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