray microanalysis in a scanning electron microscope with a cryo-stage. The lead was found to be associated with the epidermal layer and cell walls. The mechanism of the lead resistance is discussed along with the implications of selection for somaclonal variants from initiating callus cultures.

Toxic metals are major pollutants of the aquatic environment and are able to cause survival impairment of the early life stage of the aquatic organisms. They can affect the osmoregulatory system and electrolyte balance in fishes as well as the expression of genes which are essential in the formation and development of the organs at the early embryonic stages of development. There are a lack of studies concerning the toxic effects of waterborne copper and silver on the osmoregulation, electrolytes balance and expression the genes which are responsible for the formation and development of heart and metal binding proteins in the early life stage of zebrafish. The current study aimed to assess the toxic effects of waterborne concentrations of copper as an essential trace element, and silver as a non-essential trace element, on biochemical processes and the molecular biology of the early life stages (ELS) of zebrafish. The first experiment of the current study (Chapter 3) aimed: 1. to determine the time of nkx2.5 gene expression, a gene involved in cardiac development, relative to the time of embryonic development. 2. To assess the toxic concentration of the copper and most vulnerable and sensitive stage of the embryos < 1 hour post fertilization (hpf) exposed to the copper via water route. The result of the experiment showed that the expression of the nkx2.5 gene reached a maximum at 16 hpf. The first 10 hpf of the embryonic development was the most vulnerable and critical stage of the developing embryos, and characterized by increased mortality as copper concentration increased, and delayed and decreased hatching success. Exposure of embryos for 72 hpf to a concentration of 500 µg L-1 Cu increased heart rate, whereas the exposure of the embryos at the blastula stage only, showed decreases in heart rate. The third part of the experiment evaluated the protective effect of calcium as a major cation of water hardness on Cu toxicity. Embryos age < 2 hpf were exposed to copper (0, 100, 250, and 500 µg L-1), with or without added calcium (40 mg L-1). An increase in embryonic Cu accumulation was observed in live and dead embryos exposed to Cu, with and without added calcium. Calcium concentration increased with embryonic copper tissue concentration in dead embryos. Na+ and K+ concentrations were higher in live embryos compared to dead embryos, and a 4 fold decrease in Na+K+-ATPase activity was seen in live embryos exposed to copper compared to controls. There was no effect of copper on total glutathione. Expression of nkx2.5 as one of the essential genes for the formation and development of the heart increased significantly; approximately 10 fold in the presence of Cu+Ca in comparison to the unexposed control or Cu exposure alone. Whereas expression of mt2 increased significantly 6 fold compared to the control during Cu exposure without added Ca2+. The second experiment (Chapter 4) aimed to investigate the effect of the dissolved Ag+ as AgNO3 on the survival of the early life stage of zebrafish. Embryos < 2 hpf were exposed to silver 0 (no added Ag), 2.5, 5, 7.5, 10 and 15 µg L-1 Ag as AgNO3 for up to 72 h. Although, the survival was not affected by increasing concentrations of total silver, a decrease in hatching and increase in heart beat was observed. A significant increase in embryonic silver accumulation in both live embryos (at 24 and 72 hpf) and dead embryos (at 24 hpf) was observed. The accumulation of silver in 24 hpf live embryos was more significant than in dead embryos. Dead and live embryos at 72 hpf exposed to Ag had lower Na+ and K+ concentrations. Live embryos also showed a transient increase in Ca2+ concentration at 24 h. Four fold increases in Na+K+-ATPase activity, Mt2, and total glutathione concentration were seen in embryos after 72 h of exposure to AgNO3 compared to controls. In contrast, nkx2.5 gene expression was significantly decreased by 3 fold in 24 h aged embryos exposed to silver compared to controls. Due to the lack of studies that investigate the effect of silver on protein expression profiles during the early stages of development of zebrafish, the third experiment (Chapter 5) aimed to investigate the effect of silver on the changes of the expressed proteins of zebrafish embryos at the segmentation stage (24 hpf). The proteomics analysis successfully identified total of 810 proteins in the embryonic homogenate and quantified changes in their abundance in response to silver exposure. MS analysis showed the induction of new proteins which were absent in control embryonic homogenates. Also the analysis revealed there were increased expression of proteins such as zona pellucida glycoprotein, ATP synthase subunit α and β, stressed proteins such as metal chaperones and heat shock proteins, antioxidant proteins such as catalase (CAT), superoxide dismutase (Cu-Zn), glutathione S-transferase M, and glutathione S-transferase and proteins related to muscular development such as myosin heavy polypeptide 2, actin alpha 1 skeletal muscle, slow myosin heavy chain 1, actin cytoplasmic 1, and tropomyosin proteins. Overall, the thesis confirmed that the early life stages of zebrafish are sensitive to metals and that there are critical windows of toxicity during development. Metal exposure at early stages of the development initiate several disturbances in biochemical processes as well as changes in molecular biology that affect fish survival.

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The population of harbour ragworms, Nereis diversicolor, inhabiting the upper reaches of Restronguet Creek in the Fal Ria, UK, are known to be resistant to the acute, toxic effects of copper, zinc and cadmium. This research aimed to establish whether metabolic and reproductive performance costs were associated with the resistance phenomenon and what the biochemical mechanisms of resistance were, by comparing animals from the Creek with two comparable study sites in the South West of England: Froe Creek and the Teign estuary. There was a significant metabolic cost, measured as a reduction in Scope for Growth, associated with the resistance phenomenon. As no differences in energy intake or uptake were found between resistant and non-resistant animals it was concluded that this cost was covered by demand-side regulation of energetics. A reduction in the amount of biochemical energy reserves in the form of lipids and sugars was also observed in resistant animals suggesting that resource allocation had been shifted away from this endpoint and towards maintenance and activity. The perturbed metabolism and physiology of resistant N. diversicolor is shown to have detrimental consequences for their life-history. Sexually mature, resistant females were significantly smaller than non-resistant ones, indicating that they had either matured at a younger age or grown more slowly. Both total fecundity and mass-specific fecundity were significantly reduced in resistant females, likely as a direct result of the metabolic costs of resistance reducing the resources available to fuel gametogenesis. No differences were found in the energetic reserves stocked within each gamete by resistant and non-resistant animals, suggesting that this trait is heavily selected for. Synthesis of reduced glutathione [GSH] in resistant animals’ tissues appeared to be elevated. Glutathione peroxidase activity was also increased, likely to remediate the effects of Reactive Chemical Species [RCS] that result from the inevitably incomplete binding of intracellular metals. As GSH functions in metal detoxification to covalently binding metals entering resistant animals’ cells, preventing their involvement in toxic interactions and their catalysis of the production of RCS, it is proposed that together there two phenomena comprise the fundamental mechanism of resistance to metal toxicity. Ultimately, this research revealed a hierarchy of health and reproductive performance across the three study populations, with significant associations evident between measured biological endpoints and the degree of metal contamination, illustrating the consequences of anthropogenic pollution for the biology of wild animals.

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.

Epidemiological studies have linked exposure to elevated levels of airborne particulate matter with increased incidences of several types of respiratory disease, hospital admissions and morbidity. Millions of tons of airborne particulate matter are generated and released into the atmosphere each year. However, particulate matter resulting from the combustion of fuel oil and coal are of particular concern, because they are generally composed of small particles that can easily penetrate deep into the lungs, and can contain significant concentrations of toxic transition metals, such as zinc, iron and vanadium. Pulmonary toxicity (i.e. damage caused to lung tissues) of particulate matter is currently evaluated via time-consuming in-vivo testing, or via in-vitro testing. Compared to in-vivo testing, in-vitro testing offers significant advantages in terms of time savings and sample throughput. Unfortunately, the number of in-vitro testing methods are currently very limited, and do not allow a thorough investigation of the mechanisms of particulate matter toxicity. In light of these issues, the goals of the study described here were three-fold: *To adapt several in-vitro toxicity assays currently used in other applications to use in measuring particulate matter toxicity on lung cell layers; *To use these adapted assays to quantify the toxicity of numerous types of oil and coal ashes with varying particle sizes and transition metal concentrations, and; *To use the same assays to quantify the toxicities of several transition metals found in coal and oil ashes to better understand their relative contributions to overall particulate matter toxicity. Three colorimetric in-vitro assays were chosen for adaptation, and proved effective in measuring adverse cellular response to particulate matter exposure. Particle size was shown to have a large effect on the overall cytotoxicity of particulate matter; fine (less than 2.5 μm aerodynamic diameter) particles proved substantially more toxic than coarse (larger than 2.5 μm aerodynamic diameter) particles. Dose-response experiments measuring the toxic effects of the transition metals zinc, vanadium and iron revealed that zinc was the most toxic; a concentration of 0.6 mM caused a 50% drop in cellular metabolism, compared to 3 mM and 4 mM for vanadium and iron respectively.