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In recent year, Malaysia is facing a rapid development with urbanization and industrialization. The population is growing in a fast pace due to this development and thus increasing the production of waste load to the aquatic environment. Various anthropogenic activities have produced considerable quantities of solid and liquid wastes. Although Malaysia has wastewater treatment plant facilities, however, the wastewater treatment plant is not efficient enough to treat all the pollutants and thus resulted in significant amount of pollutants being discharged into aquatic systems.
Large-scale of Linear Alkylbenzenes (LABs) production started being introduced into the environment in the early 1960s when linear alkylbenzenesulfonates (LASs) were used to replace alkylbenzenesulfonates (ABSs) in detergents (Eganhouse. et al., 1983). LABs with C10-C14 normal alkyl chain are the raw material for the industrial production of LASs which have been in extensive use for the manufacturing of domestic synthetic detergents (Tsutsumi. et al., 2002).
LABs are industrially synthesized during Friedel-Crafts alkylation reaction and subsequently undergo sulfonation reaction to produce LASs. During sulfonation process, small but significant amounts of residual unsulfonated LABs are carried with LASs into detergents and subsequent discharge into the aquatic environments (Eganhouse. et al., 1983 and Takada. et al., 1987). The structure of LAB and LAS are shown in the Figure 1.
Figure 1: The structure of Linear Alkylbenzene (LAB) and linear alkylbenzenesulfonate (LAS)
Takada and Ishiwatari have proposed a ratio of internal to external isomers (I/E ratio: a ratio of the sum of 6-C 12 and 5-C 12 relative to 4-C 12, 3-C 12 and 2-C 12) as an index of the degree of LAB degradation. Apart from that, they have also demonstrated how the concentration ratio of internal to external isomers used to estimate the degree of LAB degradation (Takada. et al., 1990). Therefore, I/E ratio is useful in assessing the fate of LABs and sewage-derived particles in aquatic environments.
In my proposed study, sediment samples will be collected from selected oyster aquaculture farms of Peninsular Malaysia. The selected oyster aquaculture farms are Batu Lintang (Kedah), Pulau Betong (Pulau Pinang), Muar (Johor), Pasir Panjang (Negeri Sembilan), Kuala Setiu (Terengganu) and Merchang(Terengganu) . There are more than 100 living oyster species found worldwide. My proposed study will focus on the species of the tropical oyster Crassostrea belcheri.
In general, an oyster farm is a facility where oyster are raised and harvested for food. Oysters are mollusks and classified as bivalves. According to Fisheries and Aquaculture Department, Food and Agriculture Organization of the United Nations, mollusks production constitutes about 11 percent of the total fisheries production of Malaysia. In Sabah, experimental cultures of Crassostrea belcheri are in progress. The commercial oyster farming in Sabah in done by cooperatives. Every year, the production of oyster estimate is about 18 tons per hectare by using rack-and-raft method. However, oyster culture operations are still in limited scale in Peninsular Malaysia which is practiced on part-time by fishermen.
2.0 Problem Statement
The discharge of poorly treated or untreated sewage effluents is one of the major threats to aquatic environments. Recently, several studies have proven the bioaccumulation of LABs in some species of aquatic organisms. High concentrations of LABs in oyster aquaculture farms not only potentially degraded aquatic systems but also detriments to human health as humans are at the top of food chain.
3.0 Aim of the Research Project (Objectives)
To determine the concentration of Linear Alkylbenzenes (LABs) in sediment samples collected from selected oyster aquaculture farms of Peninsular Malaysia.
To determine the internal isomer relative to external isomer ratio (I/E ratio) of LABs in collected sediment samples in order to assess the current status of sewage pollution in oyster aquaculture farms.
4.0 Significance of the Study
At an earlier time, Linear Alkylbenzenes (LABs) were thought to be constituents of marine matter, however many researchers have proved that LABs are recognized as ubiquitous pollutants. In Malaysia, sewage and wastewater is treated before discharged into the aquatic environments but still carries significant amounts of sewage loads into aquatic environments.
My proposed study is about the distribution of Linear Alkylbenzenes (LABs) in sediment samples collected from selected oyster aquaculture farms of Peninsular Malaysia.
An assessment the current status of sewage pollution can be conducted in selected oyster aquaculture farms. The proposed study will provide information on the application of LABs as a molecular marker to assess the sewage pollution in selected oyster aquaculture farms.
5.0 Review of Research Literature
Alkylbenzenes were measured in 34 green mussels collected from India, Indonesia, Malaysia, Thailand, Cambodia, Vietnam, and the Philippines together with blue mussels collected from Tokyo Bay, Japan (Tsutsumi. et al., 2002). They discovered that the I/E ratios (a ratio of internal to external isomers of LABs) in the South and South East Asian countries (1-3) were much lower than those in Tokyo Bay (3-8). Apart from that, Alkylbenzenes with branched alkyl chains and tetrapropylene-based alkylbenzenes (TABs) were detected in mussels from Indonesia and Philippines. Based on this discovery, they concluded that sewage treatment systems in South and South East Asian countries are insufficient. They also stated that LABs are a powerful tool to assess the quantity and quality of sewage impacts on coastal environments.
Takada and Ishiwatari have conducted laboratory incubations of Linear Alkylbenzenes (LABs) to obtain experimental evidence of systematic microbial alteration of their isomeric composition (Takada. et al., 1989). They stated that the degree of LAB degradation in was quantitatively related to the change in their isomeric composition. The results indicated that the degree of LAB degradation in Tokyo estuarine and bay sediments is approximately 45%. By use of the I/E degradation diagram, they estimated that 55% of LABs entering the aquatic environment of Tokyo remain in the sediments without biodegradation. They proposed that I/E degradation diagram as an indicator of the degree of LAB degradation.
Linear Alkylbenzenes (LABs) were measured in sediments from three areas of Port Phillip Bay in Australia (Murray. et al., 1987). They found that the concentrations of LABs in sediment sample from Port Phillip Bay (0-19 µg g-1) were generally lower than those reported for other coastal sediments from the USA and Japan. The compositional profiles showed that both physical and biological processes had acted to remove LABs during deposition. Apart from that, an unusual alkyl benzene compounds were detected at one side. These compounds were probably synthesized terrestrially by the bacteria of a nearby sewage farm. They concluded that microbial oxidation and dissolution in water were the two processes acting to remove LABs during deposition.
Takada and Ishiwatari have done on the distribution of Linear Alkylbenzenes (LABs) and Linear Alkylbenzenesulfonates (LASs) in river sediments, suspended river particles, domestic wastes, and waste effluents around the Tokyo city area (Takada. et al., 1987). The results indicated that LABs are carried into aquatic environments as a result of the use of synthetic detergents around the Tokyo metropolitan area. Based on their research, they demonstrated that the isomeric composition of LABs changes systematically during biodegradation. They also concluded that the ratio of LASs to LABs decreases with progressive exposure of LAS-type detergent to the aquatic environment.
Linear Alkylbenzenes (LABs) were evaluated as waste tracers in the marine environment by determining their concentration and composition in suspended particulate matter and sediments in the vicinity of Joint Water Pollution Control Plant (JWPCP) wastewater outfall system in California (Eganhouse. et al., 1983). They found that LABs appear to be preserved in sediments for time periods of 10-20 years. In addition to residence time, a complex assemblage of surfactant related branched alkylbenzenes was also found in the waste affected sediments. The result demonstrated that LABs are potentially useful as molecular tracers of domestic wastes and under appropriate conditions, as geochronological tools.
6.0 Thesis Outline
6.1 Sampling Location
In my proposed study, the selected oyster aquaculture farms in Peninsular Malaysia are Batu Lintang (Kedah), Pulau Betong (Pulau Pinang), Muar (Johor), Pasir Panjang (Negeri Sembilan), Kuala Setiu (Terengganu) and Merchang (Terengganu). The sampling locations are shown in the Figure 2.
Figure 2: Map showing the sampling locations of the selected oyster aquaculture farms in Peninsular Malaysia
Linear Alkylbenzenes (LABs) in sediment samples will be analyzed using Gas Chromatography Mass Spectrometry (GC-MS). In general, GC-MS can be used to determine molecular weights and elemental compositions of unknown organic compounds in complex mixtures.
The Figure 3 shows the proposed research methodology for sediment samples analysis is in accordance with the Laboratory Analytical Procedures (Zakaria et al., 2002).
Sediment samples collected from selected oyster aquaculture farms
Soxhlet Extraction with dichloromethane (DCM)
Silica Gel Column Chromatography
5% H2O deactivated
65% (DCM) in Hexane
40% (DCM) in Hexane
25% (DCM) in Hexane
Fully activated Silica Gel Column Chromatography
25% (DCM) in Hexane
Alkylbenzenes (LABs & TABs)
Figure 3: Laboratory analytical procedures for sediment samples
Sediment samples collected from six selected oyster aquaculture farms in Peninsular Malaysia, which are of Batu Lintang, Pulau Betong, Muar, Pasir Panjang, Kuala Setiu and Merchang.
The following statements describe the general information on sampling locations:
Batu Lintang is situated in Kedah, Malaysia. Its geographical coordinates are 5° 38' 0" North, 100° 24' 0" East. Its original name is Kampong Pengkalan Batu Lintang.
Pulau Betong is a small island in Penang, Malaysia. Its geographical coordinates are 5° 18.868 North, 100° 11.018 East. It is it is approximately 7 acres (28,000 m2) in area and is privately owned.
Muar is situated in Johor, Malaysia. Its geographical coordinates are 2° 2' 0" North, 102° 34' 0" East.
Pasir Panjang is situated in Negeri Sembilan, Malaysia. Its geographical coordinates are 2° 26' 0" North, 101° 56' 0" East.
Kuala Setiu is situated in Terengganu, Malaysia. Its geographical coordinates are 5.543956, 102.894974.
Merchang is situated in Terengganu, Malaysia. Its geographical coordinates are 5.031379, 103.293958.
Sediment samples were collected from six selected oyster aquaculture farms in Peninsular Malaysia, which are Batu Lintang (Kedah), Pulau Betong (Pulau Pinang), Muar (Johor), Pasir Panjang (Negeri Sembilan), Kuala Setiu (Terengganu) and Merchang (Terengganu). The sediment samples were collected in the amount of sufficient quantities for the purpose of chemical analysis in laboratory. These samples were analyzed for the possibility of Linear Alkylbenzenes (LABs) adsorbed to oyster aquaculture farm sediments.
At each sampling location, the samples were collected by using Eckman Dredge Sediment Sampler and placed in the pre-cleaned stainless steel container. After that, the stainless steel container containing sediment samples were wrapped with aluminium foil. The sediment samples were stored in the zip-log bag as soon as the stainless steel containers were labeled. Next, the zip-log bag with sediment samples was stored in a cooler box with ice or dry ice at 4°C. The sediments samples were subsequently transported to laboratory. The homogenize samples were stored in freezer at -18°C for further chemical analysis.
Sediment samples collected from six selected oyster aquaculture farms in Peninsular Malaysia were use to determine the concentration of Linear alkylbenzenes (LABs) in oyster aquaculture farm sediments. Clean apparatus is an essential requirement for laboratory work. Laboratory should be cleaned prior to LABs analysis. According to Analytical Laboratory Procedure (Figure 2), 20g of homogenized wet sediments were undergo freeze-dry process to eliminate the water content in the wet sediments.
After the drying process, the sediments were placed into the Whatman Cellulose Thimbles. These sediment samples were extracted in a process called Soxhlet Extraction by using Soxhlet Extractor with 250 mL dichloromethane (DCM) solvents and thermostat control for approximately 9 hours at temperature around 40°C. The extracts containing target compounds resulted from Soxhlet Extraction process were collected and undergo copper treatment in order to eliminate the sulfur content in sediment samples that produced by microorganism that may interfere with the chromatography reading. After copper treatment process, the extracts volume can be reduced by rotary evaporation process at temperature 30°C until almost dryness.
Next, the extracts were purified and fractionated by using two-step silica gel column chromatography. The first step silica gel column chromatography is use to remove unwanted polar compounds from organic hydrocarbon. Normal hexane (HPLC grade) was transferred into 0.9cm internal diameter glass column. After that, 5% H2O deactivated silica gel were use to pack the glass column until 9cm height for the first step column chromatography using Pasture pipette. Then, 1cm height of baked anhydrous sodium sulphate (Na2SO4) is added into the glass column by using Pasture pipette. The extracts were eluted into the glass column by using Pasture pipette and the extracts in the flask were rinsed with 2mL solvent (DCM: Hexane with ratio 1:3) in the sequence of 0,4mL, 0.3ml, 0.3mL, 0.5mL and 0.5mL respectively.
The extracts were transferred into glass column by pasture pipette each time. The remaining of 18 mL DCM: Hexane (1:3) was then transferred directly into first step glass column. For each interval, the flask with solvent was rinsed in the sonicator to ensure all extracts were transfer into column. The eluted samples (contain hydrocarbon such as
n-alkanes, LABs, PCBs, PAHs and pesticides) from first step silica gel column chromatography were collected into a pear shape flask. The samples will be
roto-evaporated as nearly to dryness as possible for the use of second step silica gel column chromatography.
For the second step silica gel column chromatography, normal hexane (HPLC grade) was transferred into 4.5mm internal diameter glass column and 100% activated silica gel was used to pack the glass column until 18cm height. The eluted sample was loaded from first steps column chromatography to second step column by using pasture pipette and followed by 4mL of normal hexane solvent (HPLC grade). The pear shape flask containing the eluted samples rinsed by normal hexane and load the remaining solvent into second step glass column by pasture pipette. For each loading interval, flask with solvent was rinsed in the sonicator to ensure all extracts were transfer into column and n-alkane fraction will be collected in a small pear shape flask as first fraction. Next, load 4mL of normal hexane solvent (HPLC grade) again as the same methods shown above into glass column and second fraction (LABs) was obtained.
For this study, only the second fraction is being analyzed. LABs are the organic compound to be analyzed and the second fraction was transferred to nitrogen blowdown process and the LABs fraction was evaporated to dryness. Lastly, the samples subsequently injected into Gas Chromatography-Mass Spectrometer (GC-MS) for LABs analysis.
6.3 Limitation of the Study
There are several limitations in undertaking final year project. It is understood that the limitations are a part of project .These limitations will indirectly delay the progression of project or influence the research results. Time constraint is one of the limitations in completing final year project. Time becomes an essential project constraint when student have to make time arrangements for attending lectures and conducting the laboratory works.
Cost is one of the project constraints common to every student. Each student will be given a financial support of RM250 for final year project. At the beginning of the project preparation, I was worried about what resources are needed and how to obtain these resources. I was fortunate enough in having several masters students helping and assisting me in experimental cost management.