by Wong Pik-kwan. / Thesis submitted in: September 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 116-139). / Abstract also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENT --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF TABLES --- p.viii / LIST OF FIGURES --- p.ix / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER TWO --- LITERATURE REVIEW / Chapter 2.1 --- Introduction --- p.4 / Chapter 2.2 --- Heavy metals --- p.6 / Chapter 2.3 --- Mechanisms of metal toxicity --- p.9 / Chapter 2.4 --- Toxic effects of metals on marine organisms --- p.10 / Chapter 2.4.1 --- Cadmium --- p.10 / Chapter 2.4.2 --- Chromium --- p.10 / Chapter 2.4.3 --- Copper --- p.11 / Chapter 2.4.4 --- Lead --- p.12 / Chapter 2.4.5 --- Nickel --- p.12 / Chapter 2.4.6 --- Zinc --- p.13 / Chapter 2.5 --- Metal uptake and elimination in marine organisms --- p.14 / Chapter 2.5.1 --- Uptake of metals --- p.14 / Chapter 2.5.2 --- Elimination of metals --- p.15 / Chapter 2.5.3 --- Metal detoxification system in fish --- p.16 / Chapter 2.6 --- Heavy metals in marine fish --- p.17 / Chapter 2.7 --- Bioaccumulation --- p.20 / Chapter 2.7.1 --- Models of metal accumulation --- p.21 / Chapter 2.7.2 --- Compartment model --- p.21 / Chapter 2.7.3 --- Physiologically based pharmacokinetic (PB-PK) model --- p.22 / Chapter 2.8 --- The influence of environmental factors on bioaccumulation of metals --- p.23 / Chapter 2.8.1 --- Temperature --- p.23 / Chapter 2.8.2 --- Salinity --- p.23 / Chapter 2.8.3 --- Organic matter --- p.24 / Chapter 2.8.4 --- pH --- p.25 / Chapter 2.8.5 --- Chelators and surfactants --- p.25 / Chapter 2.8.6 --- Other metals --- p.26 / Chapter 2.9 --- Biological effects of heavy metals on man --- p.26 / Chapter 2.10 --- The use of biological indicator organisms for metal pollution --- p.28 / Chapter CHAPTER THREE --- HEAVY METAL CONCENTRATIONS IN CULTURED MARINE FISH IN HONG KONG / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.36 / Chapter 3.2.1 --- Sampling --- p.36 / Chapter 3.2.2 --- Water analysis --- p.36 / Chapter 3.2.3 --- Sediment analysis --- p.39 / Chapter 3.2.4 --- Mussel analysis --- p.40 / Chapter 3.2.5 --- Fish analysis --- p.40 / Chapter 3.2.6 --- Quality control and statistical analysis --- p.41 / Chapter 3.3 --- Results --- p.42 / Chapter 3.3.1 --- Seawater --- p.42 / Chapter 3.3.2 --- Sediment --- p.46 / Chapter 3.3.3 --- Mussel --- p.46 / Chapter 3.3.4 --- Fish --- p.50 / Chapter 3.4 --- Conclusion --- p.67 / Chapter 3.4.1 --- "Metal concentration in seawater, sediment, green mussel and fish" --- p.67 / Chapter 3.4.2 --- Accumulation of heavy metals in different tissues of cultured fish --- p.69 / Chapter 3.4.3 --- Relationship between the body weight and metal accumulation --- p.71 / Chapter 3.4.4 --- Heavy metal pollution in fish culture sites --- p.72 / Chapter 3.4.5 --- Selection of fish culture site --- p.72 / Chapter CHAPTER FOUR --- ACUTE AND SHORT-TERM EFFECTS OF COPPER(II) IONS ON SPARUS SARBA / Chapter 4.1 --- Introduction --- p.76 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.1 --- Experimental animals --- p.79 / Chapter 4.2.2 --- Determination of the 96 hour median lethal concentrations --- p.19 / Chapter 4.2.3 --- Determination of growth rate --- p.80 / Chapter 4.3 --- Results --- p.82 / Chapter 4.3.1 --- Determination of the 96 hour median lethal concentrations --- p.82 / Chapter 4.3.2 --- Determination of growth rate --- p.82 / Chapter 4.3.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.82 / Chapter 4.4 --- Conclusion --- p.91 / Chapter 4.4.1 --- Determination of the 96 hour median lethal concentrations --- p.91 / Chapter 4.4.2 --- Determination of growth rate --- p.93 / Chapter 4.4.3 --- Distribution of Cu concentration in whole body and different tissues of S. sarba --- p.94 / Chapter CHAPTER FIVE --- ACCUMULATION AND ELIMINATION OF COPPER(II) IONS TO SPARUS SARBA / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Materials and methods --- p.98 / Chapter 5.2.1 --- Experimental animals --- p.98 / Chapter 5.2.2 --- Uptake and elimination of Cu ion in S. sarba during continuous exposure to waterborne Cu --- p.98 / Chapter 5.3 --- Results --- p.100 / Chapter 5.4 --- Conclusion --- p.108 / Chapter CHAPTER SIX --- GENERAL CONCLUSION --- p.112 / CHAPTER SEVEN REFERENCES --- p.116

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

M.Sc. / The upper catchment of the Olifants River, from its origin near Bethal, to its confluence with the Wilge River, north of Witbank, as well as it tributaries, are being subjected to increasing afforestation, mining, power generation, irrigation, domestic and industrial activities. These activities have a profound effect on the water quality and the major point sources of pollution in this area include mines, industries and very importantly, combined sewage purification works, located alongside the river, which, in addition to oxidizable material contains detergents, nutrients, and metals. It was therefore necessary to determine the extent to which these activities affect the water quality of the system. The impact of these activities was therefore addressed by a Water Research Commision Project namely "Lethal and sublethal effects of metals on the physiology of fish" of which the present study investigated effects at two localities, namely in the Olifants River (locality OR1) before its confluence with the Klein Olifants River and a locality in the Klein Olifants River (locality KOR1). Apart from the field study, toxicity tests were also performed in a laboratory, in order to determine the effects of low pH and elevated aluminium concentrations on the haematology, osmoregulation and carbohydrate metabolism of the Mozambique Tilapia, Oreochromis mossambicus as the acidification of soil systems may cause the transfer of aluminium into aqueous solutions, where it may be present in different forms. During the field study, the chemical and physical characteristics of the river water were evaluated, with special attention to the concentrations of certain metals (manganese, copper, chromium, lead, nickel, zinc, iron and aluminium) in the water and sediment, as well as in fish, which are known to accumulate the elements supra and are therefore valuable as indicators of these pollutants. The two fish species used for the investigations were the African sharptooth catfish, Clarias gariepinus and the moggel, Labeo umbratus. Four tissue types were dissected, namely the muscle, liver, skin and gill tissues. The metal concentrations in these organs/tissues, as well as in the water and sediment, were determined in a laboratory with an atomic absorption spectrophotometer. Statistical analyses were performed on the results obtained from this study and the order and extent of bioaccumulation of these metals in the water and sediment were determined, as well as in the fish organs/tissues. Its dependence on the size, sex and species of the fish and the localities and seasons were investigated.

M.Sc. (Zoology) / The necessity to maintain high water quality standards has become essential not only to man, but also to conserve aquatic life forms. The Olifants, Selati and Letaba Rivers were monitored for water quality variables and metal concentration in water, sediments and muscle, gill, fat, gonads, liver, bile, blood and kidney of the sharp tooth catfish, C. qariepinus. Some of the water quality variables measured were not within the limits of those standards set for the protection of fish and other aquatic life forms. Turbidity seems to be the biggest problem, due to the Phalaborwa barrage. Metal concentrations were found, in descending order, highest in the sediment, fish and water. This pose the problem of metals being reintroduced into the water column when physico-chemical conditions are not favourable. Experimental studies were done with copper on adult C. qariepinus, due to the extensive copper mining activities in the Phalaborwa region. Haematological and enzymatic analyses revealed a interference in metabolic activity after 96 hour copper exposure. Histological studies on the gill morphology, showed an alteration in the structure with a resultant change in respiratory and osmoregulatory function. LC-50 values were determined for adult and juvenile C. qariepinus in order to set new standards for the protection of aquatic lite in a rapidly developing South Africa.

D.Phil. / The entire Olifants . River Catchment is subjected to increasing afforestation, agricultural, domestic, mining, industrial, irrigation and urbanisation activities. These activities have a profound effect on the water quality of the river and its tributaries. This is cause for concern for the water users in the upper catchment, and because the Kruger National Park, one of its downstream water users, is extremely dependent on water of a satisfactory quality to sustain its various ecosystems. It is therefore vitally important to determine to what extent activities in the Upper Catchment of the Olifants River (Mpumalanga), especially in the Witbank and Middelburg areas, influence the water quality of the river. Point sources of pollution in the upper reaches include mining and industrial activities as well as water care works located at various points along the river. These were addressed in a Water Research Commission Project (No. 608/1/97) titled "Lethal and sublethal effects of metals on the physiology of fish: An experimental approach with monitoring support". Although sixteen localities were chosen for the initial project, this study only focussed around the metal ecotoxicology at two localities (Steenkool Spruit and Witbank Dam) in the upper catchment and the effect of metals (copper and zinc) on fish blood physiology. In aquatic ecosystems water quality is an important variable and full assessment of water quality, of Steenkool Spruit (locality 3) and Witbank Dam (locality 7), included evaluation of the chemical, physical and biological characteristics at each of the localities. Water and sediment samples were collected seasonally during the study period, February 1994 to May 1995, and the chemical and physical water quality variables were measured. During the study period three metal bioaccumulation indicator species, Labeo umbratus, Clarias gariepinus and Labeo capensis, were captured from which tissue (gills, liver, muscle and skin) samples were collected. These samples as well as water and sediment samples, were analysed for aluminium, chromium, copper, iron, manganese, nickel, lead and zinc concentrations, using atomic absorption spectrophotometry. In the past routine monitoring of chemical and physical water quality characteristics left scientists and managers with a sizeable pool of data which is often difficult but not impossible to interpret. To standardise and summarise this collection of data an aquatic toxicity index (ATI), WATER2 was developed by Wepener et at (1992). The present study attempted to expand and refine WATER2, which has resulted in the establishment of a new ATI, RAUWater.

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.

Ph.D. / The current study evaluated a bio-monitoring technique developed in the USA by Adams, Brown and Goede, 1993. This project was sponsored by the Department of Water Affairs and Forestry (DWAF), to enable testing of the Health Assessment Index (HAI) under South African conditions. Testing took place in the Olifants River system, one of the most polluted river systems . in South Africa. Initially two river points were tested using Oreochromis mossambicus (Robinson, 1996), Clarias gariepinus (Marx, 1996) and Labeo rosae (Luus-Powell, 1997). The current study re-tested the HAI at the same two sample sites, namely Mamba and Balule in the Kruger National Park, using 0. mossambicus and C. gariepinus respectively. Two additional sites were tested in the upper catchment area, namely Loskop Dam and Bronkhorstspruit Dam. The current study further enabled the comparison of HAI results collected during drought and flood conditions. Results obtained after deployment of the HAI were corroborated using chemical analysis of water, sediment and biota. Water and sediment analysis was carried out by the Institute for Water Quality Studies using standard techniques. Bio-accumulation of aluminium, copper, iron, lead, manganese, nickel, strontium and zinc was assessed in the gills, liver, skin and muscle tissue of sample fish using standard Atomic Absorption Spectrometry techniques. Modifications made to the original HAI involved the inclusion of variable ranking in the assessment of fish parasites, with endo- and ectoparasites evaluated separately. Testing of this parasite hypothesis lead to the development of a Parasite Index component to the HAI. Assessment of water, sediment and fish tissue determined that the Olifants River system is indeed exposed to macro and heavy metal pollutants, which negatively affect aquatic health. Constituents posing the greatest threat are chlorides, fluorides, phosphates, total dissolved solids, copper and iron concentrations. Testing the HAI and parasite hypothesis using C. gariepinus, provided the most meaningful results. During testing of the parasite hypothesis both endo- and ectoparasite numbers conformed to the suggested idea that higher endoparasite numbers will occur at highly impacted areas, whereby ectoparasite numbers will be low. This was particularly evident in the lower catchment area, whereby comparisons between drought and flood conditions were carried out. Subsequent decreases in water quality directly after the flood were noted using water and sediment analysis. This observation reflects the results gathered using the HAT and during testing of the parasite hypothesis at all four sample sites. During statistical analysis of the HAI, using logistic regression analysis, parasite numbers, more specifically endoparasite numbers, were the most indicative of fish health. Environmental stressors (flood conditions) result in immunological responses observed in fish, and are reflected statistically using the HAI as changes in WBC %. It is suggested that endoparasites and WBC % provide the best overall assessment of fish condition. These variables should thus not be eliminated, in order to streamline the HAI evaluation procedures. Testing of this bio-monitoring technique under South African conditions provided meaningful results. This indicates that the HAI can be used to assess water quality, with existing water monitoring programmes further benefiting from its incorporation.

Tissue samples from spotted gar (Lepisosteus oculatus) and largemouth bass (Micropterus salmoides) were collected from Caddo Lake. Gar and bass livers were subjected to histological investigation and color analysis. Liver color (as abs at 400 nm) was significantly correlated with total mercury in the liver (r2 = 0.57, p = 0.02) and muscle (r2 = 0.58, p = 0.01) of gar. Evidence of liver damage as lipofuscin and discoloration was found in both species but only correlated with liver mercury concentration in spotted gar. Inorganic mercury was the predominant form in gar livers. In order to determine the role of mercury speciation in fish liver damage, a laboratory feeding study was employed. Zebrafish (Danio rerio) were fed either a control (0.12 ± 0.002 µg Hg.g-1 dry wt), inorganic mercury (5.03 ± 0.309 µg Hg.g-1 dry wt), or methylmercury (4.11 ± 0.146 µg Hg.g-1 dry wt) diet. After 78 days of feeding, total mercury was highest in the carcass of zebrafish fed methylmercury (12.49 ± 0.369 µg Hg.g-1 dry wt), intermediate in those fed inorganic mercury (1.09 ± 0.117 µg Hg.g-1 dry wt), and lowest in fish fed the control diet (0.48 ± 0.038 µg Hg.g-1 dry wt). Total mercury was highest in the viscera of methylmercury fed zebrafish (11.6 ± 1.86 µg Hg.g-1 dry wt), intermediate in those fed inorganic diets (4.3 ± 1.08 µg Hg.g-1 dry wt), and lowest in the control fish (below limit of detection). Total mercury was negatively associated with fish length and weight in methylmercury fed fish. Condition factor was not associated with total mercury and might not be the best measure of fitness for these fish. No liver pathologies were observed in zebrafish from any treatment.