Determination of Heavy Metals in Molluscs from different Fresh markets in Kuching

SHARI’ATUR RADHIAH BINTI JAFAR

(24989)

This project is submitted in partial fulfillment of the requirement for the degree of

Bachelor of Science with Honors

(Resource Chemistry)

Resources Chemistry

Department of Chemistry

Faculty of Resources Science and Technology

UNIVERSITY MALAYSIA SARAWAK

2012

II

Declaration

No portion of the work referred in this dissertation has been submitted in support of an

application for another degree of qualification of this or any other university or instituation of

higher learning.

_________________________

(Shari’atur Radhiah Binti Jafar)

Program of Resource Chemistry (2009-2012)

Faculty of Resource Science and Technology

University Malaysia Sarawak.

III

Acknowledgment

First and foremost, I would like to express my deepest appreciation to my supervisor, Dr.

Harwant Singh for being very helpful throughout this project and in ensuring the completion

of this thesis. He inspired me greatly to work in this project. I also thanks to my family

because give support to me especially in budgeting for completing this thesis. My gratitude

also goes to my friend had been very supportive and for their readiness to lend a hand

throughout this study. Besides that, I would like to thank to the lab assistant for their help and

guidance during lab work.

IV

Table of Content

Page

Acknowledgements III

Table of Contents IV

List of Abbreviations VII

List of Tables and Figures VIII

List of Appendices XI

Abstract XII

1. Introduction & Objective 1

1.1 Background 1

1.2 Objectives 2

1.3 Statement of problem 3

2. Literature Review 4

2.1 Heavy Metals 4

2.2 Heavy Metals and Health 5

2.3 Heavy Metals in Estuarine Environment 6

2.4 Importance of Marine Organisms as Edible Seafood 7

2.5 Heavy Metals in Marine Organisms 8

V

2.5.1 Intracellular Storage and Sequestration 10

2.5.2 Internal Transport 11

2.6 Mechanisms of Uptake Heavy Metal 11

2.6.1 The absorption of metals by aquatic animals involving

transfer across ephithelial cell 11

2.6.2 The cellular uptake of metals 12

2.7 Molluscs 12

2.7.1 Mollusc species as edible food 13

3. Materials and Method 17

3.1 Sample collection 17

3.2 Sample tissues preparation 19

3.2.1 Decontamination 19

3.2.2 Oven drying 19

3.2.3 Sample size reduction 19

3.2.4 Storage 19

3.2.5 Sample digestion 20

3.3 Heavy metals analysis 21

VI

3.3.1 Dilution factor 21

3.4 Statistical Analysis 22

4. Results 23

5. Discussion 34

6. Conclusion 55

7. References 56

8. Appendix

VII

List of Abbreviations

As - Arsenic

Ni - Nickel

Zn - Zinc

Fe - Ferum

Cu - Copper

Co - Cobalt

Cd - Cadmium

Cr - Chromium

Pb - Lead

mg/kg - milligram/kilogram

ᵒC - degree Celsius

HNO3 - Nitric Acid

H2SO4 - Sulphuric Acid

mL - milliliter

AAS - Atomic Absorption Spectroscopy

VIII

List of Tables

Table 1: Some common edible mollusc.

Table 2: Part of the sample was analyzed.

Table 4.1: Mean concentration of heavy metals (mg/kg) in various molluscs.

Table 4.2: Mean concentration of heavy metals (mg/kg) in various molluscs

Table 4.3: Mean concentration of heavy metals (mg/kg) in various molluscs.

Table 4.4: Result from Two-Way ANOVA, between fresh markets and molluscs

Table 4.5: Results from Post-Hoc test, test between places

Table 5.1: The concentration of heavy metals present and the Malaysian Food Act

(1983) and Food Regulations (1985).

Table 5.2: The concentration of heavy metals present and the Malaysian Food Act

(1983) and Food Regulations (1985).

Table 5.3: The concentration of heavy metals present and the Malaysian Food Act

(1983) and Food Regulations (1985).

IX

List of Figures

Figure 2.1: Bioaccumulation of heavy metals. (UW Extension, 2004).

Figure 2.2 : Blood clam (Anadara Granosa)

Figure 2.3: Razor Clam (Solen regularis)

Figure 2.4: Squid (Loligo sp)

Figure 3.1: The flow chart summarizes the steps for sample digestion ( Lau et al., 1998)

Figure 5.1: The concentration of As, Ni and Cu in the various molluscs

Figure 5.2: The concentration of Zn, Fe and Co in the various molluscs.

Figure 5.3: The concentration of Cd, Cr and Pb in the various molluscs

Figure 5.4: The concentration of As, Ni and Cu in the various molluscs

Figure 5.5: The concentration of Zn, Fe and Co in the various molluscs.

Figure 5.6: The concentration of Cd, Cr and Pb in the various molluscs.

Figure 5.7: The concentration of As, Ni and Cu in the various molluscs.

Figure 5.8: The concentration of Zn, Fe and Co in the various molluscs.

Figure 5.10: The concentration of Cd, Cr and Pb in the various molluscs.

Figure 5.11: Comparison of Ni concentration in molluscs at three fresh markets.

X

Figure 5.12: Comparison of Zn concentration in molluscs at three fresh markets

Figure 5.13: Comparison of Fe concentration in molluscs at three fresh markets

Figure 5.14: Comparison of Cu concentration in molluscs at three fresh markets

Figure 5.15: Comparison of Co concentration in molluscs at three fresh markets

Figure 5.16: Comparison of Cd concentration in molluscs at three fresh markets

Figure 5.17: Comparison of Cr concentration in molluscs at three fresh markets

Figure 5.18: Comparison of Pb concentration in molluscs at three fresh markets

XI

List of Appendices

Appendix A: Raw data from AAS.

Appendix B: Corrected concentration raw data from AAS.

Appendix C: Result above detection limits.

Appendix D: Result of concentration of heavy metals (mg/kg).

Appendix E: Mean concentration of heavy metals (mg/kg).

Appendix F: Results from Two-Way ANOVA.

Appendix G: Result from Post-Hoc Test.

XII

Determination of Heavy Metals in Molluscs From different Fresh markets in Kuching

City

Shari’atur Radhiah Binti Jafar

Resource Chemistry Programme

Faculty Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

The aim of this study was to determine the heavy metals that were present in selected molluscs from different fresh markets in Kuching area. The sampling area that were selected are Stutong fresh market,

Petanak fresh market and Batu Tujuh fresh market in Kuching. The chosen molluscs that are used are

blood clam (Anadara granosa), razor clam (Solen regularis) and squid (Loligo sp). The elements

that were analyzed throughout this project are As, Ni, Zn, Fe, Cu, Co, Cd, Cr and Pb. Atomic

Absorption Spectroscopy (AAS) model 3500, iCE Thermo Scientific are used to determined the

concentration of heavy metals present in each sample. The result obtained showed that Fe had higher concentration and detected in all samples compared to other heavy metals. Other than that, the least

amount of heavy metals found was Pb and Co.

Key words: Heavy metals, molluscs, AAS.

Abstrak

Tujuan kajian ini dijalankan adalah untuk mengkaji kehadiran logam berat yang terkandung di dalam

mollusca yang dipilih dari pasar segar di kawasan Kuching. Kawasan kajian yang dipilih adalah pasar segar Stutong, pasar segar Petanak dan pasar segar Batu Tujuh di kawasan Kuching. Mollusca

yang terpilih adalah kerang (Anadara granosa), ambal (Solen regularis), dan sotong (Loligo sp).. Di dalam kajian ini, logam berat yang dikaji adalah As, Ni, Zn, Fe, Cu, Co, Cd, Cr and Pb. Atomic

Absorbtion Spectroscopy (AAS) model 3500, iCE Thermo Scientific adalah alat yang digunakan untuk

mengkaji kadar kepekatan logam berat dalam setiap sampel.. Keputusan menunjukkan bahawa Fe

mempunyai kepekatan paling tinggi berbanding dengan logam-logam berat yang lain yang dikaji. Selain daripada itu, logam berat paling sedikit yang ditemui adalah Pb dan Co.

Kata kunci : Logam berat, moluska, AAS.

1

CHAPTER 1

INTRODUCTION

1.1 Background

Heavy metals are considered as very important and highly toxic pollutant in the

various environmental departments. Chronic contamination by heavy metals in the marine

environment is severe problem particularly in estuaries. By stimulating the lipid peroxidation

process with consequent complex sequences of biochemical reactions, heavy metals may alter

the structure of the cell membrane (Viarengo, 1989). According to Viarengo (1989), this

process is widely defined as oxidative deterioration of polyunsaturated fatty acids.

Peroxidation results in the production of lipid radicals and in the formation of a

complex mixture of lipid degradation product including malonyldialdehyde (dialdehyde of

malonic acid) and other aldahyde such as alkanals, hydroxyalkenals and ketones. There is a

growing concern that the natural cycling rates of many metals are being disturbed by

anthropogenic activities, especially the release from industrial, domestic and urban effluents of

increasing amounts of Pb, Zn, Cd, Hg and Cu (Schindler, 1991).

From food chain, marine and estuarine animals such as molluscs tend to accumulate

heavy metals in their body tissue (Netpae et al., 2009 ; Yap et al.,2009). Due to their toxicity,

persistence and ability to concentrate along the food chain, heavy metals are one of the most

serious pollutants in the environment (Beldi et al., 2006). Intensive agriculture activities and

industrial activities eventually lead to increase amount of pollutant especially heavy metals

2

and threatened the estuaries environment, because of the rapid growth of human population

(Christopher et al., 2010).

Most of living organism need small amount of essential metals such as iron (Fe),

manganese (Mn), copper (Cu), and zinc (Zn) as for essential processes such as for growth

purpose (Christopher et al., 2010 ; Culha et al., 2007). However, all this metals will cause

harmful effect when it exceeded the certain limits (Beldi et al., 2006). The non-essential metal

such as Cadmium (Cd), lead (Pb) and chromium (Cr) are toxic even at relatively low

concentration and not essential for metabolic activities (Astudillo et al., 2006).

1.2 Objectives

The aims of this study are:

1. To determine the types and concentration of heavy metals present in selected molluscs.

2. To compare the concentration of heavy metals between the sampling locations.

3. To compare the concentration of heavy metals of selected species with Malaysian

Food Act (1983) and Food Regulations (1985).

3

1.3 Statement of Problem.

Heavy metals are among the major concerns in waste water treatment (Salchi et al.,

2008). They are often derived from heavy industry, such as electroplating, battery factories

and mining operations. The problem associated with trace metals contamination were

highlighted in industry because of their large discharges and because chromium and cadmium

are especially dangerous to aquatic organisms and can be bioaccumulated in the food chain

(Medina et al., 1986; Brown and Louma., 1995; Pip., 1995). Edible seafood is regarded as

healthy and tasty food for human because it is rich in protein. It is important to determine the

concentration of heavy metal in the edible marine organisms to ensure it was safe for

consumption.

Metal can be bioaccumulated in marine organisms, adversely affecting the health of

humans who consume large quantities of seafood (Patrick et al.,2008) . It is already

known that heavy metals can accumulate in tissues during mollusc’s growth

(bioaccumulation) and would disturb the normal metabolisms of the organisms. All metals are

toxic to organisms if consume in excess. Therefore it is essential to examine the concentration

of heavy metals present in marine organism which are commonly consumed.

4

CHAPTER 2

LITERATURE REVIEW

2.1 Heavy Metals.

Heavy metals are defined as chemicals element that are relatively having high density

exceeding 6 g/cm -3 or high relative atomic weight which is greater than sodium (atomic

weight=23), such as mercury, chromium, cadmium, arsenic and lead (Duffus, 2002). In this

case, there are nine elements of heavy metals will be determined in the mollusc edible

seafood. They are lead, cobalt, nickel, magnesium, zinc, copper and magnesium that also

known as essential metals. However, the essential metals can be toxic if taken in large

quantities (Keskin et al., 2007; Tüzen, 2002).

Heavy metals can damage living things at low concentration and tends to accumulate

in the food chain. Heavy metals in sea water include hydrated metals ions, complexes with

inorganic ligands and the species with different oxidation states (John et al., 2009). Though,

lead and cadmium are known as non-essential metals. Even in the small amount, they are

toxic and harmful to human health.

Hundreds of potentially toxic substances are found in sewage effluent. These include

metals (such as mercury, arsenic, lead, chromium, copper, cadmium, copper and silver),

hydrocarbons, synthetic organic chemicals and chlorine (Povlens et al., 2001). Furthermore,

they are generally adhered to fine-grained particles in the sewage and settled with the

sediment of the riverbed.

5

Metals are present in the environment and most of them are essential for

animals and plants. According to Wagner and Boman, (2003), trace element can roughly be

divided into two groups, that are essential (Cu, Ni, Se and Zn) and non-essential (Cd,Hg and

Pb). Generally it is very difficult to distinguish between the natural metal enhancement and

that resulting from anthropogenic sources.

The term “heavy metal” was used by ecotoxicologist and environmental scientists to

refer to the metal that caused environmental problems. The metals which have been studied

extensively the last decades are : Cd, Hg, Zn, Cr, Pb, Co, V, Ti, Fe, Mn, Ag and Sn(Tin).

Some metals that get more attention are Hg, Cd and Pb because of their highly toxic properties

and their effects on the environment and the living organisms (Valavanidis A.,2010).

2.2 Heavy Metals and Health

Bioaccumulation of toxics, particularly heavy metals, by aquatic life is dangerous since

these heavy metals could be transferred directly upon consumption to human (Asuquo, 2004).

Since they contain rich protein, B vitamins and minerals such as potassium, potassium,

phosphorus and selenium (Gebauer et al., 2006). Seafood product seems to have an important

role in the prevention and modulation of neurological dysfunction, mild hypertension (high

blood pressure) and cardiovascular diseases (Valfre et al., 2003). In a study covering people

from 36 countries, consuming seafood was correlated with a reduction in the risk of coronary

heart disease as well as deaths due to stroke (Ryan McDermott, 2007).

Metals are notable for their wide environmental dispersion from such activity, their

tendency to accumulate in select tissues of the human body and their overall potential to be

6

toxic even at relatively minor levels of exposure. The toxicity of metals most commonly

involves the brain and kidney, but other manifestations occur, and some metals, such as

arsenic, are clearly capable of causing cancer. Furthermore, copper and mercury have been

found to induce conformational alteration of internal peptides and proteins. Both heavy metals

ions changed the internal viscosity of the red blood cells. These results suggest that the

possible cause of the damage of cells may be metal-protein interaction in the cells, but may

exclude the oxidative mechanism of such damage (Krzysztof, 1992).

Heavy metals differ from other toxic substances in that they are neither created nor

destroyed by human. Nevertheless, utilization by human influences the potential for health

effects in at least two major ways, first by environmental transport, that is by human or

anthropogenic contribution to air, water, soil and food, second by altering the speciation or the

biochemical form of the element.

2.3 Heavy Metals in Estuarine Environment.

The estuarine environment is characterized by having a constantly changing mixture of

salt and fresh water, and by being dominated by fine sedimentary material carried into the

estuary from sea and from the river which accumulates in the estuary to form mudflats

(McLusky, 1994). The mixtures of the fresh water and salt present challenges to the

physiology of the animals which few are able to adapt.

In general, the estuaries are the habitat intermediate between the sea, the land and fresh

water, a habitat which is a complex dynamic mixture of traditional situation and which is

almost never statics (McLusky, 1994). Fine sedimentary deposits or mud are a most

7

characteristics feature of estuaries, and indeed the estuarine ecosystem has been defined as a

mixing region between sea and inland water of such shape and depth that the net resident time

of suspended materials exceeds the flushing.

Trace metals are among the most common contaminants bound to estuarine sediments

(Polsven et al., 2001). Metals in the marine environment come from geological processes

(volcanos and erosion) and from anthropogenic source such as point sources, runoff, and

discharges from rivers and atmospheric deposition. Metal in sea water can exist in different

physicals forms, wether dissolved, colloidal, particulate and chemical forms such as ion,

inorganic complexes, organic complexes, organometallic compounds and in different

oxidations states within a given chemicals forms .

According to Mason et al., (1994), mercury is transported in the atmosphere mainly as

gaseous elemental mercury and is deposited through wet rainfall and dry particulate deposition

processes as inorganic mercury. Rapid industrialization and the discharge of heavy metals into

the marine environment has become serious problem nowadays. These anthropogenic

activities have increasingly affecting the safety level of seafood consumed.

2.4 Importance of Marine Organisms as Edible Seafood.

Edible seafood is defined as fish or crustaceans that are suitable and deemed fit for

human consumption. In other word is a fish or shellfish from the sea that can be served as

food. Since the total volume of seafood traded internationally has increased from about 10

million to around 24 million metric tons during the last two decades, seafood is widely “global

player” (Yasuada & Bowen, 2006).

8

According to Howgate et al., (1997), over two billion people rely on seafood as a

major source protein in their diets around the world. Crustaceans and shellfish are good

sources of protein. They are low in saturated fat, although some of them contain moderate

amount of cholesterol. The nutritional benefits of fish are its content of high quality proteins,

vitamins and other essential nutrients. Shellfish and crustaceans are good sources of protein

(Marti-Cid et al., 2007). They are low in saturated fat, although some of them contain

moderate amount of cholesterol.

2.5 Heavy Metals in Marine Organisms.

Asuquo (1990) stated that any marine and estuarine fin and shell-fishes are known to

accumulate contaminate from water and their food, which tend to concentrate along the food

chain (Figure 1). The bioavailability and toxicity of metals to aquatic organism depends on the

chemical form of the metal, particularly whether or not it is part of an organic compound.

Inorganic mercury in aquatic systems can be converted to methylmercury by microorganisms

in sediment ( Brezonik et al., 1991).

9

These microorganisms are then eaten by larger species and the methylmercury is transferred

up the aquatic food web (Watras et al., 1998).

Figure 2.1: Bioaccumulation of heavy metals. (UW Extension, 2004)

At each tropic level, the organisms sequester some metal, excreting less than they

consume, resulting in increasing tissue concentration at higher tropic levels, a process called

biomagnifications (Patrick et al., 2008). In addition to food chain effect, a range of other

factors also affect the levels of metals in seafood. Some factor will not directly affect human

exposure, as consumer often cannot identify seafood by gender or its habitats. According to

Dewailly et al., (2000), anthropogenic chemical contaminations, which includes Pb and Cd in

the marine environment has led to the increasing threat of high levels of heavy metals

presence in the marine food chain as many of the heavy metals accumulate the food chain.

Plankton and algae in the ocean absorb the metals.

10

Small fish feeds on these plants and larger fish will eat these small fish and due

processes incorporate the metals in their tissues. Microbial contamination by bacteria and

viruses also lead to the decrease in the safety consumption level of the seafood as well as the

increase in the chemicals contamination (Fleming et al., 2006). Microbial contamination

generally caused by sewage dumping as well as from indirect contaminated run off. Human

are exposed to microbial contamination by consuming contaminated seafood.

Marine organisms, especially molluscs, have the ability to accumulate metallic

toxicant from the environment in which they live. As such, these animals have been used

widely for monitoring contamination of marine ecosystems ( Franco et al., 2002). Shellfish

such as oyster, mussels and clams live in the estuarine areas and obtain their food by filtering

large amount of water. However, if the sewage or chemical pollutants contaminate the filtered

water, a wide variety of organism and toxin pathogenic to human can accumulate in the

shellfish alimentary tract.

2.5.1 Intracellular Storage and Sequestration

Most heavy metals upon entering a cell are sequestered rapidly by intracellular

ligand (Tessier and Tunner.,1995). Sequestration of metals in an immobilized form occurs

throughout the various tissues and organs involved in pathway for metal uptake, transport,

utilization and release. Intracellular structure involved in metal sequestration and storage are

the metallothionen and the intracellular vesicle-bound granules. Both play a significant role in

metal binding and sequestration.

11

2.5.2 Internal Transport

Heavy metals transport via the circulatory system makes possible the delivery of

metals to the internal organs and redistribution among organs. The plasma protein of

vertebrates plays the dominant role in the transport of a wide range of metals. In invertebrate

blood cells and haemocytes have been considered the primary way for transporting metals

however invertebrate’s plasma protein may play greater role in metal transport.

2.6 Mechanisms of Uptake Heavy Metal.

Many metals are retained in the organisms as a simple ion. Others particularly

cadmium and mercury can be converted into covalent organometallic compound. These will

behave in a similar fashion to covalent organic compounds described previously and will

preferentially accumulate in fatty tissue. According to Reeve (1994), the distribution of the

metals within organisms is thus very dependent on the individual metal and its detailed

chemistry.

2.6.1 The absorption of metals by aquatic animals involving transfer across

epithelial cells includes:

1. Uptake by the apical membrane such as the interface with the external environment.

2. Movement through the cell and interaction with intracellular ligands.

3. Efflux across the basolateral membrane such as the interface with the circulatory

system.

12

2.6.2 The cellular uptake of the metals can be assigned to:

1. Uptake by exposed body surfaces such as gills

2. Ingestion and take up after solubilisation in the gut.

3. The gill and intestine are primarily sites for uptake of soluble metals from the aquatic

environment.

2.7 Molluscs.

The mollusc is a large phylum of invertebrate animals. The phylum is typically divided

into nine or ten taxonomic classes. Molluscs have such a varied range body structures that is

difficult to find defining characteristics that apply to all modern group (Giribet et al.,2006).

The most abundant metallic elements in molluscs is calcium (Michael Hogan., 2010). Most

general characteristics of molluscs is that they are unsegmented and bilaterally symmetrical

(Hayward, 1996 ).

Most molluscs have muscular mouths with radulae and its mouth also contain glands

that secrete slimy mucus, to which the food sticks. Beating cilia (tiny hairs) drive the mucus

towards the stomach, so that the mucus forms a long string (Ruppert et al., 2004). Koide et al

(1982) have suggested that bivalve shells offer several advantages over soft tissues for

monitoring of heavy metals. Furthermore, it has been proposed that analysis of ancient shells

could function as “geological record” of the pollution that induced changes metal levels in the

environment (Bertine and Goldberg.,1972 ; Ferrell et al.,1973). The levels of heavy metals in