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1. GENERAL INTRODUCTION 1.1 Marine Pollution in Malaysia

The west coast of Peninsular Malaysia plays a major role in the maritime trade of the country (Naidu, 1993). It is the hub for the major urban centres, industries and plantation. These population centres are estimated to account for at least 8.5 million (70%) of Malaysias 20 million population. The rivers, which run through most of the urban/industrial areas, are the main repository of domestic and industrial wastes, sewage, organic and inorganic loadings. Siltation from land-based sources, oil and grease from shipping activities and other contaminants that result from mans economic activities also contribute to the general pollution of the aquatic environment (Department of Environment, 1997). Choo et al. (1994) published an overall assessment of the state of the coastal marine environment for the west coast of Peninsular Malaysia. The marine waters of the west coast of Peninsular Malaysia are considered to be most prone to land-based pollutants (Phang et al., 1991) since 75% of the population and 85% of industries are concentrated there (Maheswaran and Godwin, 1988). Jaafar (1991) discussed the management issues related to the marine environment of the Strait of Malacca.

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The coastal marine environment of the west coast Peninsular Malaysia, which also forms the eastern portion of the Strait of Malacca, has been the main repository of both land-based pollution and pollutants derived from shipping activities (Choo et al., 1994; MIMA, 1994). This portion of the Strait of Malacca, within Malaysian territorial waters, has also been traditionally the major fishing ground for Peninsular Malaysia, and as much as 70% of capture fisheries come from these waters (Department of Fisheries Malaysia, 1993). Landings of marine fish showed an increasing trend between 1970 and 1980, but then declined until 1986. Although from 1986 onwards there was an increase in landings, this increase was attributed to the introduction of deep-sea or offshore fishing activity. Lui (1992) hypothesised that the fisheries resource within the inshore waters of the Strait of Malacca had reached its maximum level of exploitation. There was a steep decline in fish catch per unit population from 1970 to 1990, which indicated that the fish resource was being exploited beyond its maximum sustainable yield (Sheppard, 1992). Overexploitation has been suggested as one of the main reasons for the decline in fish resource. However, the role of mainly land-based pollution and destruction of natural habitats have been suggested as other major factors responsible for the current decline in inshore fish resources (Sasekumar, 1980; Phang, 1990). It was also suggested that oil spills from tankers and bilge from shipping operations contribute to pollution originating from the sea itself (Absil et al., 1987; Maheswaran and Godwin, 1988).

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The rivers that flow into the marine coastal waters inadvertently affect critical marine habitats such as mangroves, estuarine mudflats, seagrasses and coral reefs (Peters et al., 1997). Mangrove swamps on the west coast cover 103,000 ha and comprise more than 90% of the total mangrove coverage for Peninsular Malaysia (Tang et al., 1980). The mangrove habitat is a nursery ground for many marine organisms including several commercial species of fish and shrimps (Robertson and Duke, 1987). Mangrove detritus forms a sizeable portion of the food of several fish species (Thong and Sasekumar, 1984). Sources of Pollutants The Strait of Malacca is the second busiest sea-lane in the world. In 1994, it was estimated that about 3000 vessels/day plied the maritime waters of the Strait of Malacca. This number did not include thousands of fisheries vessels navigating the waters. Rapid industrialisation coupled with chemical-dependent modern agricultural activities have led to increasing loading of xenobiotics and other contaminants to the coastal waters. Treatment and mitigating technologies currently in place have not been able to cope adequately with the rapid pace of industrialisation. Studies by Law et al. (1993) and Abdullah (1995) showed that the marine waters of the north-eastern part of the Strait of Malacca around the Langkawi Group of Islands were contaminated with significant levels of toxic dissolved/dispersed hydrocarbons. Abdullah reported levels of hydrocarbon concentrations of 1.73-1.97 mg L-1 around Langkawi Island. Abdullah et al. (1996), detected in samples of coastal sediments low concentrations of polycyclic aromatic hydrocarbons (PAHs), breakdown by-products of petroleum.

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In 1997, 117 major rivers were monitored in Malaysia, generating 4078 samples from 908 stations (DOE, 1997). Twenty-five rivers (11 in 1993, 13 in 1996) were considered badly polluted in 1997, based on water quality index (WQI) criterion (The WQI classification is based on biochemical oxygen demand (BOD), chemical oxygen demand (COD), dissolved oxygen (DO), ammoniacal nitrogen, suspended solids and pH levels). This is an increase from 12 rivers the previous year. The marine environment also deteriorated over the same period. The main pollutants were siltation from earthworks, sewage and oil & grease. In 1997, a total of 4,075 industries were identified as significant water pollution sources for the west coast of Peninsular Malaysia. The main industries were Food & Bevcrages, paper treatment, electrical & electronic and metal finishing. Other significant sources of pollution were sewage treatment plants (4,359) and animal husbandry (2,029). The main source of organic pollution load was sewage (776.4 metric tonnes BOD/day), wastes from pig-farms (304.5 metric tonnes BOD/day), the manufacturing sector (5.9 metric tonnes BOD/day) and the agro-based industries (8.4 metric tonnes BOD/day). The largest contribution of BOD loading from the manufacturing sector came from the Food & Beverages industry amounting to 1,166.4 kg/day (20%). Coastal marine water also showed considerable deterioration in 1997. Eighty-four of the 226 monitoring stations were found to have exceeded the Proposed Marine Interim Standards for of oil & grease (0 mg L -1), suspended solids (50 mg L-1) and Escherichia coli (100 MPN /100 mL-1). Copper levels exceeding the proposed standard of 0.1 mg L-1 were recorded in Sarawak. Mercury and arsenic exceeded the standards (0.001mg L-1 for Hg and 0.1 mg L-1 for arsenic) in coastal waters off Negeri Sembilan.

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Pesticides in the aquatic environment Pesticide use became widespread after WWII when DDT and other synthetic chemicals were introduced to offer an easy solution to the age-old problem of pest control in agriculture. Amongst the many xenobiotics that enter the aquatic ecosystem, pesticides pose considerable threat to the biota. In 1997, the Pesticide Board of the Ministry of Agriculture Malaysia registered 1,758 types of pesticides of which 535 (30.5%) are classified as very hazardous and 574 (32.7%) as moderately hazardous. It was estimated that 12 million kg of pesticides used in agriculture were in solid form while 8 million litres were liquid (MACA, 1997). Ninety percent pesticides used in Malaysia were for the rubber, oil palm and rice plantations, of which herbicides account for 75%, insecticides 13% and 3% were rodenticides (MACA, 1996). In 1985, the total value of pesticides sold in Malaysia was US$140 million. Of the organophosphate and carbamate insecticides, the more important were methamidophos, malathion, chlorpyrifos, fenvalerate, carbofuran and carbaryl. Diazinon and isoprocarb are used mainly for vegetables. The usage of pesticides in Malaysia is being regulated and monitored by the Pesticides Board of the Ministry of Agriculture. The Pesticides Act 1974 regulates the import, manufacture, distribution, sale and use of pesticides. The Act requires all pesticides to be registered in accordance with standards and criteria of FAO/WHO. The safety levels of pesticide residues in foods are determined by the Food Act 1983. The enforcement of this Act is under the purview of the Ministry of Health.

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Pesticides enter the aquatic environment through their application in the field. The distribution, fate and mobility of pesticides when applied in the field and the factors affecting their entry into the aquatic environment have been reviewed (Nicholls, 1988, Jaffe, 1991). Studies on pesticide contamination in Malaysia have focussed on the persistent organochlorine pesticides (OCPs). The occurrence of OCPs had been detected in Malaysias aquatic environment. Tan et al.(1990) reported contamination of Kelang River (one of the most polluted rivers in 1997) by OCPs such as aldrin (0.005-0.061 ng L 1

), endosulfan (0.009-0.256 ng L-1), and heptachlor (0.039-0.742 ng L-1). The same study

also reported the occurrence of small quantities of DDE, DDT and heptachlor in all major rivers surveyed, at levels below the critical values of the Malaysian Interim Standards for Water Quality (1992). Cavalho (1993) suggested that land-based pesticide contamination might still pose a considerable threat to marine biota as the major agricultural areas are located in coastal plains and river valleys. Contamination by OCPs in the marine biota, including fish and shellfish, has been documented (Jothy et al., 1983; Rohani et al., 1992). The study by Jothy et al. (1983) showed that OCP residue levels were found to be low in all samples analysed except for the blood cockle, Anadara granosa, from Penang and Perak where the level for total DDT ranged from 0.027-0.05 mg L -1. The OCPs detected in fish were lindane (0.001-0.012 mg L-1), dieldrin (< 0.001-0.004 mg L-1) and total DDT (0.003-0.016 mg L-1). Rohani et al. showed that OCP residues were generally low except for lindane which were detected at 180.9 and 123.7 g L-1 in the mussel, Perna viridis collected from two sites in Penang. Rohani et al. (1992) reported lower levels of OCPs compared with similar species analysed by Jothy et al. (1983). It was suggested that the lower values detected could be explained by the phasing out of OCPs in Malaysian agriculture.

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The phasing out of OCPs coincided with the introduction of the more toxic but less persistent organophosphorus and carbamate pesticides into Malaysia. However, it was suggested that the more toxic OPs and carbamates could be sufficiently persistent to exert some effect in marine biota (Cavalho, 1993). Efforts to assess impacts of OPs and carbamates on the freshwater aquatic ecosystem were initiated by measuring their biological effects especially acetylcholinesterase activity in freshwater fish (Sulaiman et al., 1989; Abdullah et al., 1993, 1994).

Heavy Metals in aquatic environment

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Heavy metals are regarded as serious pollutants in marine ecosystems because of their environmental persistence, their toxicity at low concentrations and their ability to be incorporated into food chains and concentrated by organisms (Negilski, 1976). Metals such as zinc and copper are essential trace metals required for biological processes. Other essential trace metals are Fe, Co, Mn, Cr, Mo, V, Se, Ni and Sn (Bryan, 1980). Metals may become toxic when accumulated from seawater and concentrated in the tissues of aquatic organisms. Cadmium is not known to be an essential metal for any organism. In Malaysia, monitoring of trace metals had concentrated on their levels in the tissues of fish, shellfish, and in water and sediments (Jothy et al., 1983; Liong, 1986; Devi, 1986; Din, 1992; Din and Jamaliah, 1994; Din et al., 1996). Rakmi and Salmijah (1987) estimated that 220 000 m3 of toxic waste was produced from the manufacturing industry which contained lead (5.9 mg L-1), copper (224.0 mg L-1), Cr6+ (6.6 mg L-1), Cr3+ (94 mg L-1), Ni (159.0 mg L-1), Zn (567.0 mg L-1) and Fe (2661.0 mg L-1). Results from a study carried out in 1990

involving 152 sampling stations showed that concentrations of heavy metals in coastal seawater of Cd, Cu, Cr, Pb, Hg and As surpassed the Malaysian interim marine water quality standards (DOE, 1991). Trace metal contents in estuarine and marine sediments along the coast of Penang Island showed elevated levels of As, Cd, Cr, Pb and Zn compared to control sites (Din and Jamaliah, 1994). Sungai Prai and Gertak Sanggul were shown to have the highest anthropogenic input of metals. Results from a similar study carried out in 1997 showed an improvement in heavy metals detected in the coastal marine environment, with only Hg and As exceeding the interim standard of 0.0001 g L-1 and 0.1 g L-1 respectively (DOE, 1997). Liong (1986) showed that the concentrations of Cd, Cu, Pb and Zn in various shellfish species were 0.19-1.32, 0.98-34.84, 0.06-0.42 and 19.28-471.8 g g-1 wet weight, respectively.

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Information on the toxicity of various metals on tropical marine organisms is still under development. Some studies had been carried out on the Malaysian marine organisms (Din, 1988; Zain 1990, Hamzah, 1991; Mohd. Noor, 1994; Ong and Din, 1995; Shazili, 1995; Noorzah and Eliza, 1996; Almah and Mazlin, 1996; Phang et al., 1996). Toxicity data from tropical marine organisms are required for environmental criteria formulation. The ASEAN-Canada Marine Science Programme Phase II (1992-1998) is a concerted effort by ASEAN member countries to develop criteria and standards for marine organisms and selected parameters including trace metals.

Threats from Pollution The number of health-related cases associated with the consumption of contaminated seafood, have been on the increase over recent years. A more recent event has been the first ever report of a Paralytic Shellfish poisoning occurrence in Peninsular Malaysia linked to consumption of contaminated mussels (Ismail et al., 1994). In April 1995, there was an incident of cyanide poisoning from contaminated fish reported on the Island of Pangkor (Department of Fisheries, 1995). The source of cyanide was from leaking containers, which were illegally dumped into a garbage disposal area fringing the coastline. The number of case of fish mortality have also been on the increase both for wild and cultured species (Choo et al., 1992).

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Decreasing fish production over recent years has been attributed to overexploitation from the use of increasingly efficient fishing-gear technology. But anthropogenic factors such as xenobiotic contaminants that impact marine ecosystems cannot be ruled out as contributing to this state of health of the fisheries. Studies by Law et al. (1993) have shown that juvenile shrimps are susceptible to very low concentrations of phenolics. However, more research needs to be carried out to ascertain the impacts of xenobiotics on the different life-stages of marine organisms. The water-quality monitoring programme of the Department of Environment has been providing information on the state of the environment. However, the physico-chemical data at best provide only information on contaminants in the water body, and little information on the effects of these contaminants on the marine biota. Biomonitoring for residues is being carried out at a minimal level. This is perhaps due to insufficient expertise and/or funding currently available.

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The Strait of Malacca, and the marine flora and fauna associated with it, are facing a major threat from many chemical and non-chemical pollutants. Although some

initiatives have been taken to monitor for some of these pollutants, very little information is available on the biological effects of exposure to xenobiotic chemicals. Development of toxicity tests for tropical species is being pursued in the ASEAN region under the action plan for conservation of nature. The 7-year ASEAN-Canada Cooperative Programme on Marine Science (Phase II) focuses on establishing criteria for management of living marine resources (Watson et al., 1992). More research needs to be carried out to ascertain whether these xenobiotics, singly or in a mixture, can elicit detrimental physiological changes and/or mortality in marine organisms. To this end, the recently developed concept of utilising biomarkers for environmental risk assessment is being critically evaluated on a worldwide basis.

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1.2. Relevance of Biomarkers to Environmental Risk Assessment

Since the days of the Minamoto tragedy in 1956 (Clark, 1986), environmental risk assessments have become increasingly important in many countries of the world. Governments have set up elaborate water quality monitoring programmes and regulatory frameworks to protect the aquatic environment, and, in particular, human health. Conventional risk assessment involves monitoring for certain classes of xenobiotics and other physico-chemical indicators of pollution. These are normally very costly, involving sophisticated analytical instruments and elaborate field sampling strategies (NRC, 1992). Decisions on the permitted release of xenobiotics into the environment are dependent on a process of risk assessment. Assessment of risks has always been based upon a comparison between laboratory toxicity data of surrogate species and the expected exposure in the environment.

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Furthermore, in a dynamic system such as a moving water body, contaminants are present in high concentrations for very short periods of time before dilution takes effect. This poses a real problem in acquiring a representative sample in the time window available. Other complicating factors when attempting accurate and representative sampling are the sediment type, bottom topography, water currents and water chemistry (see McCarthy and Shugart, 1990). Toxicological data derived from laboratory experiments have provided clues to the relationship between contaminants and their possible negative effects on aquatic flora and fauna. These organisms are continuously exposed to their surrounding matrix and therefore should integrate the contaminants present, even if the xenobiotics are short-lived in the environment, e.g. organophosphate insecticides and polyaromatic hydrocarbons. Exposure in itself is not reflective of the bioavailability of contaminants. When this problem is combined with the wide diversity of potential routes, species-specific differences and the pharmacodynamics of the contaminants themselves, an assessment becomes much more difficult (McCarthy and Shugart, 1990). For example, many toxicants do not bioaccumulate but are metabolised in many cases into more toxic metabolites. One example of such a compound is the organophosphate insecticide, malathion which upon accumulation is converted to the more toxic metabolite, malaoxon (Murphy et al., 1968). Because of these problems the main concern for environmentalists and regulators in recent times has been the level of uncertainty in determining the ecological effects that result from exposure to toxic chemicals released into the environment.

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Physico-chemical analyses, although valuable and necessary, do not provide all the pertinent information required for pollution assessment (Gray et al., 1992). Biological studies and ecotoxicological approaches can provide a more meaningful and realistic assessment of the state of the marine environment. This is so because biological studies provide direct information on biological effects. Over the last decade, there has been an increasing emphasis on the use of biochemical, physiological, and histological changes as well as other aberrations in organisms to estimate either exposure to chemicals or the resultant effects (Huggert et al., 1992). These changes in biological response have been termed biomarkers.

Biomarkers in aquatic environmental monitoring Biomarkers are defined as biological responses that can be related to an exposure to, or toxic effect of, an environmental chemical or chemicals. Biomarkers can be considered as intermediates between sources of contamination and higher-level effects (Suter, 1992). It is therefore necessary to show the presence of a dose-response relationship, either a predictable increase or decrease of the biomarker with increasing exposure, and that higher-level effects are predictable from such. As such, biomarkers can act as early warning of environmental effects of xenobiotics before there are serious effects upon individuals or population.

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Depledge (1993) suggested that biochemical and cellular biomarker responses on their own are of unknown ecological significance. Increased MFO induction in livers of fish from highly polluted water may signify pollution exposure, but the fish may continue to grow and reproduce normally and the MFO response may be viewed as an acclimatisation process to altered environmental conditions rather than a manifestation of an injury (Jimenez et al., 1990). Therefore, the use of a hierarchy of biomarkers has been suggested to be the most appropriate method to assess environmental risks from xenobiotic insult (Depledge, 1993). Fish in Biomarker Research Fish are suitable organisms with which to monitor aquatic contamination, as they are located at the top of the aquatic food chain. Fish are known to accumulate toxicants and they are in direct contact with polluted water via their gills and body surface. As such fish have been used in toxicological investigations of the aquatic environment. Biomarkers have been applied to the field in assessing fish health populations experiencing contaminant stress (Shugart and Southworth, 1990; Lower and Kendall, 1990) and in ecological risk assessment (Suter II, 1990). When working with biomarkers the genetic differences between individuals taken from different populations including the occurrence of genetic resistance to xenobiotics as shown in the mussel, Mytilus edulis, should always be considered (Minier and Galgani, 1995). There is also the need to consider, and therefore, plan out accordingly, differences that might ensue from species, size, age, sex, season, habitat, and geographical origin (McCarthy and Shuggart, 1990).

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Biomarkers can also be categorised according to their specificity to an exposure. One category which can be divided into two groups comprising the stressor independent proteins; the heat shock proteins which are induced by a range of compounds and metals (Sanders, 1990); and glucose-regulated proteins, which are induced when there is deprivation of oxygen or glucose. The other group is the stressor-dependent biomarkers: the mixed-function oxygenases (MFOs), Glutathione S-transferases (GSTs), the haemoxygenases (HO) and the metallothioneins (MT), where proteins are induced by exposure to specific contaminants or physical conditions. The second category of biomarkers is based on their specific responses to a class of contaminants. These are the biochemical, cellular and whole animal. Among the biochemical biomarkers are the occurrence of DNA adducts (Ericson et al., 1995), micronuclei in blood cells (Spies, 1990), and liver oncogenes (McMahon, 1990). Cellular biomarkers would include induction of MFOs by organophosphates (OPs), polynuclear aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) (Forlin and Anderrson, 1995); acetylcholinesterase inhibition (Bocquene, 1993), metallothionein induction (Kille, 1995), and vitellogenesis induction by oestrogenic-like xenobiotics (Matthiessen, 1995). Whole-animal biomarkers, which

involve measurement of physiological end points such as scope for growth (sfg) and adaptive behaviour, are strictly not biomarkers if the NRC definition is applied.

1.3. Problem Statement

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Conventional water quality monitoring programmes have been expensive and manpower intensive. The resulting data were of questionable quality (National Research Council, 1992). A great deal of work on environmental monitoring has been focused on the determination of mainly physico-chemical parameters and residue levels, especially the organochlorines and heavy metals (Peakall and Shugart, 1991). There is currently a move in the United States of America and Canada to undertake an integrated approach to environmental monitoring viz-a-viz water quality monitoring, benthos community studies and toxicity studies - the TRIAD study (Chapman et al., 1995). Though the approach is deemed to produce better quality information on the state of the environment, it will no doubt be much more expensive to implement. Malaysia has instituted, for the past two decades, a monitoring programme for water quality in both riverine and marine environments. As with the North American experience, environmental monitoring has been expensive while the data generated have been useful only to a limited degree for management decisions. Very limited information is available for residue levels in marine organisms, and less still for toxicological effects on these. It is not surprising that marine water-quality-criteria development in the tropics is still being developed. The ASEAN-Canada Cooperative Programme on Marine Science Phase II (ending 1996) has been assigned the task to generate toxicity data on marine organisms for criteria development in ASEAN (ACMSP-II, 1991-1998).

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Molecular biomarkers may provide a relatively inexpensive and precise alternative or/and complementary technique to environmental monitoring of exposure and effects. Research efforts into the benefits and viability of molecular biomarkers as a monitoring and diagnostic tool should be immediately initiated for the tropical marine environment so as to address immediately the increasing problems posed by Mans economic activities. It is in this context that the present study is proposed: the initiation of a research programme to evaluate the responses of acetylcholinesterase, the biomarker of neurotoxic effect, in the tropical seabass, Lates calcarifer (Bloch) when exposed to various man-made contaminants.

1.4. Approach for this study

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In this study, acetylcholinesterase response, a proven biomarker of effect will be studied in the tropical seabass, Lates calcarifer (Bloch). Among the major contaminants in riverine and coastal marine waters, the organophosphate and carbamate insecticides can cause serious ecotoxicological problems because of their very toxic nature. Although transfer of these contaminants to the aquatic environment is generally episodic and usually diffuse and chronic, it has been shown that very low concentrations of these contaminants could compromise the well being of aquatic animals (Galgani and Bocquene, 1996). Furthermore, recent evidence has indicated that AChE inhibition is not only caused by the organophosphates and carbamates alone the presence of other, yet unidentified, contaminants may be causing the inhibitory response (Payne et al., 1995; Huang et al., 1996). Payne et al., (1996) found muscle AChE in the trout, Salmo trutta collected from two urban rivers receiving kraft pulp mill effluents, was significantly. Similar AChE inhibition was also observed in the spotted garfish, Lepisosteus oculatus attributed to exposure to multiple contaminants in the lower Mississippi River basin (Huang et al., 1997). Therefore, AChE must not be considered as a specific biomarker just for organophosphorus and carbamate insecticide exposure (Galgani and Bocquene, 1989; Day and Scott, 1990). There are three approaches to this study involving: 1. In vitro exposures of brain AChE from unexposed fish to various contaminants, and assaying for inhibitory effects, 2. In vivo exposures of seabass to various xenobiotics under controlled laboratory conditions, and

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3. Field-exposures of feral fish at various locations as to validate the laboratory-based findings. Seabass will be exposed to sub-lethal levels of the organophosphates, diazinon and fenitrothion; the carbamate, methyl isopropyl carbamate (isoprocarb); the organochlorine, lindane; the polyaromatic hydrocarbon, benzo( )pyrene, used crankshaft oil (both acetone- and water-soluble fractions), and the trace metals, cadmium, zinc and copper. The choice of diazinon, fenitrothion and isoprocarb was based on their ease of availability and widespread use in Malaysian agriculture and horticulture. Benzo( )pyrene is normally used as an indicator of PAH contamination which might ensue from petroleum and shipping activities; while lindane, a member of the OCP family of insecticide is still being legally used in Malaysia. Used crankshaft oil and industrial effluents have been identified as significant pollutants. The addition of the trace metals is to validate their influence on AChE activity in the presence of xenobiotics. Recent developments in AChE determinations have moved from the spectrophotometric (BMC) procedure based on Ellmans microplate-based technique (Moores, 1988). It was shown that the microplate technique is much more sensitive than the spectrophotometric method, with a coefficient of variation of 6.9 - 25.5% in the former and 34.6 - 55.4% in the latter (Day and Scott, 1988). The assay technique will initially follow that of Galgani and Bocquene (1991) with the protocol being optimised for the tropical seabass, as the technique is being developed. Ultimately, it is hoped that the seabass will be utilised as a sentinel for the monitoring of environmental contamination in riverine and coastal marine waters.

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1.5. Specific Objectives

The objectives of this study are: 1. To evaluate and optimise the methodology for AChE activity for the tropical seabass, L. calcarifer. 2. To ascertain the 96-hr LC50 values of different xenobiotics and trace metals in relation to AChE activity in the tropical seabass, L. calcarifer. 3. To determine in vivo dose-response relationships between xenobiotics and the activity of AChE in the tropical seabass, L. calcarifer of different size, nutritional conditions and exposure to different salinity regimes. 4. To undertake in vitro studies on the effect of xenobiotics and trace metals, singly and in combination, on the AChE activity of the seabass. 5. To evaluate the response of AChE in caged feral seabass, L. calcarifer, exposed to waters from various coastal and estuarine locations in Penang.

1.6. References

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_______________. (1989). Heavy metals in Malaysian shellfish. Paper presented at Fisheries Research Seminar, Department of Fisheries Malaysia, June 25-29, 1989, Melaka. Annual Fisheries Statistics, 1970-1990. Department of Fisheries, Ministry of Agriculture, Kuala Lumpur, Malaysia. Choo, P.S., I. Ismail and H. Rosly. (1994). The West Coast of Peninsular

Malaysia. In: An Environmental Assessment of the Bay of Bengal Region. Staffan Holmgren (Edt). Bay of Bengal Programme. Madras, India. pp 33-51. Department of Environment. (1990). Environmental Quality Report. Annual Report : Ministry of Science Technology and the Environment Malaysia. Department of Fisheries Malaysia. (1989). Deep-sea Fisheries Resources within the Malaysian Exclusive Economic Zone Survey Report of Demersal and Pelagic Resources. Department of Fisheries, Ministry of Agriculture, Kuala Lumpur, Malaysia.

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Galgani, F. and G. Bocquene. (1991).

Semi-automated colorimetric and enzymatic

assays for aquatic organisms using microplate readers. Water Res. 25 (2): 147-150. Jimenez, B.D., (1990). A. Oikari, S.M. Adams, D.E. Hinton, and J.F. MaCarthy. Hepatic Enzymes as Biomarkers: Interpreting the Effects of

Environemntal, Physiological and Toxicological Variables. In: Biomarkers of Environmental Contamination (Editors J.F. MacCarthy and L.R. Shugart). Lewis Publishers Jothy, A. A. (1973). Coral Reefs in the Coastal Waters of West Malaysia, their Utilisation and Conservation. Department of Fisheries, Ministry of Agriculture Malaysia. Lui, Y.P. (1992). Multispecies Fish Resources and Multispecies Fisheries in the

Coastal Waters of Peninsular Malaysia, with special reference to the West Coast. Paper presented at the 6th Session of the Standing Committee on Research and Development, Colombo, Sri Lanka May 18-21 1990, FAO Fisheries Report No. 463 Suppl. Rome, Italy, 215 pp. Phang, S. M. (1990). Seagrass - a neglected natural resource in Malaysia. Proc.

12th Ann. Sem. Malay. Soc. Mar. Sci., Kuala Lumpur. Rohani, I., S.M. Chan and I. Ismail. (1992). Organochlorine pesticides and PCB

residues in some Malaysian shellfish. Paper presented at the Seminar on Pesticides in the Malaysian Environment: Impact on Agriculture and Ecosystems, Feb. 27, 1992, Selangor.

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Sasekumar, A. (1980). The present state of mangrove ecosystems in Southeast Asia and the impact of pollution: Malaysia. South China Sea Fisheries Development and Coordinating Programme, Manila. Shahunthala, D. V. (1986). Heavy metal levels in Malaysian fish. Fisheries Bulletin No. 58, Ministry of Agriculture Malaysia. Sheppard, C.R.C. (1992). Marine Parks in Malaysia: Research, development and display. Rep. Project No. ADB 862-MAL., Kuala Lumpur, Malaysia. Tan, G.H., H.F. Yeoh, K. Zain and Y. Mohktar. (1992). Pesticide use and control in Malaysia. Paper presented at the Seminar on Pesticides in the Malaysian Environment: Impact on Agriculture and Ecosystems, Feb. 27, 1992, Selangor. Tan, G.H., S.H. Goh, Vijayaletchumy, C.P. Loong and K.L. Ang. (1990). Monitoring of water-borne organic chemical pollution in the Kelang River Basin. Bull. Environ. Contam. Toxicol. Sing. N.I. Chem. 18: 67-77. Tang, H.T. H.A.H. Haron and E.K. Cheah. ((1990). Mangrove forests of Peninsular Malaysia a review of management and research objectives and priorities.

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