Changes in the fish communities of the Breede and Mbashe estuaries were investigated in relation to the environmental variables that influence the fish assemblages in these systems. The Breede Estuary (34° 24’21.6”S, 20° 51’ 08.2”E) occurs within the warm-temperate/cool-temperate transition-zone, while the Mbashe Estuary (32o 14' 55.4"S, 28o 54' 03.7"E) falls within the subtropical/warm-temperate transition-zone along the South African coastline. The Breede Estuary was sampled (seine and gill nets) during summer (January-March) over a period of 10 years (2002-2012), while the Mbashe Estuary was sampled (seine net) during spring (October) over a period of three years (2010-2012). The proportion of tropical fish species was higher in the Mbashe Estuary compared to the Breede Estuary, while the Breede Estuary contained a higher proportion of temperate species than the Mbashe Estuary. Although the abundance of individual species in both estuaries varied, the ranking of species in the Breede Estuary was stable over the 10 year period. Multivariate analysis showed that the fish communities within each reach (upper, middle, lower) of the Breede Estuary remained similar, regardless of year. In contrast, species composition in the Mbashe Estuary differed significantly between years mainly due to differences in the river flow regime during the study period. Environmental variables responsible for structuring the fish assemblage in each estuary differed. Salinity significantly impacted the spatial fish assemblage structure of the Breede Estuary, with most of the species recorded being associated with the more saline lower reaches. The abundance of tropical species near their distributional limit in the Breede Estuary appeared to be impacted by sea surface temperature (SST) as peak total abundance of these species coincided with the warmest SST, while the lowest total abundance coincided with the coldest annual SST during the study period. The fish assemblage structure in the Mbashe Estuary was impacted by differing freshwater input prior to sampling, with flooding negatively impacting the mean total abundance, species composition and diversity in this system. The variables that influenced the fish assemblage structure of both transition-zone estuaries will be affected by climate change in the future. These variables will, in turn, determine the composition, abundance and diversity of species within these important estuarine systems. In order to measure these impacts with any degree of understanding, long-term studies on the abiotic and biotic (including the ichthyofauna) features of these estuaries are required to interpret climate change trends.

The biogeography and community structure of the fishes of South African estuaries was investigated. In all, 109 systems were examined representing two broad types: temporarily closed and permanently open estuaries. Multivariate analyses of the fish communities identified three biogeographic regions. A cool-temperate region extended along the west and southwest coasts; a warmtemperate zone stretched along the south, southeast and east coasts and a subtropical region occurred along the east coast. The boundaries of these biogeographic regions were also delineated. The general physico-chemical characteristics of the estuaries within the three biogeographic regions also reflected regional differences in climate, rainfall and ocean conditions. Estuarine temperatures followed the trend for marine coastal waters, decreasing from subtropical estuaries toward cool-temperate systems. The low rainfall and runoff in the warm-temperate region together with high evaporation rates and strong seawater input resulted in higher salinities in these estuaries. These factors also accounted for the predominantly clearer waters in warm-temperate estuaries. The estuaries in the three biogeographic regions were also shown to contain somewhat distinctive fish assemblages. Temperature and salinity appeared to be the two main factors affecting the distribution and abundance of fishes in South African estuaries. Subtropical systems were characterised by fishes mostly of tropical origin as well as certain south coast endemic species. Warm-temperate estuaries were dominated by endemic taxa with some tropical species also present. The fish fauna of cooltemperate estuaries mostly comprised south coast endemic species with cosmopolitan and temperate taxa also present. Certain functional components of the ichthyofauna also exhibited slight differences between regions. Freshwater fishes were a major component of closed subtropical estuaries while estuarine resident species were more abundant in warm-temperate estuaries. Overall, estuarine-dependent marine species dominated the fish fauna of the estuaries in all biogeographic regions, signifying that South African estuaries perform a vital nursery function for this group of fishes. Slight differences were also apparent in the trophic structure of the fishes; these were related to environmental differences between regions. Zooplanktivores and fishes that feed on aquatic macrophytes/invertebrates assumed a relatively higher importance in warm-temperate systems. Overall, detritivores dominated the estuarine fish fauna in all regions, indicating that detritus forms the main energy source in South African estuaries.

The Mngazana and Mngazi estuaries are situated just south of Port St. Johns close to the biogeographical boundary between the warm temperate and subtropical zones along the east coast of Southern Africa. The Mngazana estuary is a permanently open estuary with three species of mangrove. The Mngazi estuary is a temporarily open/closed system. The ichthyofauna was sampled with seine nets and gillnets during June 2005, August 2005, November 2005, February 2006 and April 2006. The fish community structure was significantly different between the two estuaries with the differences being especially noticeable in the larger fish sampled with gillnets. There were significant differences in the fish community sampled with the seine net on both spatial and temporal scales in both of the estuaries. Certain warm temperate species were less abundant and certain subtropical species were more abundant at the Mngazana estuary than they were during a study conducted in 1975. Stomach content analysis revealed that most species had similar diets in the two estuaries with the only differences occurring among benthic feeding species. The diet of sub-adult piscivores comprised of a large proportion of penaeid prawns while the juveniles feed mainly on mysids. The zooplanktivorous fish feed mainly on copepods and mysids with the proportions varying between the species. Benthic feeding fishes showed the greatest variability in diet with plant material, detritus, crabs and copepods being the main food sources. Stable isotopes revealed that particulate organic matter and microphytobenthos were the most important carbon sources to fish in the Mngazi estuary. In the Mngazana estuary a combination of mangrove material, detritus, particulate organic matter and Zostera material were the most important carbon sources. The importance of each of these sources did, however, vary greatly between the different trophic guilds of the fish in the Mngazana estuary. The distribution of certain fish species was investigated in relation to physical parameters (temperature, salinity, turbidity and depth) and biological parameters (prey and predators) in the environment. It was found that most small fish (< 100 mm SL) were strongly correlated with turbidity where-as they showed weak correlations with their prey. Sub-adult piscivores fell into two groups, i.e. those that could tolerate turbid water and those that were only found in clear water. The group that also occurred in turbid water showed very strong correlations with their prey but the clear water group did not. It would appear that the small and thus vulnerable fish seemed to occur in areas that offered them protection rather than areas where their food supply is most abundant, where-as the piscivores were more influenced by prey distribution, as long as the physical conditions were tolerable to them.

Marine Protected Areas (MPAs), the Ecosystem Approach to Fisheries management (EAF) and Integrated Coastal Management (ICM) have been identified as possible alternatives to traditional linefish management measures, which have largely failed. Monitoring and assessment of fish communities on a long-term basis is necessary, and will provide a means to evaluate the effectiveness of such management measures. Therefore, standardised protocols and optimal sampling methods for long-term monitoring (LTM) and assessment of coastal fish communities are essential. This study aimed to identify suitable methods and develop a protocol for assessment of inshore reef fish communities. A suitable location for evaluation of proposed methods was identified in the warm temperate biogeographical region of South Africa, encompassing the well-established Tsitsikamma Coastal National Park MPA and an adjacent exploited area. Chrysoblephus laticeps (roman) was identified as an indicator species for the study, as it has been well-studied and is well represented in the area. Underwater visual census (UVC) and controlled fishing were identified as suitable methods. UVC transects were found to be superior to point counts, in terms of sampling efficiency, variability, bias and required sample size. An effort of two angler hours per fishing station was shown to provide low catch variability, while at the same time a representative catch and low overall cost and required time. The methods were incorporated in a proposed sampling protocol, and evaluated. The methods were able to detect known differences between protected and exploited communities. It is recommended that LTM within protected areas, for detection of natural change, be focused on community-level indicators, while LTM in exploited areas, aimed at detection of anthropogenic change, be focused on species-level indicators. The proposed protocol with standardised methods will allow for comparisons across a network of LTM sites and provide the opportunity for a broad-scale assessment of the effects of environmental variables on reef fish stocks. The protocol developed in this study has application in other biogeographical regions in South Africa, and other parts of the world. Shift in the focus of much marine research, in South Africa and elsewhere, to LTM, highlights the relevance and timeous nature of this study.

White steenbras Lithognathus lithognathus (Pisces: Sparidae) has been a major target species of numerous fisheries in South Africa, since the late 19th century. Historically, it contributed substantially to annual catches in commercial net fisheries, and became dominant in recreational shore catches in the latter half of the 20th century. However, overexploitation in both sectors resulted in severe declines in abundance. The ultimate collapse of the stock by the end of the last century, and the failure of traditional management measures to protect the species indicate that a new management approach for this species is necessary. The species was identified as a priority for research, management and conservation in a National Linefish Status Report. Despite knowledge on aspects of its biology and life history, little is known about juvenile habitat use patterns, home range dynamics and movement behaviour in estuaries. Similarly, the movement and migration of larger juveniles and adults in the marine environment are poorly understood. Furthermore, there is a complete lack of information on its genetic stock structure. Such information is essential for effective management of a fishery species. This thesis aimed to address the gaps in the understanding of white steenbras movement patterns and genetic stock structure, and provide an assessment of its current conservation status. The study adopted a multidisciplinary approach, incorporating a range of methods and drawing on available information, including published literature, unpublished reports and data from long-term monitoring programmes. Acoustic telemetry, conducted in a range of estuaries, showed high site fidelity, restricted area use, small home ranges relative to the size of the estuary, and a high level of residency within estuaries at the early juvenile life stage. Behaviour within estuaries was dominated by station-keeping, superimposed by a strong diel behaviour, presumably based on feeding and/or predator avoidance, with individuals entering the shallow littoral zone at night to feed, and seeking refuge in the deeper channel areas during the daytime. Conventional dart tagging and recapture data from four ongoing, long-term coastal fish tagging projects, spread throughout the distribution of this species, indicated high levels of residency in the surf zone at the late juvenile and sub-adult life stages. Consequently, juvenile and sub-adult white steenbras are vulnerable to localised depletion, although they can be effectively protected by suitably positioned estuarine protected areas (EPAs) and marine protected areas (MPAs), respectively. It has been hypothesized that adult white steenbras undertake large-scale coastal migrations between summer aggregation areas and winter spawning grounds. The scale of observed coastal movements was correlated with fish size (and age), with larger fish undertaking considerably longer-distance coastal movements than smaller individuals, supporting this hypothesis. Given the migratory behaviour of adults, and indications that limited spawning habitat exists, MPAs designed to protect white steenbras during the adult life stage should encompass all known spawning aggregation sites. The fishery is plagued by problems such as low compliance and low enforcement capacity, and alternative management measures, such as seasonal closure, need to be evaluated. Despite considerable conventional dart tagging effort around the coastline (5 782 fish tagged) with 292 recaptures there remains a lack of empirical evidence of fish migrating long distances (> 600 km) between aggregation and spawning areas. This uncertainty in the level of connectivity among coastal regions was addressed using mitochondrial DNA sequencing and genotyping of microsatellite repeat loci in the nuclear genome, which showed no evidence of major geographic barriers to gene flow in this species. Samples collected throughout the white steenbras core distribution showed high genetic diversity, low genetic differentiation and no evidence of isolation by distance or localised spawning. Although historically dominant in several fisheries, analysis of long-term commercial and recreational catch data for white steenbras indicated considerable declines and ultimately stock collapse. Improved catch-per-unit-effort in two large MPAs subsequent to closure confirmed that MPAs can be effective for the protection of white steenbras. However, the current MPA network encompasses a low proportion of sandy shoreline, for which white steenbras exhibits an affinity. Many MPAs do not prohibit recreational shore angling, which currently accounts for the greatest proportion of the total annual catch. Furthermore, EPAs within the juvenile distribution protect a negligible proportion of the total available surface area of estuaries – habitat on which white steenbras is wholly dependent. Despite some evidence of recent increases in abundance in estuaries and the surf zone in certain areas, white steenbras meets the criteria for “Endangered” on the IUCN Red List of Threatened Species, and for “Protected species” status on the National Environmental Management: Biodiversity Act of South Africa. The species requires improved management, with consideration for its life-history style, estuarine dependency, surf zone residency, predictable spawning migrations and its poor conservation status. The multidisciplinary approach provides valuable information towards an improved scientific basis for the management of white steenbras and a framework for research that can be adopted for other overexploited, estuarine-associated coastal fishery species.

Long-term interannual changes in richness, abundance, diversity and structure of the fish community in the temporarily open/closed East Kleinemonde Estuary, Eastern Cape, are described and the recruitment success of two estuary-dependent marine species assessed. In addition, laboratory experiments were conducted to assess the possible role of olfaction in the recruitment process of an estuary-dependent marine fish species. Multivariate analyses of the annual marine fish communites identified two distinct groups with more species recorded during years that succeeded spring (September to November) mouth opening events than in years following no mouth opening events in spring. Interannual community stability (IMD) and seriation (IMS) also increased from the ‘other’ to the ‘spring’ years. These results highlight the importance of the timing of mouth opening to the marine fish community in a temporarily open/closed estuary. This study also made use of long-term records of daily mouth state and linked them to the recruitment of distinct year-class cohorts in two spardis with contrasting lifehistory characteristics. Lithognathus lithognathus only recruited into the estuary in years when the mouth opened between late August and January. This was linked to the limited spawning season of this species and its inability to enter closed estuaries via wave overwash events. In contrast, recruitment by Rhabdosargus holubi juveniles appeared to be uninterrupted and was not determined by the seasonality of mouth opening. This species dominates the marine-spawning component of the East Kleinemonde Estuary and its success is attributed to an extended spawning season and its ability to recruit into estuaries during both overwash and open mouth conditions. Attraction of postflexion Rhabdosargus holubi larvae to estuary, surf zone and river water was also measured using a rectangular choice chamber. In two sets of experiments, conducted during peak recruitment periods, larvae from both the surf zone and estuary mouth region selected estuary water with a significantly higher frequency than sea water. Larvae collected in the mouth region showed a stronger preference for river water than those collected in the surf zone, thus suggesting that these fish are more attracted to freshwater influenced nursery areas once they have entered the estuary than those in the surf zone. Larvae collected in the marine environment also selected surf zone water with significantly higher frequencies than estuary water or offshore sea water, thus confirming the importance of the surf zone as an interim nursery area for postflexion R. holubi.

Under the growing demand for marine fish resources, and the apparent and expected impacts of global climate change, there is a need to conduct long-term monitoring (LTM) to ensure effective management of resources and conservation of biodiversity. However LTM programmes often suffer from design deficiencies and fail to achieve their objectives. These deficiencies stem from the fact that insufficient consideration is afforded to the design phase, with programmes selecting methods that are not suitable to address the objectives, or are not cost-efficient, compromising the sustainability of the LTM. To facilitate the establishment of LTM programmes along the southern coast of South Africa, background research needed to be conducted to identify which methods were most appropriate for LTM of reef fish. This study presents a detailed field-based assessment of the suitability and cost-efficiency of monitoring methods for long-term monitoring of reef fish in the Agulhas Ecoregion of South Africa. The approach adopted to identify the method, or suite of methods most suited for LTM, involved (i) the selection of methods considered suitable for LTM, (ii) the individual assessment and optimisation of method performance, and (iii) the comparative assessment of the fish community sampled by the different methods. The most suited method(s) were then identified as those that provide the most comprehensive assessment of the fish community and had the highest cost-efficiency. The research was conducted between January 2008 and 2011 in the Tsitsikamma and Table Mountain National Park (TNP and TMNP, respectively) marine protected areas (MPAs) within the Agulhas Ecoregion. The methods selected included fish traps (FT), controlled angling (CA), underwater visual census (UVC), remote underwater video (RUV), baited RUV (BRUV) and remotely operated vehicles (ROV). The individual assessment and optimisation was conducted with the FT, UVC, RUV and BRUV methods. The assessment of the FT method aimed to identify the optimal soak time, and whether or not the size of the funnel entrance to the trap affected the catch. The results identified that larger funnel entrances caught more fish and soak times of 80 minutes produced the highest catches per unit effort. However the data were highly variable and the method detected few of the species typical of the region. Fish traps were also associated with high levels of mortality of fish post-release. The assessment of UVC strip transect method involved directly comparing the precision of data collected by researchers and volunteers using a novel double-observer technique (paired-transects). The results showed considerable error in both the volunteers and researchers data, however the researchers produced significantly higher precision data, compared to the volunteers. The distinction between researchers and volunteers was not evident in the data for the dominant species of fish. For all observers, the abundance of a species in the sample had a significant influence on its detectability, with locally scarce or rare species poorly detected. UVC was able to sample the majority of species typical of reefs in the region, however it appeared plagued by observer and detectability biases. The assessments of RUV and BRUV were conducted simultaneously which enabled the assessment of the effect of bait on the observed fish community. In addition the optimal deployment time for both methods to maximise species richness and abundance was determined. The results showed that BRUV, and to a lesser degree RUV, were able to effectively survey the reef fish community for the region with a 50 minute and 35 minute deployment time, respectively. Baited remote underwater video was especially good at detecting the invertebrate and generalist carnivores, and cartilaginous species. On the other hand, RUV was more effective at surveying the microinvertebrate carnivores. Remote underwater video was characterised by higher data variability, compared to BRUV, and was ultimately considered a less cost-efficient monitoring method. Comparative methods assessments were conducted during two field experiments with the FT, UVC and BRUV methods in the TMNP MPA, and the FT, CA, UVC, RUV, BRUV and ROV methods compared in the TNP MPA. The objectives of the comparison were to investigate differences in the fish communities observed with the different methods, and to determine the power of the data to detect an annual 10% growth in the fish populations over a period of five years. The results from the method comparison were in turn used to conduct the cost-benefit analysis to determine the efficiency of the different methods at achieving monitoring objectives requiring population data from multiple trophic and functional groups with the community, and from species of fisheries importance. The results indicated that FT, CA and ROV were ineffective at monitoring the reef fish community, although CA appeared to provide valuable data for the dominant fisheries species. Both CA and FT required minimal initial investment however, the variability in the data translated into high annual monitoring costs, as the required sampling effort was great. The ROV required the highest initial investment and was identified as the least cost-efficient method. Underwater visual census was able to adequately survey the bony fish within the community, however it did not detect the cartilaginous species. Underwater visual census required a large initial investment and was not cost-efficient, as a many samples were required to account for the variability in the data. Remote underwater video provided a comprehensive assessment of the reef fish community, however it too was associated with high levels of variability in the data, compared to BRUV, reducing its cost-efficiency. BRUV provided the most comprehensive assessment of the reef fish community and was associated with the highest cost-efficiency to address the community and fisheries species monitoring objectives. During the course of this research stereo-BRUV has gained considerable support as an effective reef fish monitoring method. Although not tested during this research, stereo-BRUV is preferred to BRUV as it provides accurate data on the size of fish. However, the initial investment of stereo-BRUV is over three times that required for the BRUV. Although it is recommended that a baited video technique be used for LTM in the Agulhas Ecoregion, the choice between BRUV and stereo-BRUV will depend on the specific objectives of the programme and the available budget at the implementing agency.

The spatial and temporal patterns in the ichthyofaunal community composition and structure in Taylor’s salt marsh and adjacent eelgrass beds (Zostera capensis) in the Kariega Estuary, was investigated every two months between May 2006 and March 2007. Total ichthyofaunal abundances and biomass in the salt marsh ranged between 0.55 and 21.7 ind.10m-2 and between 0.03 and 1.9 g.wwt.10m⁻², respectively. There were no significant spatial patterns in the values evident (P > 0.05 in all cases) although seasonal trends were marked, with highest values consistently recorded during the warmer summer months. Investigations into the community structure showed that the ichthyofaunal community within salt marsh was composed almost exclusively of juveniles of estuarine dependant (category II) species, mainly juvenile Mugilidae (<20mm SL) that comprised up to 83% of all fish sampled. Hierarchical cluster analysis and multidimensional scaling did not identify any distinct spatial patterns in the ichthyofaunal community within the salt marsh. The absence of any spatial patterns in the community structure could be related to the absence of any significant spatial patterns in the physico-chemical (temperature, salinity and dissolved oxygen concentrations) and biological (water column and microphytobenthic algal concentrations) variables within the salt marsh (P > 0.05 in all cases). Temporal shifts in the ichthyofaunal community structure within the salt marsh were, however, evident largely reflecting the breeding cycles of individual species within the sub-region. Within the adjacent eelgrass beds, total ichthyofaunal abundances and biomass ranged between 8.4 and 49.4 ind.10m⁻² and between 2.9 and 94.5 g.wwt.10m⁻², respectively. Once again there were no distinct spatial patterns in the abundance and biomass values evident although seasonal patterns were marked. In contrast to the salt marsh, within the in the eelgrass community, there were a large number of adult individuals recorded. Again category II species, the estuarine dependent species, were numerically and gravimetrically dominant. The dominance of category II species reflects the marine dominance of Kariega Estuary. The remaining estuarine utilisation categories did not contribute significantly to abundance or standing stock totals. Hierarchical cluster analysis showed that the salt marsh and eelgrass beds represented two distinct habitats within the Kariega Estuary. Within the salt marsh, the family Mugilidae were numerically dominant contributing 83% of the total catch. Within the eelgrass beds, the sparid, Rhabdosargus holubi and representatives of the family Gobidae contributed 36.3% and 33.9% respectively to the total catch. Estuaries with a wide range of microhabitats have been demonstrated to support a more diverse ichthyofaunal community. Shallow water habitats in general are important areas for juvenile fish within estuaries. Taylor’s salt marsh provides an alternative shallow water habitat, occupied by a distinct ichthyofaunal community composition, with increased food availability and decreased predation pressure, for a wide range of fish species.

Freshwater fish species have been introduced into freshwater systems around the world, primarily for aquaculture, ornamental fish trade and sport fishing. Their introduction into estuarine systems is uncommon however, instances do occur and their establishment success and impacts on these estuarine systems is not well documented. The extent of invasion by four freshwater fishes in a RAMSAR listed estuarine system, the Wilderness Lakes, Western Cape was investigated. This thesis determined the relative abundance and distribution of alien fishes in relation to the native fish biota, their establishment success in the system, what factors inhibited their establishment and whether the introduction of alien fishes negatively impacted the native fish biota. The distribution and abundance of fishes were assessed primarily using fyke nets, seine nets and gill nets in each of the lakes, interconnecting channels and the Touw Estuary. The fish fauna was made up of euryhaline marine species comprising 46%, native estuarine species comprising 18%, catadromous species comprising 7% and freshwater alien species comprising 29% of the total biomass sampled. A total of 26 species were sampled in the system, three of which were considered alien; Oreochromis mossambicus, Gambusia affinis and Cyprinus carpio, and Micropterus salmoides were not sampled but confirmed in the system. Establishment success was determined by evidence of: a sustainable breeding population, a wide distribution, abundant in the sampling area, and all size classes of fish sampled. Gambusia affinis and Oreochromis mossambicus have been recorded in the system for a minimum of 13 and 26 years respectively. They were widely distributed and highly abundant and are established in the system. Micropterus salmoides was first recorded in the system in 1985 but abundances have remained low with fish appearing to be limited to Island Lake and Langvlei. Reproduction appeared to be limited by higher salinity and these factors indicated that this was a casual species which relies on repeated introductions for population maintenance. Cyprinus carpio spawned successfully in 2010 and was widely distributed but abundances were still low with a total of 15 fish being sampled throughout the system, and was thus in the establishing phase. As these are considered freshwater alien species, the physico-chemical parameters in the estuarine environment inhibiting the establishment success of the alien fishes were investigated. Gambusia affinis and O. mossambicus were not limited by the physical environment, and while O. mossambicus cannot tolerate temperatures below 11⁰C, temperatures only dropped below its tolerance for a total of two days between February 2010 and February 2011. Cyprinus carpio and M. salmoides were restricted by salinity in Rondevlei and Langvlei but could tolerate salinity in Island Lake and the Touw Estuary during closed mouth phases. While adults appeared to tolerate the salinity in the system, egg and larval development could be affected thus reducing the viability of the population. The abundance of alien fishes did not negatively impact the abundance of native fish species. The interactions between the native and alien fish biota tended towards biotic acceptance where, as alien abundance increased so did native fish abundance. The impacts of the alien fishes on the native fish biota were assessed by comparing the fish community from a study completed in 1985 to the findings of this study. From these two studies there were no apparent negative impacts on the native fish biota and the fish community composition would most likely be structured by estuarine mouth opening events.

The aims of this study were to determine the potential use of biomarkers at multiple levels of biological organisation together with a fish community bioindicator to assess the estuarine health status of three temporarily open/closed estuaries. The estuaries investigated were the East Kleinemonde (EK), Old Woman's (OW) and Mtana (MTN), all of which are situated in the Eastern Cape Province. Three biomarkers, the acetylcholinesterase (AChE) assay, lipid peroxidation (LPx) assay, liver histopathology and a condition factor were used to determine sub-organism health and one bioindicator, the Estuarine Fish Community Index (EFCI), was used as a bioindicator of community health. The estuarine-dependent marine species Rhabdosargus holubi was selected as an indicator species for the sub-organism level analyses. The results from the community analyses indicated that the EK and OW estuaries were in 'good' condition, while the MTN was found to be in 'moderate' condition. Histological analyses revealed that R. holubi from all three estuaries showed signs of pathological changes to the liver, with the fish from the MTN eliciting the highest occurrence of these changes. The LPx assay found that R. holubi from both the OW and MTN showed signs of oxidative damage in the liver tissue, but those from the EK did not appear to be affected. The AChE assay showed that only the fish from the OW had been affected by anticholinesterase compounds. A laboratory study was undertaken using R. holubi as a positive control for the AChE and LPx assay. The fish were exposed to 3 μg/L chlorpyrifos, a known cholinesterase inhibitor, for six hours and their tissues were examined for changes to LPx levels and AChE activities. AChE activity was significantly inhibited (Mann Whitney U test, Z = 3.65, n = 38, P < 0.001) by the exposure, but LPx levels were not significantly affected. A composite index incorporating the biomarkers at different biological levels of organisation was developed. The index was designed to assist managers and scientists to determine whether the ichthyofauna of a system was being affected by environmental stressors and what management interventions could be undertaken to ameliorate the water quality in an estuary. The index was applied to the three estuaries investigated during the present study and both the OW and MTN were assessed to be in need of immediate management intervention. The fish in the OW were found to be stressed at all the sub-organism levels measured and the reason for this was hypothesised to be as a result of golf course activities in this adjacent estuary. A number of management actions are proposed to reduce the sub-organism stress observed in the fish from the OW. The livers of fish from the MTN were shown to be under stress; however the causative agent of this stress was unknown because there is no formal development in the MTN catchment. However, a possible contaminant source is proposed and management interventions to alleviate the stress on the biota of the MTN are suggested. The EK does not require immediate management intervention, however, continuous routine monitoring is recommended to ensure that conditions do not deteriorate. Shortcomings of the index were outlined and a number of suggestions were made in terms of other measures of biological health which could be incorporated into the index.