KULLIYYAH OF SCIENCEINTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

FINAL YEAR PROJECT PROPOSAL

NAME: MUHAMMAD AFIQ B. HAMZAH

MATRIC NUMBER: 0629991

DEPARTMENT: DEPARTMENT OF BIOTECHNOLOGY

CONCENTRATION: BIOTECHNOLOGY (MARINE)

TITLE: HEAVY METALS CONCENTRATION IN MOLLUSC (BIVALVE)

ALONG THE COASTAL AREA OF LANGKAWI ISLAND

SUPERVISOR: PROF. DR. KAMARUZZAMAN B. YUNUS

Co-SUPERVISOR: NIL

INTRODUCTION

Aquatic environments are often at risk of exposure to pollutants, either from

specific and non-specific sources. Most of the pollutants released to the aquatic

ecosystems are heavy metals which are frequently present at elevated concentration,

as a result of industrial discharges, domestic sewage, non-point runoff and

atmospheric precipitation (Tao et al., 1998). The environment impact of introducing

heavy metals (like Hg, Pb, Cu and Zn) into estuaries and coastal areas has been an

issue of great concern and significance. Toxicity of metals is well known for

centuries. Heavy metals like copper, zinc and iron are essential metals for fish and

shellfish since they play an important role in biological systems. Some others such as

mercury, cadmium and lead are non-essential metals and have no known role in

biological systems as they are toxic even in trace amounts (Ong et al., 2007).

However, if the essential metals are taken in excessively, it can also be a threat to the

organisms. Accumulation of pollutants in fishes can be passed to human through the

food chain which can causes severe health effects. In modern history, mass poisonings

by metals especially Hg in many countries as for example the Minamata case in Japan,

and some serious case in Arabian Gulf and Indian Ocean had lead the scientific

community to intensification of research of the toxic effect of heavy metals to living

organisms. These cases often occur at developed areas of human activities. The

studies will be conducted at Langkawi Island as it is well known as a tourist attraction

site. The booming of tourism industries led to exponential development of resorts and

hotels to accommodate such activities. As more buildings are built, the amount of

sewage disposed also increase. Thus, it is prominent to monitor the impact of the

pollution occurred and it can be done by measuring the heavy metals presence in the

mollusc (bivalve) along the coastal area. This will act as a bioindicator to determine

the level of pollution.

OBJECTIVE:

i. To determine the concentration of heavy metals (Pb ,Cu and Zn) in the mollusc (bivalve - mussels, cockles, etc) along the coastal of Langkawi Island.

ii. To determine the degree of heavy metal contamination in marine commercial mollusc (bivalve) of these areas.

iii. To evaluate concentration of heavy metals in mollusc (bivalve) with respect to national standard for human health.

LITERATURE REVIEW

Heavy Metal

Heavy metals are defined as any metallic chemical element that has an atomic

number over 20 which relatively are high in density and are toxic or poisonous at low

concentrations. Heavy metals are natural components of the Earth's crust and they

cannot be degraded or destroyed. Some heavy metals are neurotoxin, for instances

lead, mercury, nickel, zinc, cadmium, chromium and manganese (Stewart, 1975).

However, as trace elements, some heavy metals for example, copper and zinc are

essential to maintain the metabolism of the human body. Yet, at higher concentrations

they can lead to poisoning as heavy metals tend to bioaccumulate in the affected

organisms. Bioaccumulation means an increase in the concentration of a chemical in a

biological organism over time, compared to the chemical's concentration in the

environment. Compounds accumulated in the living things are taken up and stored

faster than they are metabolized or excreted. According to Kamaruzzaman et al.

(2007), heavy metals from natural and anthropogenic sources are continually released

into aquatic system and they are serious threats because of their toxicity, long

persistence, bioaccumulation and biomagnifications in the food chain.

Copper (Cu)

Copper with the symbol Cu is group IB element in periodic table with an atomic

number of 29 and atomic mass of 63.546 (Hill and Petrucci, 2002). Copper is an

essential substance to human life, but in high doses it can cause deleterious effects to

human health. According to Shahnaz and Dayanthi (2006), copper is an essential

metal and important component of the respiratory pigment haemocyanin in

crustaceans. Copper sources are normally from drinking water from copper pipe,

metal plating, industrial and domestic waste, mining and mineral leaching. Copper-

containing compounds have been used in Florida as fungicides, herbicides, and soil

amendments, resulting in elevated Cu in the aquatic ecosystem (Rogevich et al.,

2008).

Zinc (Zn)

Zinc, with its molecular formula is Zn, has a molecular weight of 65.38 and a

density of 7.14 g/cm3. It has a melting point and boiling point at 419.5°C and 908°C

(EPA, 2005). Zinc is an essential trace element for all living organisms but an excess

or a deficiency in zinc uptake might lead to stimulation or retardation of cancer in

human and certain animals. Zinc and copper are used in small amounts as fertilizers in

some soils deficient in these elements. It is said that the primary anthropogenic

sources of zinc in the environment are from the metal smelters and mining activities

(ATSDR, 1995), while the production and uses of zinc in brass, bronze, die castings

metal, alloys, rubbers, and paints may also lead to its release to the environment

through various waste streams (EPA,2005).

Lead (Pb)

The symbol of lead is Pb and its atomic number is 82 with atomic weight of 207.2,

a melting point at 327.502 °C and the boiling point is about 1740 °C (Hill and

Petrucci, 2002). Lead is usually stored in the human bones, brain and teeth. One of the

few negative effects of lead to human is impairment of mental and physical

development. Lead can reacts with the red blood cell membrane to increase its

mechanical fragility. It also can cause nervous system impairment and muscle pain,

and can be passed throughout generations. Lead enters aquatic environment by a

number of pathways. The earth’s crust, geologic weathering phenomena and volcanic

activity account for natural sources, but most waterborne lead derives from human

activities such as mining and smelting, coal burning, cement manufacturing and is

used in gasoline (Rogers et al., 2003).

Heavy Metals in Mollusc (bivalve)

Metals deposited in the aquatic environment may accumulate in the food chain

and cause ecological damage and even form threats to human health (Bervoets et al.,

1999). Aquatic microflora and microfauna, which constitute fish food, are capable of

incorporating and accumulating heavy metals into their living cells from their

environment. Mostly all type of bivalve is filter-feeder organism. They are benthic

organisms which mean they live on the ocean floor. They feed by consuming the

nutrient-rich water and sediment. Their filter-feeder mechanism act by separating the

essential nutrients from the unwanted debris like soil. However, their mechanism do

not separate the heavy metals present in the environment thus consuming them as part

of their diets. This will lead to bioaccumulation of the heavy metal in the organism.

Benthic organisms tend to accumulate more heavy metal due to the higher metal level

in the sediment compared to the water (Vigh et al., 1996).

Green Mussels as bioindicator

Bioindicator organisms have been used in pollution monitoring studies for

detecting pollution in certain ecosystem. Bioindicators are organisms that are

used to assess pollution by either measuring the organism’s tissue content or by

their sensitivity towards pollution. The organisms chosen as bioindicators should

show tendency of accumulating the pollutant in their body or sensitivity towards

certain pollutants. Bioindicators not only reflect chemical exposure but also have

the capacity to integrate many of the physical, chemical and biological stressors

that operate in aquatic ecosystems (Ham et al., 1997). Green mussels have been

long established as a bioindicator for heavy metals in Thailand and Hong Kong.

Mussels were suggested long time ago to be good biomonitoring agents due to

their wide geographic distribution, sedentary lifestyle, stable population, easy

sampling, bioaccumulation and correlation with the average pollutants of the

environments, tolerance of salinity, resistance to stress of high accumulation of

wide range of chemicals and they can provide an assessment of bioavailability.

However, in Malaysia only limited studies has been carried out on the biology,

ecology and bioaccumulation of heavy metals by green mussels along the

Malacca Straits only. So, the study on the bioaccumulation of green mussels in

the coastal water of Langkawi Island could enable us new array on this

commercial importance species.

HYPOTHESIS

There might be accumulation of Copper (Cu), Zinc (Zn), and Lead (Pb) in mollusc

(bivalve) caught along the coastal area of Langkawi Island.

METHODS

The place where the study will take place is the coastal area of Langkawi Island.

Langkawi Island is located in the state of Kedah and part of the Malacca Straits. This

place fall under West Coast of Malaysia.

Sampling Procedure:

First, three locations will be selected as sampling site prior to the sampling. Next,

three species of bivalve (to be determined at the sampling site) of the same size will be

collected at each site. Samples will be collected in labelled bags or boxes. Then,

samples will be kept at storing temperature (0oC to -20oC) for transportation to lab for

further analysis.

Laboratory Work:

For extracting the tissues needed for heavy metal testing, first, the bivalves will be

thawed under running water at the lab. Next, the bivalve’s shell will be opened to get

the organs and meat inside. The organs and meat of the bivalve will be weighed using

analytical balance. The samples will be put into different plate and labelled according

to the species. Later, the samples will be dried in oven at 70oC for 72 hours. After the

sample dried, they will be stored inside a dry place until further use.

Acid Digestion:

Before digestion, the Teflon beakers will be cleaned thoroughly with a detergent

solution, rinsed with tap water, soaked in 5% nitric acid for at least 24 hours, and then

rinsed with metal-free water (EPA, 2000). Then, 1.0g of the bivalve’s tissues will be

heated in Teflon beaker with mixed concentrated acids of Hydrogen Peroxide (H2O2),

nitric acid (HNO3), hydrochloric acid (HCl) and sulphuric acid (H2SO4) in the ratio of

1:1 (Kamaruzzaman et al, 2008). The beakers will be kept heated at 100°C for two to

three hours and then, hydrogen peroxide (H2O2) will be added as to breakdown any

recalcitrant lipid material that remains in the solution. Next, the acids will be added

constantly until clear, light yellow solutions were obtained. The digestions of samples

were done along with a control (mixed of acids added) and a standard reference

material from green mussel for each batch of digestion. After cooling, the clear

solutions will be filtered and transferred into 50mL Falcon tubes and 5% of nitric acid

(HNO3) will be made up to 50mL. Lastly, the tubes will be sealed and kept in the

refrigerator prior to sample analysis.

Statistical Analysis:

The samples will be analysed by using inductively coupled plasma mass spectrometer

(ICP-MS). ICP-MS is used for quick and precise determination of copper, zinc and

lead in the tissue samples. It can detect the heavy metals in ppb value an in 100 times

dilution. Several analysis of the data will be done which are Metal recovery

measurement (the recovery for quality assurance), Formula for measurements

(calculation based on dry weights), and formula of calculations of heavy metal

concentration in bivalve’s tissue according to the equation below:

µg metal = [(reading/1000) x (weight of test tubes x 1000) x (diluted volume)]

g dry weight [(dry sample weight) x 1000]

EXPECTED RESULTS

1) Record of concentration of heavy metals in mollusc (bivalve) along the coastal

area of Langkawi Island

2) Determine the level of pollution in Langkawi Island coastal area

3) Report and publication for the Final Year Project journal

4) Direct cooperation with various related agencies and departments

5) New methodology for future research and development

REFERENCES

ATSDR (Agency for Toxic Substances and Disease Registry). (1995). Toxicological profile for zinc. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA. Available online at http://www.atsdr.cdc.gov/toxprofiles.

Bervoets, L.R. and Verheyen, R. (1999). Accumulation of metal in the tissues of three spinned stickelback (Gasterosteus aculeatus) from natural sea water. Ecotoxicology and Environmental Safety. 48, 117-127.

Ham, K.D., Marshall, S.M. and Peterson, M.J. (1997). Application of multiple bioindicators to differentiate spatial and temporal variability from the effect of contaminant exposure on fish. Ecotoxicology and Environmental Safety. 37, 53-61.

Hill, W.J and Petrucci, R.H. (2002). General Chemistry: An integrated approach, Prentice Hall, United States of America.

Kamaruzzaman B. Y. et. al. (2008). Levels of Heavy Metals in Green-Lipped Mussel Perna veridis (Linnaeus) from Muar Estuary, Johor, Malaysia. Pakistan Journal of Biological Sciences, 11 (18): 2249-2253.

Kamaruzzaman, B.Y., Zaleha, K., Ong, M.C. and Willison, K.Y.S. (2007). Copper and zinc in three dominant brackish water fish species from Paka Estuary,

Terengganu, Malaysia. Malaysian Journal of Science. 26, 65-70.

Ong, M.C., Effendy, A.W.M. and Kamaruzzaman, B.Y. (2007). Determination of Pb, Cu, Cd and Zn in fish sample of the Mengabang Telipot River surrounding University Malaysia Terengganu by ICP-MS. Malaysian Journal of Science. 26, 71-78.

Rogers, J.T., Richards, J.G. and Wood, C.M. (2003). Ionoregulatory distruption as the acute toxic mechanism for lead in rainbow trout (Onchorchynchus mykiss). Aquatic Toxicology. 64, 215-234.

Shahnaz, K. and Dayanthi, N. (2006). Sensitivity of juvenile freshwater Cryfish Cherax destructor (Decapoda: Parastacidae) to trace metals. Ecotoxicology and Environmental Safety. 68, 463-469.

Stewart H. (1975). Heavy metals in: Pharmacological Basis of Therapeutics 5th ed. Goodman L.S. and Gilman A. (ed). Macmillan. New York. Pp 924-941.

Tao, S., Liang, T., Cao, J., Dawson, R.W. and Liu, C. (1998). Synergistic effect of copper and lead uptake by fish. Ecotoxicology and Environmental Safety. 44, 190-195.

U.S. Environmental Protection Agency (EPA). (2005). Toxicological review of zinc and compounds.

U.S. EPA. 2000. Guidance For Assessing Chemical Contaminant Data For Use in Fish Advisories. Volume II: Risk Assessment and Fish Consumption Limits. U.S. EPA, Washington, DC.

Vigh, P., Mastala, Z. and Balogh, K.V. (1996). Comparison of heavy metal concentration of Grass Carp in a shallow eutrophic lake and fish pond. Chemosphere. 32, 691-701.

GANTT CHART

2009 2010

Project activities Aug Sept Oct Nov Dec Jan Feb Mar Apr May

Title Finalisation

Proposal

Proposal

presentation

Sampling

Laboratory Work

Data Analysis

Paper Presentation

Report Submitted

Kuantan Langkawi Island = ±700 km

Langkawi Coastal Area (Sampling Site) = ± 10 km