Ph.D. (Zoology) / Please refer to full text to view abstract

M.Sc. / Please refer to full text to view abstract

There are many types of chemicals/contaminants present in the environment, ranging from synthetic chemicals to trace metals that are required for life. When an organism is exposed to a toxicant, adverse effects are generally first at the sub-organismal level. There is thus a need for physiological and biochemical indicators of organismal health and sub-lethal toxicant effects. A bio-indicator, or biomarker, is a response of a living organism to environmental changes or because of exposure to contaminants. The main objectives of this study were to evaluate and investigate the possible use of biomarkers as indicators of sub-lethal (chronic) effects induced by certain chemicals/pollutants on the Mozambique tilapia, Oreochromis mossambicus. The biomarkers were evaluated as a possible sensitive and reliable measure of effects due to exposure to pollutants. This study was divided into laboratory experiments and field evaluations. The laboratory experiments consisted of the evaluation of biomarkers in exposure studies on 18-24 day old O. mossambicus juvenile fish, to determine the sub-lethal effects induced by chemicals. Acetylcholinestrase (AChE), Ethoxyresorufin-O-deethylase (EROD) and glucose levels acted as the battery of biomarkers evaluated. Thirty (30) juvenile fish were exposed for 24 and 96-hours to sub-lethal concentrations of cadmium, zinc, pentachlorophenol (PCP) and cyanide. For the field evaluation, mature Oreochromis mossambicus of both sexes, were caught during the summer and winter at the Rust de Winter Dam, Loskop Dam and Hartebeespoort Dam. Rust de Winter Dam acted as the reference site. The biomarkers evaluated during the field studies include: erythrocyte AChE, ƒx-Aminolevulinic acid dehydratase (ALAD) and plasma glucose levels, EROD and liver glycogen. The laboratory (exposure) test used during this study was not sensitive enough to obtain reliable, repeatable results. But standardised procedures were established for possible use in future studies. During the field studies, ALAD, EROD and plasma glucose showed significant results and differences between the reference and polluted sites. More accurate results could be obtained at impoundments with higher levels of pollution. Although, erythrocyte AChE and liver glycogen did not show significant results during this study, they might also show more accurate and reliable results at higher levels of pollution. In future, chemical water analysis should be carried out during field evaluation to determine what chemicals/toxicants are present in the aquatic environment. Biomarkers will show the level of effects of the toxicant on the organism, while chemical analysis will determine the specific pollutant present in the water. / Prof. J.H.J. Van Vuren

M.Sc. (Zoology) / There has been an increasing concern worldwide regarding the possible adverse effects of pharmaceutical supplements present in our aquatic ecosystems and whether or not they modify the physiological functioning in humans and wildlife. Trenbolone acetate (TBA) and zeranol (Z) for example, are two commonly used synthetic anabolic growth promoting hormones in cattle production. TBA is metabolized into trenbolone-β and excreted as both trenbolone-α and -β. In liquid manure trenbolone-β has a half-life of over 270 days and Z, 120 days. Therefore if released into the surrounding environment there is the possibility for long-term severe ecological impacts i.e. fish reproduction and general health. The aim of this study was to determine the physiological effects of several growth promoting hormones used as growth promoting hormones in cattle production on the Mozambique Tilapia – Oreochromis mossambicus. The growth promoting hormones assessed in this study were; Trenbolone acetate, Methyltestosterone, Diethylstilbestrol and Zeranol. The aim was accomplished by making use of histology (gills, liver and gonads) and three biomarker assays; Glutathione-S-transferase (GST), Uridine-Diphosphate Glucuronosyltransferase (UDPGT) and Cellular Energy Allocation (CEA). Stock solutions of Trenbolone acetate (14 μg/l and 15 μg/l), Methyltestosterone (7 μg/l and 7.5 μg/l), Zeranol (2.8 μg/l and 3 μg/l) and Diethylstilbestrol (0.28 μg/l and 0.29 μg/l) were prepared. Fish were exposed under controlled conditions for a period of 24-hours, 4-, 15- and 30-days respectively using a flow-through system. The aquarium water was changed (45 L removed and replaced with 45 L of prepared growth hormone containing bore-hole water) every 48 – 72 hours to remove all waste material thus ensuring the aquariums were clean. Upon performing the necropsies, gills, liver and gonads were removed and examined using standard histological techniques. Muscle tissue was used to determine the CEA, liver and kidney tissue was used for both GST and UDPGT assays. The results obtained from the histology revealed that the gills and liver were not severely affected by exposure to the growth promoting hormones although possible exposure related alterations were evident. The gonads results indicated that exposure to the growth promoting hormones severely affected the morphology and functioning of the organs to the point where reproduction is questionable. The results obtained from the hepatosomatic index (HSI) and gonadosomatic index (GSI) revealed no significant differences (p<0.05) although a trend of increasing HSI and decreasing GSI was evident in the male fish exposed to the androgens. With regards to the biomarker assays there were minor decreases in CEA in the exposed fish but no significant differences (p<0.05) could be established. The GST assay revealed that Zeranol prompted a significant increase (p<0.05) in GST activity in the kidney after 4- and 15-days of exposure while the liver displayed no change in GST activity. The UDPGT assay revealed minor fluctuation in UDPGT activity in both the kidney and liver throughout the study, however, no significant differences (p<0.05) could be established. To conclude, exposure to these growth promoting hormones at the selected concentrations and exposure periods severely compromised the fish’s reproductive capabilities thus challenging the fish’s fitness. Further studies examining the energy metabolism and xenobiotic detoxification pathways of the Mozambique tilapia and other indigenous fish species are recommended to better comprehend the effects that these growth promoting hormones may possess.

M.Sc. (Zoology) / The catchment area of the Olifants River is being subjected to increased agricultural and mining activities, industrial development and urbanisation. As a result of this, the water quality 'of the Olifants River and some of its tributaries (e.g. Selati River) has been deteriorating since 1983. This causes reason for concern as one of the downstream water users in the Olifants River catchment is the Kruger National Park, which requires of good quality to sustain its terrestrial and aquatic ecosystems It was therefore necessary to determine what extent activity midstream-or--tlie I Olifants River, especial y in the Phalaborwa area, influenced the water quality of the Olifants River. In this study, an evaluation was done of the water quality (physically and chemically of the Lower Olifants River inside of the Kruger National Park, as well as the Lower Selati River, a tributary of the Olifants River which flows through the Phalaborwa area. Special attention was paid to the metal concentrations in the water, sediment and fish. Water and sediment were sampled every alternate month from April 1990 to February 1992 at six sampling sites along the Lower Olifants River and one in the Selati River. Standard methods were used to determine the physical and chemical characteristics (e.g. pH, TDS, etc.) of the water. The fish species Barbus marequensis was also sampled every alternative month, but only at three sampling sites along the Olifants River and one in the Selati River. Organs and tissues that were dissected, included the gills, fat, liver, gonads, gut, muscle, skin, blood, vertebrae, kidney and bile, as well as the gut contents. In February 1992, additional sampling was performed at Pioneer Dam, a natural reference point used in this study. Atomic absorption spectrophotometry was used in the laboratory to determine the Cr, Cu, Fe, Mn, Ni, Pb, Sr and Zn concentrations in the water, sediment and fish samples. (The water quality of the Selati River was found to be stressful to aquatic life, especially .) with regard to the sodium, fluoride, chloride, sulphate, potassium, total ~dissolved salts 1 and metal concentrations (except strontium). The Selati River also influenced the ': water quality of the Olifants River after the Selati-Olifants confluence. Most of the :. time the water quality of the Lower Olifants River in the KNP did, however, comply With the recommended guideline limits, except for the metal concentrations. The high metal concentrations detected in the water (in some cases sub-lethal levels) indicated some degree of metal pollution, but, due to the hardness of the Olifants River water, conditions were not necessarily toxic to the aquatic life. The accumulated metals in the organs and tissues of marequensis gave a good indication of the metal levels to which the fish were exposed. More metals were taken up by the fish from April 1990 to February 1991 than from April 1991 to February 1992. The various metals were . distributed differently to the tissues of B. marequensis, with the highest zinc concentrations being accumulated by the skin and ovaries; the highest copper and iron by the liver, kidney and gut; the highest chromium and nickel by the blood; and the highest manganese, lead and strontium by the vertebrae and gills. These results afforded guidelines as to the types of tissue which should be sampled as the most suitable for the analysis of the various metals in polluted waters.

M.Sc. / Developments in the catchment area of the Olifants River, such as poor agricultural practices and overgrazing, caused increased amounts of erosion material to enter the river. This leads to the silting up of the Phalaborwa Barrage, situated in the river, on the border of the Kruger National Park. Because of the occasional flushing of the Barrage, in order to retain storage capacity, the Olifants River inside the Kruger National Park is subjected to very high sediment loads. This has led to poor water quality and subsequent fish mortalities. The objective of this study was therefore to investigate the possible effects of silt loads released from the Barrage on the survival of selected life stages of Oreochromis mossambicus. Silt was collected from the Phalaborwa Barrage, and the silt/fish interaction was invesigated under controlled laboratory conditions. Oreochromis mossambicus was exposed to various silt concentrations for different exposure periods to determine the LC50 and sub-lethal concentration ranges. After determination of sub-lethal values, 0. mossambicus was exposed to these concentrations to determine whether they have any effects on the oxygen consumption and selected haematological parameters of exposed fish. Lethal levels for suspended sediment are interrelated with age differences of the fish, particle size of the suspended sediment and concentration. Increased suspended sediment disrupted activity and respiratory patterns, changed orientation responses and ultimately resulted in the death of the fish. The gills and the opercular cavities of the dead fish were usually clogged with a thick layer of mucus and silt. The laboratory experiments revealed that LC5 0 values for 0. mossambicus ranged from 26,78 to 54,20g silt/1 for larvae, 35,10 to 39,20g silt/1 for juveniles and 32,93 to 35,43g silt/1 for adult fish. The larval stage was generally less tolerant of suspended silt than juveniles and adults. Sub-lethal levels for adult fish ranged from 0 to 27g silt/l, where no mortalities occurred. Exposure of adult fish to sub-lethal concentrations of silt did alter routine oxygen consumption by causing increased rates (9 to 330% from initial) of oxygen consumption. This indicates that sub-lethal levels of suspended silt concentrations result in an increase in oxygen consumption by the fish due to an increase in metabolic rate associated with increased activity and stress. Silt exposure Exposure of adult fish to sub-lethal silt concentrations (<20g silt/l) resulted in increases in haemotocrit, red blood cell counts, white blood cell counts and blood glucose concentrations, but decreases in haemoglobin and mean corpuscular volume when compared to that for the control fish. These differences became more apparent as the silt exposure concentration increased with statistically significant differences (P<0.05) between most of the selected

Contamination of aquatic ecosystems (for example, dams, lakes, rivers, streams) with heavy metals (according to McDonald and Wood (1993), the metals that are of greatest concern in fresh waters are Hg, Pb, Cd, Cu, Zn, AI, Mn and Cr (approximately in order of decreasing toxicity)) has been receiving increased worldwide attention, and the literature has many publications on the topic. These amplified levels of contamination have become a global problem in both developed and developing countries and the impact of them on aquatic environments is severe due to the inability of water to disperse contaminants. To meet this threat, ecotoxicologists face a difficult challenge on how to assess the long-term impacts of metals which have already been released into the environment and which persist in sediment, water and biota. In recent years, the field of biomarker study has evolved and expanded rapidly to address this void. Biomarkers examine molecular and biochemical responses in exposed individuals in an effort to assess the status of an impacted environment and they can also serve as a basis for a Rapid Risk Assessment of Fish Health (RRAFH) employed by water quality managers. This effort to incorporate biomarkers into RRAFH research will eventually be worthwhile. Moreover, with this multidisciplinary approach, cause-effect relationships may be examined. It was in this context that the present study evaluated three biomarkers (metallothionein (MT), Na+K+ ATPase and heat shock/stress protein 70 (HSP 70)) to determine the integrated physiological responses in Oreochromis mossambicus to one metal (copper (Cu)) at a sub lethal level over the short term (96, 48, 24, 16, 12, 6, 4 and 2 h respectively). The information gained from these tests was valuable in suggesting what the baseline levels of biomarkers in O. mossambicus would be, as studies of metabolic adjustments to stress in fish have been focused on salmonids. The demand for sensitive, specific and rapid biological assays was also addressed by the development of an HSP 70 ELISA. This assay with, with additional refinement, will also prove to be a valuable tool in the management of Cu pollution in this species. Evaluation of the biomarkers in the gills of O. mossambicus indicated that this species has relatively high levels of MT in their gills compared with other species of fish. Exposure to sub lethal Cu concentrations [Cu] caused fluctuating, but not significant, levels in Na+K+ ATPase and HSP 70 during the exposure time, and the actual [Cu] in the gill seems to be the biggest factor when interpreting results. Significant decreases were found in MT levels in the gill tissue at 2 h and 12 h of exposure. HSP 70 results also indicated non-significant decreases to Cu exposure. Data from the gills analysed in the current study suggest that down-regulation in the biomarker response might be a better indicator of Cu exposure or effect in O. mossambicus. These data also suggest that further studies should rethink the amount of Cu that the fish under investigation would find sub-lethal as this metal seems to be very well regulated in this species of fish and seems frequently to non-significantly effect the chosen biomarkers. Evaluation of the biomarkers in the liver of O. mossambicus indicated that MT levels present in the liver most likely fall within the in range of normal baselevels for tilapia living in a Cu-rich environment and that HSP 70 in the liver only responds significantly to increased aqueous [Cu] at 16 h of exposure. This study also confirms current knowledge that the toxicity assessment of any pollutant, including Cu, cannot be exclusively based on one biomarker and that the true value of these biomarkers only becomes evident when they are used not as individual indicators but as integrated responses that influence one another. All these results furnish useful data for future research into the sub lethal effects of a pollutant such as Cu. The next step would be to test the usefulness of these physiological methods for detection of similar responses in natural fish populations inhabiting Cu polluted waters. This will provide a database relating the presence of Cu to biological effects at a molecular level. / Prof. J.H.J. Van Vuuren

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Forming part of a conservation programme, this study was concerned with two species of Cichlid fish (Oreochromis mossambicus and O. ni/oticus), which were brought into contact with each other by unnatural ways. They are now hybridizing to some extent and there is also evidence that the foreign O. ni/oticus may out compete the native O. mossambicus. To cast light on what the current distribution is of both these species and the hybrids in Southern Africa, it is important to identify specimens very accurately. In attempting to find genetic markers to distinguish between two species of Cichlids we tested 20 microsatellite dinucleotide (CAn) repeats during a preliminary study and found five of these promising to exhibit little intra-specific genetic diversity but large genetic variation between species. We amplified these five loci in 145 individuals from 10 populations, which included the two species and their hybrids. Exact sizes of the fragments were determined using an automated DNA sequencer. Between the two species, allele sizes were overlapping, but when data were analyzed by statistical models, the differences could be seen for populations, however on individual level there was overlap between the species. The hybrids were found to be intermediate positioned between the two pure species. Our attempt to assign individuals to populations provided doubtful results. Thus, using this set of markers, populations can be ascribed to one of these species, but not individuals by themselves. / AFRIKAANSE OPSOMMING: As deel van 'n natuurbewarings program, word daar in hierdie studie twee spesies van vis ondersoek was in kontak met mekaar gekom het op onnatuurlike wyse. Hierdie twee visspesies vanuit die CICHLIDAEfamilie (Oreochromis mossambicus en 0. ni/oticus) kan hibridiseer wanneer hul saam voorkom, maar dit is ook bekend dat die uitheemse O. ni/oticus die inheemse O. mossambicus kan bedreig in terme van leefruimte, kos en broeispasie. Om die voorkoms van hibriede tussen die twee spesies te ondersoek in Suider Afrika se varswater opvangsgebiede, is dit baie belangrik om individue baie akkuraat te identifiseer. In hierdie poging om genetiese merkers te vind wat die twee spesies van mekaar onderskei, het ons 20 mikrosateliet di-nulkleotied (CAn) herhalende volgordes op verskillende loci ondersoek. Vyf daarvan het belowend voorgekom om as spesie spesifieke merkers te dien. Die fragmente op die vyf loci is ge-amplifiseer in 145 individue vanuit 10 populasies. Presiese groottes van die fragmente is bepaal met behulp van 'n ge-outomatiseerde DNA volgorde bepaler waarna genotiepes vir elke individu toegeken is. Tussen die twee spesies het alleel groottes oorvleuel, maar wanneer data geanaliseer word met behulp van statistiese metodes, was verskille tussen die spesies duidelik op populasie vlak. Die hibriede het intemediêr tussen die twee spesies voorgekom. Dus met behulp van hierdie stel merkers kan onderskei word tussen die twee spesies op populasie vlak, hoewel individue nie op sig self identifiseer kan word nie.

Thesis (MPhil (Animal Sciences. Aquaculture))--University of Stellenbosch, 2005. / This paper contains literature on tilapia culture, feeding and nutritional factors of prime consideration to survival and growth of Oreochromis mossambicus. Results are presented for a three-phase laboratory experiment on survival of O. mossambicus fry in an attempt to evaluate the use of live Spirulina platensis. The experiment was conducted on fry in a closed system in an effort to maximize the use of live Spirulina and also optimize growth and production. Fry were tested for tolerance levels of salinities, 0-35 g/lt, and showed favourable survival rates up to 15 g /lt salinity without being fed. Manipulation of input in freshwater turned high fry mortalities with increasing rates without difference for physical form of Spirulina. Growth was not significantly affected by types of input. However fry grew well at 0-40% rates with considerable survival performance. It is noted good quality of water that allow improved survival and growth of fry in a closed system may be assured with rates of input up to 5 or 10% of bodyweight. These input rates can guide use of live Spirulina in saline water tilapia culture if Spirulina proves good productivity at the consistency of fry tolerance to the salinity levels established in this paper.

Assignment (MPhil)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Many species of tilapia such as Oreochromis mossambicus are euryhaline, able to adapt to different salinity waters. Their ability to withstand high salinity levels has given rise to the possibility of using tilapia as baitfish for tuna line fishery. The purpose of the study was to determine the survival rate of tilapia O. mossambicus during direct transfer from freshwater to the salinity levels of 0, 15, 20, 22.5, 25, 27.5, 30, 32.5, and 35 ppt. The data was analysed through means of univariate ANOVAand regression analysis. O. mossambicus showed no mortality to all salinity regimes up to 25 ppt. Mortality was observed at 27.5 ppt, with 100% mortality at 35 ppt. LC 50 and LC 90 were found to be 30.5 and 34.2 ppt, respectively. The results indicate that tilapia (0. mossambicus) will survive a direct transfer to salinities up to 25 ppt. acclimation will be required in the event of transfer to salinity levels above 25 ppt, in order to prevent significant levels of mortalities. / AFRIKAANSE OPSOMMING: Meeste van die tilapia spesies soos Oreochremis mossambicus het die vermoë om by water van verskillende soutgehaltes aantepas. Dit is hierdie vermoë om hoë sout vlakke te weerstaan wat die moontlikheid vir gebruik as lewende aas in die tuna langlyn visvangbedryf moontlik maak. Die doel van hierdie studie was om die oorlewingsvlak van tilapia, O. mossambicus te bepaal by die oorplasing van varswater direk na soutwater by vlakke van 0, 15, 20, 22.5, 25, 27.5, 30, 32.5, en 35 dele per duisend. Die data is verwerk deur gebruik te maak van eenvariant ANOVAen regressie analises. O. mossambicus het geen mortaliteite tot gevolg gehad by al die oorplasings van vlakke tot en met 25 dele per duisend sout nie. Mortaliteite is wel gevind vanaf 27.5 dele per duisend, met 100 % mortaliteite by 35 dele per duisend. LC 50 en LC90 was gewees 30.5 en 34.2 dele per duisend onderskeidelik. Die resultate toon aan dat tilapia (0. mossambicus) sal oorleef by direkte oorplasing na soutwater by vlakke van tot en met 25 dele per duisend. Tilapia wat na hoër vlakke as 25 dele per duisend oorgeplaas wil word, sal eers geleidelik moet akklimatiseer om mortaliteite te beperk.