Cyanobacteria or blue green algae are known for their extensive and highly visible blooms in rivers or dams. One of the most important cyanobacteria is Microcystis aeruginosa which can synthesize various microcystins that can affect the health of terrestrial and aquatic animals. Commercial Nile crocodile (Crocodylus niloticus) farming in South Africa is based on keeping breeders (adult males and females) in big dams on farms (captive-bred approach). Unfortunately, cyanobacterial blooms in the breeder dams are a concern to farm owners, managers and veterinarians. This research project focussed on the monitoring of microcystins in the Hartbeespoort Dam and a crocodile breeding dam over a period of nine months. A commercial, but expensive, Abraxis ELISA kit was compared to a much cheaper and robust Norwegian-developed ELISA to detect microcystins in fresh water. Another objective was to determine if microcystins were present in the contents of crocodile eggs and dead hatchlings. Water samples were collected monthly from August 2014 to April 2015 at two sites, the Hartbeespoort Dam (control site) and the breeding dam of a commercial Nile crocodile (Crocodylus niloticus) farm. In addition, various water quality parameters including nitrate, phosphorous, chlorophyll a, oxygen saturation, pH and total dissolved solids (TDS) were determined to assess eutrophication. During the crocodile hatching season microcystin concentrations in unfertilized eggs, egg-shell membranes and in the yolk and liver of dead hatchlings were determined using liquid chromatography-mass spectrometry (LC-MS). Water quality parameters showed that there was no significant difference between the two dams' (the Hartbeespoort and the breeding dam) eutrophic state i.e. phosphates, TKN and nitrates; they both seemed to be becoming more eutrophic as the nutrient supply to the dam was increasing. Furthermore, microcystin concentrations during peak summer months were generally higher at the Hartbeespoort Dam compared to the crocodile breeding dam. The two ELISAs as performed on water samples "as is" and following an adsorbent disk/methanol extraction method were positively correlated; however, the correlation between the two assays was much stronger when using the adsorbent disk/methanol extraction as compared to using water "as is". Besides dissolved oxygen all the other water quality parameters were not significantly different (p > 0.05) between the two sites. Microcystin concentrations (MC-LR, MC-RR, MC-YR) in the crocodile egg and hatchling samples collected from batches with a good hatching rate (? 90%) ranged between 0 - 1.76 ng/g, with the highest concentration in the eggshell membranes. Microcystin concentrations in samples collected from batches with a bad hatching rate (? 10%) ranged from 0 - 1.63 ng/g with the highest concentration detected in the hatchling yolk. Although the "tissue" concentration levels were probably underestimated with the extraction method employed for LC-MS as the percentage recovery from spiked samples were very low. Bayesian analysis suggests that the liver, yolk and unfertilized egg all have similar microcystin concentrations, while the membranes have (with moderate to high certainty) higher microcystin concentrations. In conclusion, when using the Norwegian ELISA it seems as though the use of a resin-containing adsorbent disk followed by methanol extraction is more reliable than analysing water "as is". Following methanol extraction the results of the two ELISAs were strongly correlated, which suggests that the two ELISAs provide comparable results. There appears to be no difference in microcystin concentrations among good and bad clutches across all tissue types or within a specific tissue type. Vertical transmission of microcystins to the Nile crocodile egg does occur, but due to the small sample size, final conclusion cannot be made if microcystin affects Nile crocodile hatchling mortality and/or hatching of eggs. Future studies will include a longitudinal study to be done since a single season of breeding is insufficient to conclude that microcystins do not contribute to the low hatching rate in Nile crocodiles. / Dissertation (MSc)--University of Pretoria, 2016. / Paraclinical Sciences / MSc / Unrestricted

Diatoms have a successful history of use in assessments of wetland biological condition. In North America and across Europe, diatom assemblages are used for routine wetland condition assessments to meet the statutory requirements of the European Water Framework Directive and the National Aquatic Resource Survey by the US Environmental Protection Agency. In South Africa, the use of diatom assemblages as indicators of wetland condition may be a promising alternative to the traditional biotic assemblages employed, such as macroinvertebrates or macrophytes, which have proven to be ineffective. We present a preliminary investigation on the feasibility of diatoms in wetland biological assessments in South Africa by evaluating the use of diatoms as indicators of biological condition for depressional wetlands in the Mpumalanga Highveld region of South Africa. Depressional wetlands typically found in this region are either temporary (seasonally inundated) or permanent depressions. Temporary depressional wetlands are expected to be affected by natural environmental disturbances (e.g., seasonal fluctuations in water-level which may cause changes in water chemistry) as compared to relatively stable permanent ones. Establishing whether diatoms are suitable indicators of natural environmental disturbances in temporary depressional wetlands in this region is necessary for further investigations of anthropogenic disturbances. We sampled epiphytic diatoms from three least human-disturbed temporary depressional wetlands during various stages of inundation and showed that the species composition of epiphytic diatom communities were strong indicators of temporally changing environmental conditions. Using the same diatom and physical and chemical data, we also demonstrated that simplifying the taxonomy by using the functional composition (ecological guilds, life-forms) of the epiphytic diatom communities, can assess temporally changing environmental conditions as effectively as the species composition. Moreover, these functional groups provide valuable ecological information that is not available from the species data. Acid mine drainage (AMD) is the predominant stressor in permanent depressional wetlands of the Mpumalanga Highveld region, where coal mines utilise these wetlands for storage of AMD, which has severe impacts on the structure and function of the ecosystem. In order to develop an approach for impact assessment and management of depressional wetlands in the region, we developed an epiphytic diatom multimetric index (MMI) for AMD impacted permanent depressional wetlands. This is also the first diatom index to quantify AMD impacts in wetland habitats. Data collected from 34 sites that represented a range of conditions along an AMD gradient within the Mpumalanga Highveld was used to select responsive diatom metrics which we combined into a multimetric index. We developed separate MMIs for classes of depressional wetland types in order to account for natural variation among diatom assemblages, and compared their performance with an MMI that did not account for natural variation. To account for natural variation, we classified reference sites based on diatom typologies and hypothesised that by using this approach, we would improve MMI performance. Overall, all MMIs performed considerably well, although grouping sites by diatom typology to account for natural variation improved MMI performance, especially the precision, responsiveness and sensitivity to disturbance. We conclude that diatoms have strong potential for use in wetland ecological assessments in South Africa. The experimental and statistical approaches used in this study should expand our knowledge of diatom ecology and further advance the research and development of diatom bioassessment. / Thesis (PhD)--University of Pretoria, 2017. / Paraclinical Sciences / PhD / Unrestricted

Coal mining and coal utilization in Mpumalanga have increased over the years due to national reliance on coal as a source of power generation. In general, this has caused significant deterioration of water quality wherever streams are impacted by acid mine drainage (AMD). The aim of this research was to assess the use of in vitro bioassays as a complement to, or potential future replacement of, waste effluent testing in whole animals from AMD impacted watersheds subjected to passive and active treatment, correlating observed changes with water chemistry analysis. To accomplish this goal, water samples were collected and in vitro bioassays carried out to investigate generation of reactive oxygen species by the water samples and cytotoxicity against Vero kidney cells, C3A liver cells and trout RTgill-W1 cells. Primary fish gill cultures were established and used as sensitive in vitro models for assessing possible contaminants in water, measuring the induction of cytochrome P450 (CYP) 1A and resultant increase in 7-ethoxyresorufin-o-deethylase activity as a potential biomarker in fish gill cells exposed to polycyclic aromatic hydrocarbons. The genotoxic potential of AMD water on commercially available cell lines was also determined. / The study site was an impacted stream located downstream of a coal mine discharge point whose effluent flowed away from the mine. Water chemistry results suggested high AMD impact evidenced by acidity, elevated sulphates, increased conductivity and presence of heavy metals. Al, Fe, Zn, Mn and Si were the major metals of potential concern in the AMD impacted stream; sulphates and major ions like Ca, K, Na and Mg were present at levels above target water quality range (TWQR) for effluents in receiving stream. The AMD impacted stream caused increased generation of reactive oxygen species (ROS) detectable in vitro in selected cell lines (Vero, C3A and RTgill-W1 cell lines), an indication of oxidative stress. In-stream, active treatment with caustic soda was efficient at reducing metal burden, with subsequent reduction in ROS generation in fish gill cell lines. For in vitro cytotoxicity tests, passive and active treated AMD water was cytotoxic to cell lines (Vero and RTgill-W1), with the fish RTgill-W1 cells exhibiting greater sensitivity compared to the mammalian Vero cells. Mitochondria played a larger role in observed loss in cellular viability (increased vacuolization, mitochondrial membrane swelling and damage), which was detected using mitochondrial specific stains, and by transmission electron microscopy (TEM). Increased dose- dependent cytotoxicity was observed in the fish gill and mammalian cell lines. Cells exposed to water samples (AMD and reference sites) revealed significant differences (p < vi 0.05) between the AMD impacted watershed and a relatively pristine site (reference site) where exposure to the same cells maintained approximately 100% viability at all concentrations for up to 72h exposure. The observed differences in effect in this study demonstrate that the effluent from the coal mine negatively impacted surface water quality, resulting in toxicity to cell lines, therefore creating an environment that would not be conducive for the survival of biological aquatic communities and potentially of concern for downstream human end users. / The induction of cytochrome P450 (CYP) 1A and resultant increase in 7- ethoxyresorufin-o-deethylase activity in primary fish gill cultures exposed to polycyclic aromatic hydrocarbons B[a]P, a known AhR agonist contaminant associated with coal mining, showed that there was as increase in EROD activity which was not observed using the RTgill-W1 cell lines. Gill epithelial cells isolated from the gills of Tilapia fish (Oreochromis mossambicus) bear close similarities to fish gills in vivo and their capacity to respond to the presence of AhR indicates that they may serve as a simple, cost-effect screening tool for assessing PAHs and dioxin-like compounds in fresh water. / For genotoxicity evaluation, the Ames test performed without metabolic activation using bacterium Salmonella typhimurium TA98 and TA100 strains revealed no indication of genotoxic activity in any of the water samples. Genotoxicity assessment of all water samples using the comet assay however exposed DNA damage to Vero and RTgill-W1 cell lines. A significant reduction in DNA damage was observed following active treatment. The results suggest that neither treatment technologies employed were efficient at removing all potential genotoxicants so further improvements are required. The comet assay proved sensitive enough to detect genotoxicity in reference water samples despite no known untoward effluent inputs at the site, suggesting potential for this assay to be integrated into an environmental monitoring framework. / The results obtained support the use of in vitro bioassays for evaluating toxicity of industrial effluent through biological responses in test systems elicited following exposure, improving ability to detect AMD polluted water. This could be beneficial when assessing the degree and extent of impact of AMD in natural water sources, and the possible environmental impact resulting from hazardous elements present in effluent water. In conclusion, these results suggest that in vitro techniques involving cell lines and primary cultures from fish may serve vii as simple, rapid and cost-effective tools for assessing risk and potential toxic effects of contaminants in AMD waters. / Thesis (PhD)--University of Pretoria, 2016. / The National Research Foundation / Department of Paraclinical Sciences (University of Pretoria) / Schlumberger Stichting Fund, Netherlands / Paraclinical Sciences / PhD / Unrestricted