Investigating the biosynthesis of long-chain (C20–24) polyunsaturated fatty acids (LC-PUFA), physiologically important compounds including arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in fish is crucial to identify dietary requirements for essential fatty acids (EFA). Moreover, knowledge of the C20–24 LC-PUFA biosynthetic capability of farmed fish species enables us to understand their ability to utilise commonly used raw materials such as vegetable oils, which naturally lack LC-PUFA but include C18 PUFA that are metabolic precursors of LC-PUFA. Studies have shown that the potential of a species for LC-PUFA biosynthesis is associated with the complement and function of fatty acyl desaturase (fads) and elongase of very long chain fatty acid (elovl) genes existing in that species. The present study therefore aimed to investigate these genes in the African catfish (Clarias gariepinus), the most commercially important farmed fish in sub-Saharan Africa. A fads2, a fads6 and four elovl (elovl2, elovl4a, elovl4b, elovl8) cDNAs were cloned and functionally characterised by heterologous expression in yeast. The Fads2 was a bifunctional desaturase enzyme with ∆6∆5 and ∆8 activities, and thus catalysing all the desaturation reactions required for ARA and EPA biosynthesis from C18 precursor fatty acids. Moreover, the C. gariepinus Fads2 enzymes also desaturated 24:5n-3 to 24:6n-3, a ∆6 desaturation required for the biosynthesis of DHA through the so-called “Sprecher pathway”. Functional characterisation of Fads6 by heterologous expression in yeast did not reveal its function. With regards to elongases, the C. gariepinus Elovl2 demonstrated the ability to elongate C20 and C22 PUFA and thus complements the Elovl5 with elongase capability towards C18 and C20 PUFA. The Elovl8 was capable of only limited elongation of C18 and C20 PUFA. Elovl4a and Elovl4b, enable the biosynthesis of very long-chain (>C24) fatty acids, compounds with major roles in vision and fertility of vertebrates. The present study confirmed that C. gariepinus possess all the enzymatic capabilities required for the biosynthesis of ARA, EPA and DHA and, therefore, its physiological EFA requirements could be satisfied with dietary provision of C18 PUFA.

M.Sc. / A comparative study was undertaken in the Olifants River catchment, to determine the water quality at two dam sites, namely, Bronkhorstspruit Dam (control) and Loskop Dam (polluted) in the upper Olifants River system and two river point, namely, at Mamba and Balule in the Kruger National Park, in the lower Olifants River system. Data was obtained during the physical and chemical analysis of water and sediment samples, and during bioaccumulation studies using Atomic Absorption spectrometry, testing for aluminium, chromium, copper, iron, lead, manganese, nickel, strontium and zinc. These tests were conducted on the liver, skin, muscle and gills of Clarias gariepinus. The information collected from Mamba and Balule, was compared with a previous study carried out primarily in the Kruger National Park by Marx, (1996). The study undertaken by Marx (1996) was carried out during a drought period, which allowed for the comparison with results recorded after flooding during the present study. This current research project therefore allowed for the comparison between two different water sources, namely, that of dams and rivers, and under varying environmental conditions. The Aquatic Toxicity index (ATI) developed by Wepener et al. (1992) was employed to facilitate the comparison between the water physical and chemical parameters that were measured, at each sample site, with a single variable being calculated for each sample site per survey. The water quality (ATI values) at the two control sites namely, Bronkhorstspruit Dam and Balule deteriorated substantially after the floods. High water levels due to the floods had a dilution effect on the concentration of pollutants however, toxicants were washed in from upstream and due to surface runoff. The removal of the purifying reed beds upstream of Balule and at the inflow to Bronkhorstspruit Dam, resulted in the release of latent sludge containing metals and organic pollutants into the water column. The ATI value's obtained for the two control points were similar or even higher than those obtained for the two polluted sites namely at, Loskop Dam and Mamba for the autumn and winter sampling periods. During following surveys the water quality at all four sample sites improved, returning to pre flood values by the last survey in summer, namely reflecting similar values as those recorded by Marx (1996) at Balule, Mamba and Loskop Dam for the same period. Sediment concentrations recorded a similar trend to that for water at all four sample sites throughout the study. The bioaccumulation study indicated that the gill concentrations recorded were generally the highest, followed by the liver concentrations. From this one may deduce that gills were the dominant site for metal absorption by the fish, with the excretion of metals also taking place via this route. Liver concentrations are an indication of the activation of protective _mechanisms in the fish sampled, the high concentrations recorded indicate the high physiological response the fish have had to the various levels of exposure. The low concentrations recorded in the muscle indicates the effectiveness of the liver in the detoxification of the fish, indicating that only limited storage of the various metals tested for took place at this site. Skin concentrations were quite high, these concentrations represent the products of an excretory process, which takes place via the skin. The order of concentrations recorded during the present study, were similar to the orders recorded by Marx, (1996) and other authors, however, the order of the skin and muscle concentrations were often reversed. This may be due to the increased excretion of these metals via the skin, resulting from increased exposure to these metals after the floods. Thus the increased exposure

M.Sc. (Zoology) / The freshwater indicator fish species, Clarias gariepinus, was used to assess and compare the health status of fish from the Hartbeespoort Dam (HBPD) and the Groot Marico Bosveld Dam (GM) which was used as a reference site. HBPD is known to be polluted and renowned for its extreme eutrophic state. GM receives water from the Marico River and is said to be in an unmodified natural ecological state. Adult Clarias gariepinus were sampled using gill nets; during low-flow (n=17) and high-flow (n=20) seasons from the HBPD, and once from the GM (n = 20). A histology-based fish health assessment was done using a necropsy based health assessment index and a qualitative and semi-quantitative histological assessment on selected target organs namely the gills, liver, kidney and gonads. Focus was also placed on reproductive health aspects by staging the gonads according to their reproductive development and assessing the motility of activated sperm using computer assisted sperm analysis (CASA). Water samples were analyzed for selected physical parameters and for selected metals. The results showed distinct macroscopic differences in the livers and testes comparing the two sites. Fish from HBPD had fatty livers and the macroscopic morphology of the testes showed abnormalities regarding the interstitial tissue, possibly due to the proliferation of connective tissue. The macroscopic abnormalities of the livers were reflected in the histological assessment, which concluded steatosis, vacuolation, hepatocyte nuclear alterations and the presence of large numbers of melanomacrophage centers (MMCs). Of these alterations, only MMCs and intracellular deposits were found in fish from the GM. When comparing the HBPD low-flow (LF) and high-flow (HF) sampling trips, the fish from the low-flow had more alterations present in all organs. While the CASA results showed that the motility, velocity and progression of sperm were lower in fish from the HBPD for all parameters, results from GM showed the ideal trend expected from the moment of sperm activation until degeneration. The velocity and progression were significantly (p value <0.05) different between HBPD samples and fish from the GM. The water quality showed increased concentrations of selenium, as well as the endocrine disrupting chemicals (EDCs) nonylphenol and di-n-butyl phthalate. According to the selected parameters assessed, it seems like the water of the HBPD has increasing detriment upon fish health.

D.Phil. / The endocrine system regulates important physiological processes in an organism. Numerous natural and synthetic chemicals are released into the environment and can disrupt the normal functioning of the endocrine system, influencing physiological processes even at relatively low concentrations. These chemicals are known as endocrine disrupting chemicals (EDCs). These compounds are highly persistent in nature due to a resistance to biodegradation, and because of their hydrophobicity. EDCs bio-accumulate especially in the fatty tissue of aquatic and terrestrial organisms. Developmental and reproductive disorders are potentially harmful effects of exposure. EDC sources include agricultural and urban runoff, and industrial effluent discharges that eventually pass through sewage- and wastewater treatment plants. These chemicals degrade at various rates and to varying extents during treatment processes, forming many new chemicals. Methods to predict the occurrence of endocrine disruption include the occurrence of intersex, and changes in spermatogenesis and secondary sexual characteristics (SSCs) of supposedly exposed organisms. The urogenital papilla (UGP) is a SSC whose development and morphology is hormonally controlled and may be influenced when exposed to EDCs. The sharptooth catfish (Ciarias gariepinus) is often used in environmental toxicity studies. Sex is determined by the external dimorphic UGP found only in males. Intersex has been observed in this species in South African freshwater systems. A field study was carried out in the Rietvlei (RVD) and Marais (MD) dams in the Rietvlei Nature Reserve (RNR). MD acts as a sedimentation dam before water enters RVD, and is theoretically more polluted. The RNR system receives effluent from various domestic and industrial sources including agriculture, industries, informal settlements and municipal sewage treatment plants. Four sampling surveys, two low and two high flow, were carried out over a period of two years. Supposedly male catfish (n=97) were collected and identified as male or possible intersex based on the UGP. Water and sediment was collected for target EDC and endocrine disrupting metal (EDM) analyses. Fish length and mass, and UGP length and width were measured. Laparotomy was performed and gonads were inspected macroscopically for alterations. A section of the gonad and the entire UGP was removed and processed according to standard techniques for histological analysis. The gonadosomatic- (GSI), hepatosomatic- (HSI) and UGP length (UGPLI) indices were calculated. Fish fat was removed and analysed for target EDCs.

M.Sc. (Environmental Management) / Please refer to full text to view abstract

M.Sc. / The Vaal Dam subcatchment is located in the upper reaches of the Vaal River. As a result the water quality is reasonably good. In contrast the Vaal River Barrage catchment includes the PWV area, resulting in poorer water quality. During this study, a fish Health Assessment Index (HAI) successfully tested in previous studies on the Olifants River System was applied to the Vaal River System. The aim was to determine if the HAI could distinguish between the Vaal Dam and Vaal River Barrage with regards to water quality. Surveys were conducted bimonthly from November 1998 to February 2000. Physical water quality variables were measured. Water and sediment samples were also collected and analyzed (Institute for Water Quality Studies, Department of Water Affairs and Forestry) to verify the HAI results. Rand Water Board and the Department of Water Affairs and Forestry also made water quality data available. Twenty sharptooth catfish, Clarias gariepinus, were collected from both localities with the aid of gill nets. Fish were checked for external parasites on the boat. On land fish were weighed and measured, after which blood was drawn and slime smears made. Blood and slime smears were examined under a light microscope for parasites. The HAI examination was performed after severing the spinal cord. Internal parasite numbers were recorded. From the parasite data collected infestation statistics were calculated. Four variations of the Parasite Index (PI) were incorporated in the HAI and results compared. During each survey gill arch, gill filament, muscle, skin and liver tissues were collected from each fish. These were analyzed for strontium, aluminium, chromium, manganese, iron, lead, copper, zinc and nickel concentrations using atomic absorption spectrophotometry. Differences in water and sediment trace metal concentrations between localities were small. Metal concentrations in fish tissues recorded from both localities were also almost identical. Possible explanations for this trend were discussed in the relevant section. Highest metal concentrations were generally recorded in gill tissue followed by liver, skin and muscle. Physical water quality variables (salinity and conductivity), and macro water analysis (e.g. phosphate and nitrate) indicated that water quality at the Vaal River Barrage was poorer. The HAI confirmed this. Higher index values were recorded from the Vaal River Barrage, with the converse being true for the Vaal Dam. Regression analysis indicated that plasma protein, haematocrit and the index value obtained using the Inverted Parasite Index, most successfully predicted (70 %) from where a randomly chosen fish were collected. When viewing index values obtained using the four versions of the PI, all four distinguished between localities. The discriminatory ability of the Inverted PI was slightly higher than that of the other PI's. It is concluded that the HAI distinguished successfully between the Vaal Dam and Vaal River Barrage on the grounds of water quality. Poor fish health correlated with decreasing water quality (salinity and eutrophication).

M.Sc. / The catchment area of the Olifants River has, over a number of years, been exposed to extensive mining, agriculture and urbanisation activities in the Witbank-Middelburg and Phalaborwa regions which has largely contributed to the deterioration of water quality. Of major concern is the influence anthropogenic activities have on the aquatic ecosystem of the Olifants River within the Kruger National Park and how me health of fish residing In these waters is affected. To determine fish health, a biological monitoring method, the Health Assessment Index (H41), was tested for the first time In South Africa. Four surveys were conducted at Mamba and Balule In the Olifants River, Kruger National Park during 1994 (February, May, July and November). An additional survey was conducted in February 1995 at Loskop Dam. Samples of water and sediment were taken for analysis of metals and physical and chemical water parameters. A maximum of 20 Clarias gariepinus fish were sampled at each location. Evaluation of the fish was done according to guidelines set in the HAI and parasite population composition (prevalence, abundance, mean intensity) was determined. Organ and tissue samples including gills, liver, muscle and skin were analysed for the bioaccumulation of chromium, copper, Iron, manganese, nickel, lead, strontium and zinc, using atomic absorption spectrophotomeby. Metal concentrations in the water at Mamba and Balule were within guideline limits, whereas concentrations at Loskop Dam were above guidelines. As reflected by the application of the Aquatic toxicity Index, Loskop Dam presented with the poorest water quality followed by Mamba then Balule. It was found that certain physical and chemical variables namely fluoride, potassium, sulphate and total dissolved solids concentrations at Mamba and Balule were relatively high, particularly during drier months. Metals accumulated in organs and tissues, with the highest concentrations In the gills followed by the liver, skin and muscle. The discriminant analysis, utilising metal bloaccumulation, discriminates between water quality at Mamba and Balule, revealing a 100 % classification probability for each survey. Values obtained In the application of the HAI indicated that variables with good predictor accuracy were plasma protein, all parasites, endoparasites, liver, white blood cell counts, ectoparasites, skin, fins and gills. The discriminant function for the HAI generally Indicated variables similar to those exhibiting high predictor accuracy. The discriminant function showed relatively low classification probability for each survey. In case 1, where separate endo- and ectoparasite variables were Included in the determination of me discriminant function, probability for me entire study ranged between 47.5 % and 84.2 %. In case 2, where endo- and ectoparasite variables were given a refined score rating system, probability ranged between 62.5 % and 100 %. The low classification probability Indicates either the Importance of repetitive testing for this technique or a total departure from it. Results showed that fish populations with higher HAI values are found in water of poorer quality (Mamba), while healthier fish populations i.e. with lower RAI values are found In water of better quality (Balule). Parasite data shows a similar tendency by indicating that ectoparasites are abundant in water of higher quality, while endoparasftes increase in water of poorer quality. The HAI reflects the condition of fish populations in relation to their aquatic environment, therefore, the HA/ gives an indication of water quality and should be used as a first level screening tool. If complemented by a parasite survey, distinguishing between endoand ectoparasites, the results from the HAI will be enhanced.

M.Sc. / There are 39 known species of Lamproglena to date. A review of all species including the species name, the author, as well as an accompanying sketch and information regarding the distribution, pathology and host preference is given. Lamproglena clariae is a copepod gill parasite found on Clarias gariepinus. It has been observed that they cause extensive gill tissue proliferation, which may interfere with respiration. It is uncertain on which tissue type Lamproglena feeds, as red blood is never apparent in the gut of Lamproglena monodi, whereas in the cases of both L. clariae and L. barbicola they undoubtedly feed on blood, proven by the frequently blood-filled gut. Even thought the mouthparts are not fully understood as yet, it is deduced that feeding involves only the maxillae and maxillipedes. Many poecilostomes lacks mandibles. Also, in the lernaeids the mandible is appendage is the smallest and from the 39 known Lamproglena species, the mandibles are described in only eight species. This study examines the digestive tract and the structure of the mouthparts using both light and scanning electron microscopy and tracts the nerves to establish if L. clariae does possess mandibles. Specimens were collected from Clarias gariepinus in the Vaal Dam after the fish were euthanised and the gills removed. The samples were fixed in an aceto formaldehyde alcohol solution and preserved in 70% ethanol prior to dehydration and embedding in resin. Serial section were made at 5 pm and stained with a trichrome stain. A schematic as well as graphic reconstruction of the digestive system is presented. The digestive system consist of three predominant parts, the fore, mid and hindguts. The foregut has a cuticularised epithelium. The oesophagus has a muscle layer iv which stretched and contracts in order to aid feeding and it extends into the midgut via a funnel-like structure. There are four designated zones within the midgut. The hindgut is a simple, cuticle lined tube. Cell structures are discussed. The bilobed protocerebrum in L. clariae is present. The deutocerebrum innervates the antennules. The tritocerebrum consist of two lobed parts and innervates the antennules. The ventral nerve cord is situated posterior to the cephalon. It protrudes dorsally, from where the posterior commissures innervates. The anterior commissures innervates closer to the tritocerebrum. The nerves leading to the mandibles are innervated from the anterior commissure. The more posteriorly located commissure innervates the nerve to the seta located between the antennae and the maxillae. Both the nerves to the maxillae and the endopodite to the maxillae are innervated from this commissure. Mandibles were left out of previous species descriptions of L. clariae. Other Lamproglena species descriptions, showing the mandibles, revealed slender looking mandibles located in close proximity to the mouth opening. This is contrary to the stout mandibles of L. clariae. Future research topics are highlighted and discussed

Invasive species are of particular concern as they have the potential to alter community structure and food web relationships within their invaded habitats. African sharptooth catfish Clarias gariepinus, a generalist predator, was introduced through an inter-basin water transfer scheme into the Great Fish and Sundays Rivers, Eastern Cape, South Africa, where it threatens the native riverine biota. This thesis assessed its impact from a trophic perspective. Patterns in catfish distribution and abundance revealed an upstream to downstream gradient that was associated with spatial distribution of most species within the mainstream, and a mainstream to tributary gradient that was associated with the spatial distribution of native minnows. The catfish was predicted to occur widely within the mainstem habitats and to decrease progressively along the mainstrem to tributary gradient with the physico-chemical environment being a good proxy for predicting both its occurrence and abundance. The results suggest the catfish proliferated within mainstem habitats where invasion resistance was possibly reduced due to alteration of flow. Population dynamics and size structuring of two native cyprinid minnows Pseudobarbus afer and Barbus anoplus, threatened by catfish, were examined within uninvaded headwater streams in relation to their proximate physical habitats. Their habitats were characterised by seasonal variation in physico-chemical conditions and a spatial variation in substrata compositions. No evidence of differences was found between seasons for density and capture probability for either species. The population size and density for P. afer was found to increase with increasing proportion of boulders. In comparison, B. anoplus population size and probability of capture increased with increasing proportion of bedrock and bank vegetation, respectively. Size structuring was explained predominantly by seasonality and habitat variables for P. afer and B. anoplus, respectively. Stable isotope ratios of carbon and nitrogen were used to compare the spatial variation in both the community-wide and catfish-specific niches and to estimate catfish prey sources from different habitats within the invaded systems. Aquatic community and catfish niches were statistically different among localities, suggesting that each locality had a distinct community-wide trophic structure. Dispersion metrics indicated no evidence of differences in the clustering among individuals, but provided evidence of differences in path trajectories for the comparisons of catfish populations that suggested dietary plasticity within different localities. Dietary studies revealed both ontogenetic shift and omnivory that suggested that catfish may exhibit less pronounced top-down effects within its invaded habitats. Manipulative experiments were used to test the response of benthic macroinvertebrates within two rivers that were differentially impacted by catfish as a presstype disturbance. Macroinvertebrates were non-responsive to catfish presence within a system where catfish had previously been established. In contrast, excluding catfish in this system indicated a response that suggested the importance of refuge within invaded habitats and the possible recovery pattern of certain macroinvertebrate taxa. By comparison, introduction of catfish within previously uninvaded localities provided evidence of direct catfish impact through elimination of conspicuous taxa. Acoustic telemetry was used to investigate catfish movement patterns within an invaded lentic habitat and provided evidence that habitat utilisation was non-random. The shallow and structured river mouth habitat, which was most utilised, was probably the most ideal for its breeding and feeding. This inferred potential overlap with native species and suggested the risk of predation and competitive interference. Catfish also exhibited both nocturnal and diurnal activity patterns that were probably related to feeding.

Im Rahmen der Studien zur Wirkung von Bleinitrat auf die Embryonalstadien des afrikanischen Welses Clarias gariepinus wurde zunächst der Einfluß der Besamung auf den Härtungsprozess des Chorions untersucht, um die Bedeutung des gehärteten Chorions als Schutzfunktion im Hinblick auf Schadstoffeinwirkung zu klären. Das Studium der Embryonalentwicklung war erforderlich, um das Ausmaß der Änderung der Normalentwicklung unter dem Einfluß von Bleinitrat bewerten zu können. Im Rahmen der toxikologischen Untersuchungen der Wirkung des Bleinitrats auf die Embryonalstadien wurden folgende biologische Marker (Biomarker) betrachtet: Änderungen in der Entwicklung und der Schlüpfrate, morphologische und histologische Änderungen, sowie biochemische Veränderungen (Änderungen von Stoffwechsel-Enzymaktivitäten) und molekulare Veränderungen (Erfassung von DNA-Schädigungen). Die Exposition der besamten Eier mit Bleinitrat führte zu einer Verlängerung der Inkubationszeit und zu starken Mißbildungen. Der Rückgang der Häufigkeiten der Mißbildungen mit der Zeit ließ die Annahme zu, daß die mißgebildeten Embryonen starben. Im Gegensatz zu den morphologischen Mißbildungen wurden histopathologische Effekte nur bei Embryonen gefunden, die den höchsten Dosierungen (300 µg/l und 500 µg/l Bleinitrat) ausgesetzt waren. Nach dem Schlupf war das Muster der Enzymaktivitäten nach Exposition mit Bleinitrat uneinheitlich; die Aktivität von G6PDH nahm zu, die von LDH nahm ab und die von PK zeigte unregelmäßige Fluktuationen. Die Embryonalstadien zeigten signifikante Dosis-abhängige Antworten über die Zeit, da das Ausmaß der DNA-Schädigungen signifikant mit den Bleinitrat Konzentrationen anstieg. Vor dem Schlupf konnten bei den Embryonen nach Bleinitrat Exposition keine Änderungen in den Enzymaktivitäten gefunden werden und nur geringe DNA-Schädigungen, d.h die toxischen Effekte waren sehr gering. Eine Erklärung könnte die schützende Wirkung der Eihülle gegenüber Schadstoffen sein. Die gewählten Biomarker stellen sensitive Detektionsmethoden für Bleinitrat dar. So könnten sie sich als sinnvolle Bioindikatoren für Ägypten erweisen, da dort zunehmend Umweltverschmutzung mit Blei und Bleiakkumulation in Lebensmitteln zu verzeichnen ist. / In order to study the embryo-toxic effects of lead nitrate of the African catfish Clarias gariepinus, we first had to study the effect of fertilization on the hardening process of the chorion to clarify the role of the hardened chorion on the protection of the embryo from the pollutants. Also we had to study the embryonic development of C. gariepinus for providing us with a model for comparison when normal patterns of development are altered due the exposure to lead nitrate. The present toxicological work focuses on lead toxicity in different developmental stages of C. gariepinus considering different biological markers (biomarkers) comprising changes in the development and hatching rate, morphological and histological changes, biochemical changes (alteration of metabolic enzymes activity) and molecular changes (monitoring of DNA damage). Exposure of fertilized eggs to lead nitrate prolonged the incubation period and caused severe morphological malformations. Since the frequencies of the morphological malformations decreased with time, we conclude a lethal impact and selected mortality of abnormal embryos. Unlike the morphological malformation, histopathological changes were only recorded in embryos exposed to the highest dosages (300 µg/l and 500 µg/l lead nitrate). In the post-hatching stages, the patterns of the enzymes activities after lead exposure varied, G6PDH increased, LDH decreased and PK showed fluctuations. Embryonic stages revealed significant dose-related DNA damage response over time, since the degree of DNA damage increased significantly with higher lead concentrations. No specific response in the activities of the selected enzymes and low DNA damage were recorded in the pre-hatching stage after exposure to the lead nitrate doses. This means the lead nitrate had a minute toxic effect on the pre-hatched embryos. We conclude that, low susceptibility in pre-hatching stages is most probably a consequence of the chorion, which seems to protect the embryos from a range of external pollutants. The selected biomarkers were sensitive detection methods for low-level toxicity of lead nitrate. Thus, these are useful tools for biomonitoring, urgently required in Egypt with regard to increasing environmental deposition of lead and bioaccumulation in human food recently observed.