Ph.D. / Heavy metals like copper are toxicants commonly found in mining, industrial, agricultural and waste effluents in South Africa¡¯s riverine systems. In high concentrations these metals could have a negative effect not only on river systems, but also on fish populations. It is therefore important to determine the effect of these heavy metals on the fish at an early stage, before the fish show any sign of being exposed to the pollutants and before any bodily functions are affected. It is known that polluted water, especially heavy metal pollution like copper, can cause physiological and biochemical alterations in fish. Histopathology is a mechanism, which can provide an indication of fish health by determining early injury to cells and can therefore be considered an important tool to determine the effect of pollutants like copper on fish tissue. At present very little is known about the effect of toxicants on the histology of fish tissue of freshwater fishes in South Africa. This is important in gonads, where many variables not only in different fish species, but also in the number of developing germ cells in the testes exist. The large number of fish species and the variation in their anatomy and reproductive patterns make knowledge of the anatomy of all fish species difficult. In order to perform a histopathological analysis, it is important to be familiar with the normal histology of the fish organs that are being investigated. It was therefore necessary to describe the histology of selected organs in South Africa¡¯s freshwater fish species. The Mozambique tilapia, O. mossambicus was chosen as test organism and copper was selected as toxicant. The normal morphology and histology of the testes of O. mossambicus were described in order to serve as a baseline study and to provide histological parameters for further toxicological work on the gonadal histopathology of fish. Fish were exposed to sub-lethal concentrations of copper (CuCl2) for a short-term (96 hours) in experimental flow-through systems under controlled laboratory conditions (23 ¡¾ 1¨¬C). An external investigation of each of the 30 control and 40 copper exposed fish was performed. Standard length, body mass and gonadal mass were recorded and the maturity of each fish was obtained histologically. A light and electron microscopic examination was performed in order to describe the histology and histopathological changes. The histopathological changes were obtained by comparing the histology and ultrastructure of the testis of the control group with the information obtained for the testes that were exposed to copper. Fish obtained from the control group that were run simultaneously with every exposure group were used to describe the normal histology of the testes of O. mossambicus. The aim was to describe the normal morphology and histology of the testes of O. mossambicus so that the information obtained during this investigation could serve as a baseline study when dealing with toxicity studies. The different germ cells of the spermatogenesis process were also described with a view to assess not only the reproductive pattern, but also to provide histological parameters which might form a basis for any toxicological work on the gonadal histopathology of fish. Identification of reproductive biomarkers requires an understanding of all the principal cellular components of the testes and their normal range of variation. The cellular components that were described are the Sertoli and Leydig cells as well as the primary and secondary spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa. The information on the GSI, gonad reproductive stages as well as the histological examination provided the baseline which was needed for comparison of the testes of fish that were exposed to copper. The cellular and sub-cellular responses at light and electron microscopic level were examined to determine early cell changes and damage in the testes which were caused by different sublethal exposures of copper. There were no differences in the gross structure of the testes of the control group and the group that were exposed to copper. The testes of the fish that were exposed to 0.16, 0.40 and 2.0 mg Cu/§¤ all revealed disorganisation of the lobules, disintegration of the spermatogonia as well as disintegration of the interstitial tissue. The exposure of fish to sublethal concentrations of copper induced histopathological changes in the testes of all three groups of fish that were exposed to copper. The results also showed that the intensity of the histopathological changes increased with an increase in the concentration of copper. As a biomarker of exposure to toxicants, histopathology represents a useful tool to asses the degree of pollution. The results from this investigation showed a decrease in the GSI after being exposed to copper, when compared with the control groups, which were not exposed to copper. The information on the effect of copper on the GSI, gonad reproductive stages as well as the histological examination also provides information on gonadal health and maturational stage. This information however provides structural, rather than functional information regarding gonadal health. The results showed that the GSI and gonadal staging should be used together with the gonadal histopathology in order to provide a baseline for comparison when dealing with other toxicological studies. The information obtained during this investigation showed that fish histology and histopathology could be used as a biomarker to provide information on the effect of heavy metals on fish health. It is however suggested that the information obtained during this investigation should be extended to field investigation in order to be validated as a reliable histopathological biomarker. / Prof. J.H.J. van Vuren Prof. G.J. Steyn

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

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Numerous workers studied the impact of pollutants and agricultural chemicals, containing heavy metals such as copper (Cu), on soil microbes. It was found that elevated soil Cu levels do have a detrimental effect on soil bacterial populations however the filamentous fungi seemed to be less affected. Most of these studies were conducted in soils containing already relatively high Cu levels and the effect of this heavy metal on the nonfilamentous fungi (i.e. yeasts) was never investigated. The aim of this study was therefore to determine the impact of elevated Cu levels on filamentous fungi and yeasts occurring in soils containing relatively low natural Cu levels. A synthetic selective medium containing glucose as carbon source, thymine as nitrogen source, vitamins, minerals and chloramphenicol as anti-bacterial agent (TMV-agar), was used to enumerate ascomycetous and basidiomycetous Cu resistant yeasts in a sample of virgin soil containing ~ 2ppm Cu. Media that were used to enumerate Cu resistant filamentous fungi were malt extract agar, malt extract agar with streptomycin sulfate, maltyeast- extract-peptone agar with chloramphenicol and streptomycin sulfate, benomyl–dichloran-streptomycin medium for the enumeration of hymenomycetous fungi and two selective media for the isolation of mucoralean fungi. Cu resistant fungi able to grow on all of the above mentioned solid media supplemented with 32 ppm Cu occurred in the soil sample. To obtain an indication of the level of Cu tolerance of fungi present in this soil sample, a number of fungal isolates were screened for the ability to grow on a series of agar plates, prepared from glucose-glutamate-yeast extract agar, containing increasing concentrations of Cu. It was found that filamentous fungi and yeasts that were able to grow on this agar medium containing up to 100 ppm Cu were present in the soil. A series of soil microcosms was subsequently prepared from the soil sample by experimentally contaminating the soil with increasing amounts of copper oxychloride, were after fungal populations in the microcosms, including Cu resistant fungi, were monitored using plate counts. At the end of the incubation period, after 245 days, fungal biomass in the microcosms was compared by determining the concentrations of the fungal sterol, ergosterol, inthe soil. Generally, Cu had little impact on the numbers of filamentous fungal colony forming units on the plates, as well as on the ergosterol content of the soil. The numbers of filamentous fungi in the soil, including the Mucorales and hymenomycetes, seemed to be less affected by the addition of copper oxychloride than the numbers of soil yeasts able to grow on TMV-agar. The focus of the next chapter was on the response of yeasts in different soils to elevated levels of Cu in the soil. TMV-agar was used to enumerate yeasts in soil microcosms prepared from four different soil samples, which were experimentally treated with copper oxychloride resulting in Cu concentrations of up to 1000 ppm. The selective medium supplemented with 32 ppm Cu was used to enumerate Cu resistant yeasts in the microcosms. The results showed that the addition of Cu at concentrations ≥ ~1000 ppm did not have a significant effect on total yeast numbers in the soil. Furthermore, it was found that Cu resistant yeasts were present in all the soil samples regardless of the amount of Cu that the soil was challenged with. At the end of the incubation period, yeasts in the microcosms with zero and ~1000 ppm additional Cu were enumerated, isolated and identified using sequence analyses of the D1/D2 600-650bp region of the large subunit of ribosomal DNA. Hymenomycetous species dominated in the control soil, while higher numbers of the urediniomycetous species were found in the soil that received Cu. These observations suggest that urediniomycetous yeasts may play an important role in re-establishing overall microbial activity in soils following perturbations such as the addition of Cu-based fungicides. / AFRIKAANSE OPSOMMING: Vele navorsers het al die impak van besoedelingstowwe en landbouchemikalieë wat swaarmetale soos koper (Cu) bevat, op grond-mikrobes bestudeer. Dit is gevind dat verhoogde Cu vlakke ‘n nadelige effek het op grond-bakteriese populasies, maar dat die filamentagtige fungi geneig is om minder geaffekteer te word. Meeste van hierdie studies is gedoen met gronde wat alreeds relatief hoë Cu vlakke bevat het en die effek van hierdie swaarmetaal op die nie-filamentagtige fungi (d.i. giste) is nooit ondersoek nie. Die doel van hierdie studie was dus om die impak van verhoogde Cu vlakke op filamentagtige fungi en giste in gronde, wat natuurlike lae vlakke van Cu bevat, te bepaal. ‘n Sintetiese selektiewe medium wat glukose as koolstofbron, timien as stikstofbron, vitamiene, minerale asook chloramfenikol as anti-bakteriese agent bevat (TMV-agar), is gebruik om askomisete en basidiomisete Cu weerstandbiedende giste in ‘n monster ongeskonde grond, bevattende ~ 2dpm Cu, te tel. Media wat gebruik is om Cu weerstandbiedende filamentagtige fungi te tel, was mout-ekstrak agar, moutekstrak agar met streptomisiensulfaat, benomiel-dichloran-streptomisien medium vir die tel van hiemenomiseetagtige fungi en twee media vir die isolasie van mukoraliese fungi. Cu-weerstandbiedende fungi wat op al die bogenoemde media, aangevul met 32 dpm Cu, kon groei, het in die grondmonster voorgekom. Om die mate van Cu-weerstandbiedendheid van fungi wat in die grondmonster voorkom, te bepaal, is ‘n getal fungus-isolate op agarplate, voorberei met glukose-glutamaat-gis ekstrak agar, bevattende verhoogde konsentrasies Cu, nagegaan. Daar is gevind dat daar filamentagtige fungi en giste in die grond voorkom wat die vermoë het om op media bevattende 100 dpm Cu te groei. ‘n Reeks grond mikrokosmosse is dus voorberei vanaf die grondmonster deur om dit eksperimenteel te kontamineer met verhoogde hoeveelhede koper oksichloried, waarna die fungus-populasies asook die Cu-weerstandbiedende fungi in die mikrokosmos gemoniteer is deur middel van plaattellings. Aan die einde van die inkubasie periode, 245 dae, is die fungus biomassa in al die mikrokosmosse bereken deur die konsentrasie van die fungus sterool ergosterool te bepaal en dit met mekaar te vergelyk. Oor die algemeen het Cu min impak ten opsigte van diegetal filamentagtige fungi kolonie vormende eenhede die plate, asook op die ergosterool inhoud van die grond gehad. Dit wil voorkom of die getal filamentagtige fungi in die grond, insluitende die Mucorales en die hymenomisete, minder geaffekteer is deur die toediening van koperoksichloried as die aantal grondgiste wat op die TMV-agar kan groei. Die fokus van die volgende hoofstuk was dus op die reaksie wat giste in verskillende grondtipes gehad het op verhoogde Cu in die grond. TMV-agar is gebruik om die getal giste te bepaal in die grond mikrokosmosse van die vier verskillende grondmonsters, wat voorberei is deur om dit eksperimenteel met koper oksikloried te kontamineer tot en met Cu konsentrasies van 1000 dpm. Die selektiewe medium wat gesupplementeer is met 32 dpm Cu, is gebruik om Cu weerstandbiedende giste in die mikrokosmosse te bepaal. Die resultate toon dat die toevoeging van Cu by konsentrasies ≥ ~1000 dpm nie enige beduidende effek op die totale gis getalle gehad het nie. Daar is ook gevind dat daar Cu weerstandbiedende giste in die grond monsters voorkom gekom het ten spyte van die hoeveelheid Cu wat tot die grond toegevoeg is. Aan die einde van die inkubasie periode is die giste wat die die mikrokosmosse bevattende nul en ~1000 dpm Cu getel, geïsoleer en geïdentifiseer deur gebruik te maak van DNA volgorde bepaling van die D1/D2 600-650 bp areas geleë in die groter subeenheid van die ribosonale DNA. Hymenomisete spesies het in die grond kontrole gedomineer, terwyl hoër getalle uredinomisete spesies in die grond met addisionele Cu gevind is. Die resultate dui daarop dat uredinomisete giste dalk ‘n belangrike rol kan speel in die hervestiging van die oorwegende mikrobiese aktiwiteit in grond na skoktoestande soos die aanwending van Cu-gebaseerde fungisiede.

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The detection of anthropogenic impacts in our estuaries has become a critical social, political and scientific concern in recent years. Work has focussed on the effects of these impacts on the spatial and temporal patterns of biotic assemblages and searched for bioindicators and biomarkers of pollutants that may act as early warning signs. The estuaries in temperate Southeastern Australia have a diversity of biotic assemblages living in the soft sediment. One of the most abundant macroinvertebrates is the little studies mussel, Xenostrobus securi that is found living with an assemblage of benthic biota including amphipods, crabs, isopods and tanaids, bivalves and gastropods. Two commercial fish species bream and mullet also inhabit these estuaries. Numerous stormwater drains can be found entering the estuaries through the mangrove forests lining the shores. Along with inputs of freshwater, stormwater drains are thought to be responsible for the entry of heavy metals into estuaries. These heavy metals have the potential to alter the patterns of biotic assemblages and bioaccumulate in the tissues of miacroinvertebrates, mussels and fish living within temperate estuaries. Over the time of this study the concentrations of heavy metals in the sediment tissues and shell of X.Securis varies spatially and temporally. Although this study adds substantially to current knowledge there is still more that is needed to establish X. Securis as a bioindicator. Questions remain about uptake, depuration and response to environmental gradients of heavy metals in X. Securis. Before X Securis can be used routinely in monitoring heavy metal contamination these questions need to be further investigated / Doctor of Philosophy (PhD)

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Heavy metal contamination of mangroves is of critical concern due to its accumulative and adverse effects in aquatic ecosystems. This study was undertaken to investigate the effects of mercury (Hg ), lead (Pb ), copper (Cu ) and zinc (Zn ) on plant responses, specifically growth and productivity, in Avicennia marina (Forssk.) Vierh. A. marina plants were grown for twelve months in pots contaminated with Hg +, Pb +, Cu2+ and Zn2+ at concentrations of 0, 40, 80, 120 and 160 ppm (1 ppm = 1 (agmf1). Accumulation and distribution of the heavy metals in shoot and root tissues were determined using atomic absorption spectroscopy (Perkin-Elmer Model 303) while secretion of the heavy metals from leaves was studied using scanning electron microscopy and energy dispersive X-ray microanalysis. I hypothesized that heavy metals have deleterious effects on plant growth and that they are absorbed by roots and secreted from salt glands present on the leaves. SEM X-ray microanalyses confirmed secretion of Cu + and Zn + ions as well as salt (NaCl) from glandular structures on both the adaxial and abaxial surfaces of leaves; however Hg2+ and Pb2+ were not detected in the secretion. Ion concentrations were significantly higher in plant roots than in shoots, particularly at 160 ligml"1 for all heavy metals. In addition, toxic levels of Hg and Pb were detected in the shoot tissue; however, Cu2+ and Zn2+ were within the normal ion concentration in the shoots. Plant height, number of leaves, biomass accumulation and chlorophyll content were significantly lower at 160 ugml" than the control values for all heavy metals. Carbon dioxide exchange, transpiration and leaf conductance generally decreased with increasing metal concentration. CO2 exchange at a concentration of 160 (J-gmf1 was significantly lower than the control for all metals. CO2 exchange at 160 ugml"1 for Hg2+, Pb2+, Cu2+ and Zn were 49.6 %, 55 %, 47.6 % and 63.6 % respectively lower than the control values. Photosystem II (PS II) quantum yield, photochemical efficiency of PSII (Fv/Fm) and electron transport rate (ETR) through PS II generally decreased with increasing concentration for all heavy metals. XV This study has shown that A. marina experiences dose-dependent stress responses to Cu2+, Zn2+, Hg2+ and Pb2+ in shoot and root tissue at a concentration of 160 lagmi"1, evidenced by decreases in growth and photosynthetic performance. The results also ~)A- "7-1- 9-1- "J-\-indicate that CuZT, Znz\ HgZT and PbZT are taken up by roots and transported to shoots. In addition, only Cu and Zn are secreted via the glands while Hg and Pb accumulate within the shoots. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2008.