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

Cu is one of the most toxic elements that affect fish populations when the fish are exposed to concentrations exceeding their tolerance. To investigate the effects of elementary Cu on aspects of bioconcentration, histology and behaviour, O. mossambicus were exposed to 0 and 0.75 ± 0.20 mg/l of Cu for 96 hours (short-term study), and 0, 0.11 ± 0.02, 0.29 ± 0.02, and 0.47 ± 0.04 mg/l of Cu for 64 days (longterm study) under controlled conditions in the laboratory. For the long-term study fish were sampled for gills, liver, and kidney Cu accumulation analysis after 1, 32 and 64 days of exposure and after 1, 2, 4, 16, 32, and 64 days for gills, liver and spleen histology analysis. Cu accumulation was concentration-duration dependent with the highest accumulation capacity in the liver. A multifactor linear model was developed for the relationship between exposure dose, exposure duration and Cu accumulation in the organs with the liver model: Log L = 3.35 + 0.85W + 0.31T (r² = 0.892) giving a better fit than the gills: G = −35.09 + 10.58W + 17.58T (r² = 0.632). Where L = Cu accumulation values in the liver, G = Cu accumulation values in the gills (both in μg/g dry mass); W = exposure dose in water (mg/l); and T = exposure time (days). Using this model Cu accumulation in organs can be estimated when exposure concentration and duration is known. This model should be tested under different conditions to determine the potential of the model in monitoring Cu toxicity in the environment. Lesions were observed in the liver, gills and spleen in all Cu treatments at all exposure concentration and exposure durations. However, the incidence and the degree of alteration was related to the concentration of Cu and duration of exposure. The sequential appearance of lesions in the order of, hepatic vacuolar degeneration, fatty degeneration and necrosis indicated a gradual increase in liver damage with larger duration of exposure time and increasing Cu concentration. The initial lesions in the gills were manifested as hypertrophy and hyperplasia of the gill epithelium causing increase in the thickness of the secondary lamellae, mucous cell hypertrophy and proliferation, mucous hypersecretion, proliferation of eosinophilic granule cells and hyperplasia of interlamellar cells. With increase in exposure time, necrosis of the eosinophilic granule cells, lamellar oedema, epithelial desquamation and increase in severity of lamellar hyperplasia were observed. These lesions indicated an initial defence mechanism of the fish against Cu toxicity followed by advanced histological changes that were related to Cu concentration and duration of exposure. Changes in the spleen were haemosiderosis, increase in the white pulp and macrophage centres, reduction in the red pulp, and necrosis suggesting that fish exposed to environmentally relevant levels of Cu may be histopathologically altered leading to anaemia and immunosuppression. Regression analysis was used to quantify the relationship between the total activity of the fish, and duration of exposure. There was a gradual decline in fish activity related to Cu concentration and duration of exposure before introducing food into the tanks. There was a constant activity after introducing food in the tanks at the control and 0.11 ± 0.02 mg/l Cu exposure levels irrespective of exposure time. Analysis of covariance (ANCOVA) was used to test for the difference in slopes between treatments. There was no significant difference (p > 0.05) between slopes of the control and 0.11 ± 0.02 mg/l Cu, and between 0.29 ± 0.02 and 47 ± 0.04 mg/l Cu before and after introducing food in the tanks. The slopes of both the control and 0.11 ± 0.02 mg/l Cu were significantly different from those of 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu (p < 0.05). There were significant differences in the mean opercular movements per minute between treatments (p < 0.05). There was hyperventilation at 0.11 ± 0.02 mg/l Cu i.e. 87 ± 18 opercular movements per minute (mean ± standard deviation) and hypoventilation at 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu i.e. 37 ± 34 and 13 ± 6 opercular movements per minute compared to the control. Hypo- and hyperventilation were related to the lesser and greater gill damage, respectively. In conclusion Cu accumulation and effects on histology of the liver, gills and were related to the concentration of Cu in the water and duration of exposure showing a gradual increase in incidence and intensity with larger duration of exposure time and increasing Cu concentration. The fish were initially able to homeostatically regulate and detoxify Cu. However, as the exposure continued, the homeostatic mechanism appears to have failed to cope with the increasing metal burden causing advanced histological changes.

M.Sc. / The organochlorine insecticides were amongst the first pollutants shown to cause adverse population effects. The potential adverse effects of these pollutants on wildlife are a cause for great concern. Severities of their effects were sometimes surprising given the low levels of the compounds in environmental compartments such as surface waters and soils. High lipophilicity combined with chemical stability and very slow biodegradation are characteristic features of these toxic Persistent Organic Pollutants (POPs). Regional declines in fish, bird as well as invertebrate populations resulting from long term exposure to POPs such as 1,1-bis (4-chlorophenyl) -2,2,2-trichloroethane (DDT) and its stable metabolite 1,1-bis (4-chlorophenyl) -2,2-dichloroethene (DDE), could be related to some biochemical, endocrine and physiological effects in individuals. Some POPs have been suggested to have negative effects disrupting physiological processes and resulting in alterations of homeostasis, reproduction, development and behavior. Such adverse effects upon populations may be avoided if the potential of chemicals to cause them is recognized before problems arise. The aim of this study was to determine whether or not the ongoing spraying of DDT in the Limpopo Province is negatively affecting the health of aquatic species found in surface water of the area. Extensive research has shown that biomarkers have been very effective in the trace determination of a number of adverse effects caused by metals, and thus, are also being used for POPs. A battery of biomarkers (EROD, CAT and CEA) were used, both in the field and in a controlled laboratory environment, in order to try and determine the long term effects of exposure to low environmentally relevant levels of DDE in the selected area. DDT levels in the biota, water and sediment samples were also measured to determine the possible levels of exposure. Dose-response relationships were most successfully determined by the EROD and the CEA biomarkers in this study. In a controlled laboratory study, a definite effect was noted on the Mozambique Tilapia with increasing concentrations of DDE. In the natural environment, dose-response relationships to DDE exposure were more difficult to quantify as additional chemicals and natural environmental stressors also affect the results.

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

Among the 155 species of fish recorded so far in the St Lucia estuarine lake, Oreochromis mossambicus and Ambassis ambassis are the two most prominent. Although originally endemic to southern Africa, O. mossambicus is now one of the most widely distributed exotic fish species worldwide. Together with A. ambassis, they have become the dominant fish species in the St Lucia estuarine lake since the closure of the mouth in 2002 and are, therefore, a crucial component of the food webs throughout the system. After a decade dominated by dry and hypersaline conditions, the St Lucia system has changed dramatically in terms of prevailing environmental conditions, as a result of higher than average rainfall at the end of 2011 and the onset of a new wet phase at the start of 2012. In response, A. ambassis, which prefers lower salinity regimes, has expanded its distribution range throughout the estuarine lake. Stable δ¹⁵N and δ¹³C isotope analysis was used in conjunction with gut content analysis to elucidate the diet of these species at sampling localities spanning the geographical range of the system and determine whether these species shift their diet in response to environmental or climatic shifts. From both studies it is evident that from a temporal and spatial scale these two species adopt similar, yet very different, dietary tactics. Oreochromis mossambicus was shown to adopt a generalist feeding strategy, opportunistically feeding on dietary items that are available thus allowing this species to alter its diet according to the environment that it inhabits. Trophic positioning of this species was found to be controlled by salinity in St Lucia as dietary composition differed greatly between sites. In contrast, Ambassis ambassis displayed a more specialist dietary composition, feeding predominantly on zooplankton. However, this species also opportunistically supplements its diet with additional sources when available. Trophic position of A. ambassis was higher in the dry season owing to the increased productivity of the system during the wet season. The success and dominance of both species in the St Lucia system can therefore be attributed to their dietary strategies. Under extreme environmental conditions, O. mossambicus has the added advantage of its wide tolerance of different environmental conditions, particularly salinity, thus allowing it to proliferate. / M.Sc. University of KwaZulu-Natal, Durban 2014.

Water is one of the most important resources in South Africa. The increased pressure on river systems in SA as a result of human activities and industrial development is evident from the systematic deterioration of the Olifants River (Mpumalanga). While the number of water users grows daily, the river is seen as a convenient disposal site. It is necessary to continually monitor the river to be aware of its status. Regular monitoring also supplements the existing data on water quality, biotic communities and possible points of pollution. The study had the following aims: Firstly to contribute to a larger project on the effect of pollutants on the physiology of fish populations in the Olifants River. This study concentrates on the bioaccumulation of certain metals (i.e. chromium, copper, iron, manganese, nickel, lead, strontium and zinc), in the gills, liver and muscle of Labeo congoro. Standard methods were used to prepare the organs and tissues for metal analysis using atomic absorption spectrophotometry. The following conclusions were reached subsequent to statistical analysis of the results: • The highest mean concentrations of all metals were recorded in the liver of Labeo congoro. • The lowest mean metal concentrations were calculated in the muscle, except for chromium, the concentrations of which were the lowest in the gills. • The highest mean concentration of each metal was recorded in organs / tissues of fish sampled at locality 2. • None of the three localities can be singled out to indicate the lowest mean concentration of each metal. Summary iii The second aim of this study was to determine the 96-hr LC50 (lethal concentration at which 50% of the test population dies) of nickel. Oreochromis mossambicus was used as test organisms for these laboratory studies. A flow through system was used to ensure that the organisms were exposed to the same concentration of nickel for 96 hours. Subsequent to the 96-hr LC50 determination, test organisms were subjected to sublethal exposure of nickel to determine the effects of the various concentrations of the haematology and blood coagulation processes of fish. The following conclusions were drawn from the statistical data processing: • The 96-hr LC50 of nickel for Oreochromis mossambicus is 50 μg.l-1. • The exposure to sublethal concentrations of nickel did effect certain haematological variables • No statistically significant differences in the blood coagulation variables at different sublethal concentrations of nickel were confirmed The results of this study can be used to supplement the database on the water quality and general status of the Olifants River (Mpumalanga). The LC50 of nickel can be used as one of the variables in water quality studies. / Prof. Johan van Vuren

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Fish infections caused by pathogenic Flavobacterium spp. are a major problem in the aquaculture industry worldwide, often leading to large economic losses. Thirty-two Flavobacterium spp. isolates, obtained from various diseased fish species and biofilm growth, were characterised genetically using 16S rRNA gene sequencing, 16S rRNA gene PCR restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) element PCR, plasmid profiling, whole cell protein (WCP) and outer membrane protein (OMP) analyses. The biofilm-forming capability of five genetically heterogeneous Flavobacterium spp. study isolates was investigated using a modified microtiter-plate adherence assay, as well as flow cell studies. Experimental infection studies with Mozambique tilapia (Oreochromis mossambicus) were carried out in order to determine the virulence of the Flavobacterium spp. study isolates. 16S rRNA gene sequence analysis showed the Flavobacterium spp. study isolates were closely related, and 97% sequence similarity was shared with published F. johnsoniae sequences. A high degree of genetic heterogeneity was displayed by the Flavobacterium spp. study isolates following RAPD-PCR, REP-PCR and OMP analysis, however, based on the results obtained by plasmid profiling and WCP analysis, the isolates appeared genetically very homogeneous. The biofilm phenotype was displayed by all five Flavobacterium spp. isolates tested and varied from weakly to strongly adherent. No specific correlation was observed between the RAPD, REP and/or OMP profiles and degree of adherence displayed by Flavobacterium spp. isolates. However, a specific WCP profile (profile B), exhibited by 48% of the Flavobacterium spp. isolates, was linked to strong adherence. Experimental infection studies showed that Flavobacterium spp. isolates displayed variable levels of virulence, which could not be linked to biofilm formation, nor specific genotypes. This is the first reported isolation and characterisation of Flavobacterium spp. isolated from diseased fish in Southern Africa, and there appears to be significant diversity amongst the isolates which is not geographically linked nor host related. / AFRIKAANSE OPSOMMING: Visinfeksies veroorsaak deur Flavobacterium spp. is problematies in die akwakultuur industrie wêreldwyd en lei tot groot ekonomiese verliese. Twee en dertig Flavobacterium spp. isolate, geïsoleer vanaf verskye geïnfekteerde visspesies en biofilm groei, was geneties gekarakteriseer met behulp van 16S rRNS geenvolgorde, 16S rRNS geen PKR restriksie fragment lengte polimorfisme (RFLP), toevallig geamplifiseerde polimorfiese DNS (TGPD) PKR, herhaalde ekstrageniese palindromiese (HEP) element PKR, plasmied profilering, heelsel protein (HSP) en buite membraan protein (BMP) analise. Die vermoë van vyf geneties heterogene Flavobacterium spp. isolate om biofilms te vorm was ondersoek met behulp van ‘n gemodifiseerde mikrotiterplaat vashegtings toets asook vloei-sel studies. Eksperimentele infeksie studies was uitgevoer op bloukurpers (Oreochromis mossambicus) om die virulensie van die Flavobacterium spp. studie isolate te toets. 16S rRNS geenvolgorde analise het getoon dat die Flavobacterium spp. studie isolate naby verwant was, en het 97% ooreenstemming getoon met gepubliseerde F. johnsoniae volgordes. TGPD-PKR, HEP-PKR en BMP analise het ‘n hoë graad van heterogeniteit tussen die Flavobacterium spp. studie isolate aangetoon, egter, op grond van plasmied profilering en HSP analise, was die studie isolate geneties baie homogeen. Die biofilm fenotipe was getoon deur al die getoetsde Flavobacterium spp. isolate en het gevarieer van swak tot sterk vashegting. Geen spesifieke korrelasie was waargeneem tussen die TGPD, HEP en/of BMP profiele en graad van vashegting vertoon deur Flavobacterium spp. isolate nie, maar ‘n spesifieke HSP profiel (profiel B), getoon deur 48% van die Flavobacterium spp. isolate, was verbind met sterk vashegting. Eksperimentele infeksie studies het getoon dat Flavobacterium spp. isolate varierende grade van virulensie vertoon het en wat met biofilm formasie of spesifieke genotipes geassosieer kon word nie. Hierdie is die eerste gedokumenteerde isolasie en karakterisering van Flavobacterium spp. geïsoleer van geïnfekteerde vis in Suider Afrika, en daar is beduidende diversiteit tussen die isolate wat nie geografies of gasheer geassosieerd is nie.

Thesis (MSc. (Zoology)) -- University of Limpopo, 2014 / The Olifants River is presently one of the most threatened river systems in South Africa. The Upper and Middle catchments are heavily impacted by anthropogenic activities, however little is known of the status of the river in the Lower catchment in the Limpopo Province. Thus the aim of this study was to evaluate the impact of water quality on the health of Oreochromis mossambicus and Schilbe intermedius at the Phalaborwa Barrage. This was achieved through assessing the water (seasonally) and sediment quality (bi-annually) at three sites in the Phalaborwa Barrage (inflow, middle and the wall). Ten specimens of each of the two selected fish species were seasonally collected using gill nets of different mesh sizes from May 2010 to January 2011. The water, sediment and the dorsal muscle tissues from both fish species were analysed for selected metals at an accredited laboratory by means of ICP-OES spectrometry and the human health risk factor upon consumption of fish contaminated with metals was assessed. Fish health was determined at the field laboratory by applying the HAI, PI and IPI. In addition, the condition factor (CF) was determined for the selected fish species during all seasons. All water constituents with the exception of turbidity during spring (wall), anions (Cl, F and SO4) and cations (Ca, K, Mg and Na) fell within the target water quality range (TWQR) for aquatic ecosystems throughout the study. Inorganic nitrogen concentrations showed eutrophic (spring) and oligotrophic conditions (autumn, winter and summer), while phosphorus indicated eutrophic (autumn, winter and spring) and hypertrophic (summer) conditions throughout the study. There was no significant difference in anions (F=0.07; df=3; p=0.97), cations (F=0.57; df=3; p=0.64) and nutrients (F=0.79; df=3; p=0.56) detected in water. The metals that were detectable in the water samples were: Al, Fe, Sn, B, Sr, Se, Ba, Mn, Sb and As, while in sediment all metals (Fe, Al, Ti, Mn, Ba, B, Zn, Ag, V, Cr, Ni, Sr, Sn, Co, Cu, Cd, Pb, As and Sb) were detected with the exception of Se. The average metal concentrations detected above the TWQR are; Al, Fe, Se, Sn, Sb and As, in the sediment only Cr and Cd were above suggested quality guidelines. There was no significant difference in metals detected in water (F=0.05; i df=3; p=0.98) and sediment (F=1.62; df=1; p=0.21). Higher HAI values were recorded for S. intermedius than O. mossambicus, indicating that O. mossambicus was in better health than S. intermedius. For S. intermedius, parasites contributed the most to the total HAI followed by abnormal liver conditions, haematocrit levels (internal variables) and abnormal gills (external variable). While for O. mossambicus, parasites, liver, haematocrit (internal variables), gills, eyes, fins and skin (external variables) contributed to the HAI values. There was no significant difference in HAI values for O. mossambicus (F=1.9; df=3; p=0.15) and S. intermedius (F=2.58; df=3; p=0.66) throughout the study. The PI and IPI for ectoparasites for both fish species, were higher than the PI for endoparasites. There was a significant difference for PI values of O. mossambicus (F=0.07; df=3; p=0.041) and S. intermedius (F=3.65; df=3; p=0.019) during the study. Oreochromis mossambicus had higher CF values than S. intermedius and both fish species had values that indicated good fish. Metals that were detectable in O. mossambicus muscle tissue included: Ba, B, Zn, Fe, Sr, Al, Mn, Cr, Ag, Pb, Ni, Cu, Se, V, Sb, Ti, Sn and Co, while for S. intermedius Ba, B, Zn, Fe, Al, Cr, Sr, Mn, Cu, As, Pb, Se, Sn, Ag, Ti, Ni, Sb and Co were recorded. There was no significant difference in bioaccumulation levels between the two fish species (F=1.412; df=3; p=0.250). The human health risk assessment indicated that fish muscle tissues are unsuitable for continuous (weekly/daily) consumption. As some carcinogenic metals i.e. Pb, Cr and Sb for O. mossambicus and Pb, As and Cr for S. intermedius were detected at HQ levels of more than 50 times the recommended value of one. In conclusion, the Phalaborwa Barrage is impacted by metals (Al, Fe, Se, Sn, Sb and As) and occasionally with nutrients. The water and sediment quality and metal bioaccumulation have confirmed the latter statement and to a lesser extent the HAI, PI and IPI. The human risk assessment done indicated that continuous consumption of fish by humans, especially O. mossambicus may have adverse effects on the health of humans.

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.

Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005