M.Sc. / The Olifants- and Luvuvhu rivers both flow through the Kruger National Park (KNP). The Olifants River (OR) is a major tributary of the Limpopo River with water quality being less than desirable due to high concentrations of pollutants as a result of the activities in the upper catchment. The crocodile population of the river declined from 1000 in 2008 to 347 in 2009 due to pansteatitis. Labeobarbus marequensis in the upper catchment also showed symptoms of the disease (Templehoff, 2010). This raised concern about the health of the fish in the OR, particularly in the section flowing through the KNP. The Luvuvhu River (LR) is also a tributary of the Limpopo River with main land uses in the catchment being agriculture, mining and communal lands. Of concern is the finding of DDT residues in water, sediment, and domestic and indigenous biota upstream of the sites sampled in this study (Barnhoorn et al., 2009) which raised concerns about the health of fish in the lower reaches of the river flowing through the KNP. Therefore, the aim of this study was to assess the health status of [9] selected fish species from the OR and LR in the KNP using a histology-based health assessment protocol (HBHA). The species selected for this study included Hydrocynus vittatus, Labeobarbus marequensis, Labeo cylindricus and Labeo rosae. The OR was sampled in September 2009 and May 2010 while the LR was sampled in November 2009 and April 2010. A necropsy was done to determine if there were any internal or external macroscopic alterations with any alterations noted on a score sheet. Blood was taken for haematocrit (Hct), leukocrit (Lct) and total plasma protein (TP) determination. As part of the HBHA, a necropsy-based health assessment index (HAI) was applied using a modified protocol of Adams et al. (1993) where the necropsy and blood parameters were used to calculate the mean and sum HAI for the different species sampled on each sampling trip. Biometric indices including the condition factor, hepatosomatic index, splenosomatic index and the gonadosomatic index were calculated. Age was determined using otoliths for tigerfish and scales for the other species. Samples of selected organs (liver, kidney, gill, testes or ovaries) were taken for histological analysis. Microscope slides were assessed qualitatively to identify any histological alterations present. These results were semi-quantitatively assessed according to the protocol of Van Dyk et al. (2009a) from which an organ index and fish index were calculated. The organ index is an indication of the number and severity of histological alterations in a particular organ of a selected species. The organ indices were classed according to the classes of Van Dyk et al. (2009a) with Class 1 (<10) being tissue with slight histological alterations; Class 2 (10-25) being tissue with moderate histological alterations. The fish index is a sum of all of the organ indices for any given fish sampled.

PhD / An important component of fish health is an optimally functioning reproductive system. The Luvuvhu River Catchment in the Limpopo Province, South Africa, is a tropical, high-risk malaria area where 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), an endocrine disrupting chemical (EDC), has been used annually since 1945 as a malaria vector control. DDT is known to affect testes morphology and motility of fish sperm. As such, testicular histology and sperm motility (kinematic) parameters were studied as reproductive indicators of the reproductive capacity for two wild, indigenous fish species (Oreochromis mossambicus and Clarias gariepinus) from the currently DDT sprayed area. Three field studies were carried out over two years (2007 – 2008), including two high flow (HF) periods and one low flow (LF) period [HF 1 (March 07), LF (October 07), HF 2 (February 08)]. Both species were sampled from three sites on the Luvuvhu River for testicular histology and computer assisted sperm analysis (CASA), during all three field studies. The sites included a reference site outside the DDT sprayed area, Albasini Dam (AD), and two exposed sites within the DDT sprayed area, Xikundu Weir (XW) and Nandoni Dam (ND). CASA, based on open-source software, was used for the first time in South Africa to assess sperm kinematic parameters of indigenous fish species in field conditions. These included percent motile sperm (% MOT), curvilinear velocity (VCL μm s-1), velocity of an average path (VAP μm s-1), straight line velocity (VSL μm s-1), linearity (LIN %), progression (PROG μm), and average efficiency (AVE. EFF.). Water and sediment samples were collected during all field studies from the three sites for metal and EDC analysis. Controlled laboratory studies were also carried out on the sperm of both species, externally sourced from aquaculture farms equipped to breed and raise fish in toxicant free water. The laboratory studies involved in vitro exposure of spermatozoa to two different, but environmentally relevant, concentrations of both DDT (DDT 1: 0.27 μg L-1; DDT 2: 0.5 μg L-1) and 1,1-dihloro-2,2-bis(p-chlorophenyl)ethylene (DDE) (DDE 1: 0.11 μg L-1; DDE 2: 1.0 μg L-1) with the aim to provide data to support the possible outcomes found in the field studies using CASA. Furthermore, peroxidation of sperm lipids was assayed by production of malondialdehyde (MDA) after in vitro exposure of spermatozoa to DDT and DDE. DDT and its metabolites were found in varying concentrations in the water from all three sites (0.1 μg L-1 – 1.2 μg L-1). Levels of dieldrin (3.5 μg L-1) and lindane (9.4 μg L-1) residues were also found at XW in HF 2. The histological results revealed alterations to testis tissue of both species at all three sites. The testes were assessed through the identification of alterations and an organ index was calculated: Testes Index (IT). The index is indicative of the histological response in the respective tissue type. O. mossambicus at XW had the highest mean IT value during LF (7.45 ± 5.73) and for all field studies combined (5.47 ± 4.63), primarily due to the occurrence of testicular oocytes (intersex), where the frequency of prevalence was 72.73% and 58.82% respectively. These results were statistically higher than the laboratory control (C) group. The CASA results showed statistical differences primarily for O. mossambicus, where motility parameters were lower at XW when compared to AD. Laboratory exposures found a decrease in sperm motility (% MOT) between the control (C) group and the DDT 1, DDE 1 and DDE 2 exposed groups for C. gariepinus. No significant differences were seen for lipid peroxidation (MDA). On the other hand, no significant differences were seen in CASA parameters between the control and exposed laboratory groups for O. mossambicus, but there was an increase in MDA production from the control to the DDT 1 exposure group.