M.Sc. / Heavy metals occur naturally in the environment and are found in varying levels in all ground and surface waters. Some heavy metals are essential elements for the normal metabolism of organisms, while others are non-essential and play no significant biological role. Anthropogenic activities do, however, cause an increased discharge of these metals into natural aquatic ecosystems. Aquatic organisms are exposed to unnaturally high levels of these metals. Fish are relatively sensitive to changes in their surrounding environment. Fish health may therefore reflect and give a good indication of the health status of a specific aquatic ecosystem. Early toxic effects of pollution may only be evident on cellular or tissue level before significant changes can be identified in fish behaviour or external appearance. Histological analysis appears to be a very sensitive parameter and is crucial in determining cellular changes that may occur in target organs, such as the liver. The liver is a detoxification organ and essential for both the metabolism and excretion of toxic substances in the body. Exposure to heavy metals may cause histological changes in the liver. Fish liver histology could therefore serve as a model for studying the interactions between environmental factors and hepatic structures and functions. In this study, the effect of two heavy metals, cadmium (Cd) and zinc (Zn), on the histology of the liver of the South African freshwater fish species, Oreochromis mossambicus, was investigated. The aim of this study was to determine the toxic effect of cadmium and zinc on the histology of the liver, by identifying significant histological changes in the liver tissue, after exposing the fish to two concentrations of a mixture of cadmium and zinc, over both short and long-term exposure periods. Seventy two, adult O. mossambicus specimens were selected for the study. Two experimental exposures were executed under controlled conditions by means of a flow-through system in an environmental room. For each of the two exposures, twenty-four fish were exposed to different concentrations of cadmium and zinc. The remaining twenty-four specimens were used as a control group. The two respective metal concentrations selected for each exposure were 5% and 10% concentrations of both cadmium and zinc calculated from known LC50 values for cadmium chloride and zinc chloride. Liver samples were fixated in 10% neutrally buffered formalin and prepared for light microscopy analysis using standard techniques for Haematoxylin and Eosin (H & E) and Periodic Acid Schiff (PAS) staining. The liver histology of all seventy two specimens - including the forty eight exposed specimens and twenty four control specimens - were analysed, compared and documented. Although histological analysis can provide a clear indication of the degree of damage caused in the tissue(s) or organ(s) of exposed specimens, the need arises to quantify the histological results in studies where the effects of the exposing substance(s) are compared, to illustrate the possible decrease or increase in histological changes over time or the effect of two different concentrations of the same exposure substance on the histology of the liver. The histological results in this study were quantified in terms of a histological index. An index value representing the specific histological characteristics of the liver was assigned to each individual specimen indicating either normal histological structure (index value of 0-2) or a possible pathological response (index value of 3-6). Histological changes were identified in specimens exposed for 12, 18, 24, and 96 hours to both the 5% and 10% concentrations of cadmium and zinc, indicating a toxic response after the short-term metal exposures. Similar histological changes were identified in both the 5% and 10% exposed livers. These histological changes included hyalnization, vacuolation, cellular swelling and congestion of blood vessels. The liver histology of fish exposed over a long-term period of 672 hours, did, however, appear relatively normal in both the 5% and 10% exposure groups, indicating an adaptative, regenerative response. According to the results obtained, it was clear that exposure period did influence the degree of histological changes identified. The two metal concentrations did however seem to have similar histological effects and no definite variation could be identified in terms of 5% and 10% metal concentrations used. It can therefore be concluded that low concentrations of cadmium and zinc exposure caused histological alterations in the livers of exposed specimens and therefore allows the liver of O. mossambicus to be used as a biomarker of prior exposure to cadmium and zinc. / Dr. G.M. Pieterse

The mussel bed and its multitude of inhabitants form a life community typical of our intertidal, rocky, open-coast areas. These animals are essentially immobile, are conveniently available at low tide, and have been well defined in their consumer order. Certain main members from this community were chosen with the intention that they would represent an index of heavy metal pollution for a given area under different seasonal and other variable conditions. Samples for monitoring were collected from two different sites. The first site was immediately outside the entrance to San Francisco Bay and located between Seal Rocks and Phelan Beach State Parks. This location was chosen to represent a water mass of supposed maximum pollution. The Golden Gate can be assumed to be the funnel through which flows all waters from the San Joaquin and Sacramento River drainages and from the San Francisco Bay area itself. The second site, immediately north of the Dillon Beach township, located at the juncture of Bodega and Tomales Bays, was chosen since it might represent a water mass of minimum pollution. This area is not immediately near any large urban influence, industrial activity or subject to heavy auto traffic. Therefore, at the outset it was hoped that the “immobile” consumer order within the mussel bed community would reflect the relative pollution of two supposedly different water masses.

The aim of this study was to assess sensitivity of early life stage zebrafish to cadmium (Cd). Embryos and larva were exposed to Cd before the formation of skeletal elements. Exposure times were selected to determine whether Cd perturbed developmental processes that lead to skeletal deformities, and to characterize the types of skeletal deformities that occurred. Embryo/larva were exposed to 3μM, 10μM, 30μM, 100μM, 300μM, and 1000μM Cd at 12-36 hours post fertilization (hpf), 36-60hpf, 60-84hpf, and 144-168hpf in one series of experiments. This experiment was conducted at circumneutral pH. A second series of experiments with these same Cd concentrations were also conducted at pH 6 with embryo/larva exposed at 12-36hpf, 36-60hpf, and 60-84hpf. The fish that survived the exposures were raised for 40-60 days whereupon the surviving fish were overdosed with MS222, and X-rayed. The later early life stage fish were more sensitive to Cd toxicity; the chorion appeared to protect the earlier exposed embryos (zebrafish hatch 48-72hpf). Embryo/larva were more resistant to toxicity from dissolved Cd (pH 6) than particulate Cd (circumneutral pH); absence of functional gills during early life stages perhaps explained resistance to dissolved Cd. Notochord lesions (typically lethal within two weeks) occurred when embryos were exposed 12-36hpf. There was no evidence for sensitivity of early life stage zebrafish to Cd induced skeletal deformities that occurred within 40-60 days of Cd exposures. / Graduation date: 2004

M.Sc. / The contamination of water by toxic compounds is one of the most serious environmental problems today. These toxic compounds mostly originate from industrial effluents, agriculture runoff, natural sources (e.g. heavy metals in water from rocks and soil erosion) and human waste. The contamination, which is both “organic” and “inorganic” has an impact on the environment and human health. The demand for water and the pressure to re-use this valuable resource has increased the need for improved techniques and materials to remove pollutants from water. The Nanomaterials Science research group at the University of Johannesburg has focused on developing synthetic polymers that can be employed in water treatment and pollutant monitoring. Recently, cyclodextrins (CD) and carbon nanotubes (CNTs) have been included in polymers for this application. For example, CD-co-hexamethylene-/toluene-diisocyanate polyurethanes and CNT-modified equivalents have been developed and have been successfully applied in removing organic contaminants from water to very low levels.1 Calixarenes are synthetic analogues of cyclodextrins that can be exploited via chemical modification to express a range of properties. In the present study, calixarenes, thiacalixarenes and carbon nanotube-based polymeric materials incorporating these molecules have been synthesised, characterised and tested for removing both organic pollutants (such as p-nitrophenol) and inorganic pollutants (Cd2+, Pb2+) from water. Lead(II) and Cadmium(II) are a threat in South Africa because of their toxicity, and while p-nitrophenol is much less of a problem it represents a useful model organic pollutant. The absorption capacity of the polymers towards heavy metals and organic contaminants was tested by mixing the polymer with synthetic water containing known concentration of the contaminants at about 10 mg/L. Atomic absorption spectrometry (AAS) and ultraviolet-visible spectrometry (UV-vis) were used to determine the levels of heavy metals and organic contaminants, respectively. The target pollutants (Cd2+, 1 see KL Salipira MTech dissertation, University of Johannesburg 2008 Pb2+ and p-nitrophenol) were all successfully removed from water by the various polymers, however the degree of removal and loading capacities of the polymers differed. This information gives some insight into what functional components are needed for making successful adsorbents. It was observed, for example, that ptert- butylcalix[8]arene/hexamethylene diisocyanate (C8A/HMDI) had a higher adsorption capacity towards p-nitrophenol and Pb2+ than towards Cd2+, and also a higher capacity than the corresponding calix[4]arene polymers with smaller calixarene cavities.