0 Introduction

Seaweeds are marine macro-algae that are one of the living renewable resources of the marine environment and well known for its potential food and therapeutic applications (Tseng, 2004). According to Awang et al. (2008), Sabah and Sarawak have a great potential for the cultivation of seaweed. The seaweeds found in Sabah such as Kappaphycus alvarezii, Ulva reticulate, Gracilaria changgi, Caulerpa lentillifera and Sargassum polycystum species.
Seaweeds are one of the best sources of essential trace metals. Essential trace metals are metals needed by human body in little quantity. These metals can be determined by using atomic spectroscopy techniques. Consumption of seaweeds can increase the amount of dietary fiber and lower the development of some chronic illnesses (Southgate, 1990). The followings are some essential trace metals found in seaweeds and their benefits to human (Alexander, 2014):

Chromium: Regulates blood sugar and glucose metabolism.
Cobalt: Helps in the formation of red-blood cells.
Copper: Formation of red-blood cell and connective tissue. Acts as a catalyst to keep and discharge iron to help the formation of hemoglobin. Contributes to the central nervous system function.
Iron: Required for red blood cell formation and function. Important for brain function.
Molybdenum: Helps for normal growth and development.
Zinc: Crucial for more than 200 enzymes involved in metabolism, reproduction, digestion, and wound healing.

However, some researchers reported that some seaweed are contaminated and contained high amount of essential trace metals more than the amount needed to human body. These are due to the area and conditions of seawater. According to Noda (1993) the chemical composition of seaweed may vary due to the species, geographic area, seasons of the year and temperature of water.
This study focused on the essential trace metals found in different type of seaweeds. The results of the concentration of the essential trace metals are compared to dietary reference intakes (DRI). This study is significant to show the role of essential trace metals and enhance the awareness on the importance of essential trace metals to human health.

Problem Statement

Seaweeds can be classified into three main groups which are Phaeophycae, Rhodophyceae and Chlorophyceae. The compositions of essential trace metals in these groups are differed. Some groups contain high amount of certain essential trace metals and vice versa. These essential trace metals are important for maintaining health. These metals take part in enzymes, hormones and cells in the human body. Inadequate intake of essential trace metals can cause symptoms of nutritional deficiency. There are many researches that have been done on seaweeds. Most of them discussed more on the composition of macronutrients in seaweeds. Therefore, this research focused on essential trace metals. There is necessity to know the composition of essential trace metals in these seaweeds because they supply nutrition and give benefit to human’s health.
3.0 Objectives of study

To determine the concentration of essential trace metals like vanadium, zinc, iron, copper, manganese, chromium and molybdenum in seaweed by using inductively coupled plasma mass spectrometry (ICP-MS).
To compare the concentration of essential trace metals found in seaweed with the dietary reference intakes (DRI).
To compare the concentration of essential trace metals in different types of seaweeds and determine which seaweed has the highest value of essential trace metals.

4.0 Literature Review
4.1 Introduction
Many researches have been made to determine the composition of seaweeds. In the previous researches, they discussed more on the composition of macronutrients in seaweeds and lack of micronutrients. This research focused on essential trace metals found in seaweeds. This research is significant as these metals provide and supply nutrition values to human. This research is carried out in order to determine the concentration of essential trace metals like vanadium, zinc, iron, copper, manganese, chromium and molybdenum in seaweed, compare the concentration of essential trace metals found in seaweed with the dietary reference intakes (DRI), compare the concentration of essential trace metals in different types of seaweeds and determine which seaweed has the highest value of essential trace metals.
4.2 Definition of essential trace metals
Essential trace metals are metals that are required for human body in small amounts generally less than 100 milligrams per day (Cesar, 2005; Araya et al., 2006). These trace elements are minerals which human body cannot produce by itself but can be found in diet. Essential trace metals include vanadium, chromium, manganese, iron, cobalt, copper, zinc and molybdenum (Gadd, 1992).
Figure 1 shows the position of essential trace metals in the periodic table. These metals are transition metals and majority of them are located in period 4, showing the relationship between the size of nuclei and the availability of electron of the element to interact with organic molecules present in biological systems (Cesar, 2005).
Each essential trace metal has their own specific functions depend on their chemical structures and is important for life. Although these metals are needed for only 0.02% of the total body weight, they are vital as trace bioactive substances and active centers of enzymes (Osamu, 2004). A man required essential trace metals about 50 micrograms to 18 milligrams per day and these metals function as catalytic or structural components of larger molecules (Mertz, 1981).

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Essential trace metals do not exist by themselves but exist with each other. Excess of one trace metal can cause imbalances in other elements and lead to diseases. To be well-absorbed in the intestine, most trace metals need to be in ionic form. Diet, concentrations of trace element in water, interactions of drug-nutrient are crucial to stabilize the concentration of essential trace elements in the body (Alexander, 2014).
Figure 1: Periodic Table with white background indicating elements essential for human and bold characters representing the essential trace metals and non-metals (Cesar, 2005).
4.3 Recommended daily intake of essential trace metals
A certain daily intake of food supplements is needed by humans. The essential trace metals are important as enzymes, hormones and cells in the body. Inadequate intake and excessive consumption of essential trace metals can cause symptoms to human body. Table 2 summarizes recommended daily intakes of essential trace metals and its effects of excessive consumption.
Table 2: Recommended dietary allowances of essential trace metals (Dietary Reference Intakes, 2001).

Essential trace metal

Recommended Dietary Allowances per day

Adverse effects of excessive consumption

Zinc

2 mg – 12 mg

Decreased copper status

Manganese

0.003 mg – 2.6 mg

Increased blood concentration and neurotoxicity

Iron

6 mg – 27 mg

Gastrointestinal distress

Chromium

5.5 μg – 45 μg

Chronic renal failure

Copper

200 μg – 1,300 μg

Gastrointestinal distress, liver
damage

Molybdenum

2 μg – 50 μg

Reproductive effects

Vanadium

Not Determined

Renal lesions

4.4 Classification of seaweed
Seaweeds can be classified into three main groups based on their brown, red and green pigmentations. The groups of seaweed are Phaeophycae, Rhodophyceae and Chlorophyceae respectively. Sargassum polycystum species is in Phaeophycae group whereas Kappaphycus alvarezii and Gracilaria changgi species are in Rhodophyceae group and Ulva reticulate and Caulerpa lentillifera species are in Chlorophyceae group (Awang et al., 2014).
The sizes of seaweeds are different according to their group. Brown seaweeds are often large. They are usually having length about 20 meters long and the thickness is about 2 to 4 meters, whereby the smaller species is about 30 to 60 centimeters. Red seaweeds are usually smaller in size, generally ranging from a few centimeters to about one meter in length. The colors of red seaweeds are not always red. They might be purple, brownish red, but botanists classified them as Rhodophyceae because of other factors. Green seaweeds are small, with a similar size to the red seaweeds (Fisheries and Aquaculture Department, 29 October 2014).
4.5 The study of essential trace metal in different type of seaweeds
Seaweeds rich in about 8% to 40% of essential trace metals required for human body (Indegaard & Ostgaard, 1991). Some reports said that the metal contents in seaweeds were higher than edible land plants (Ortega et al., 1993). Concentration of essential trace metals may differed with each other because of several factors includes genetic species, sea conditions, seasons, habitats, maturity, geographical locations and environmental parameters of the seaweed (Ito & Hori 1989; Fleurence, 1999; Krishnaiah et al., 2008).
According to past researches, the commonly essential trace metals found in seaweeds were copper, cobalt, iron, manganese and zinc. Copper plays an important role in metabolism by allowing enzymes to function properly (Harris, 2001). Copper is vital for maintaining the strength of the skin, blood vessel, epithelial and connective tissue throughout the body. Cobalt is essential for the production of vitamin B12 that is necessary to ensure an adequate number of red blood cells are produced in the body (MedlinePlus 2014, 23 October 2014). Zinc is important for growth, cell division, immune system, vision and also helps to accelerate the renewal of the skin cells (Vallee & Falchuk, 1993). Iron is essential as oxygen and electron transport forhemoglobin synthesis of erythrocytes, oxidation–reduction reactions, and cellular proliferation (Yutaka et al., 2008). Manganese is important for the formation of bones, connective tissues, brain and nerve functions (Barbara, 2013).
4.5.1 Kappaphycus alvarezii species
The trace amount of manganese, iron, zinc, cobalt, chromium and copper were determined in this species that was collected from Palk Bay Mandapam, Tamilnadu in South India region. The concentrations of these metals were 10.6 ppm, 438.7 ppm, 25.5 ppm, 3.9 ppm, 52 ppm and 31.9 ppm respectively (Nageswara, 2013). Another sample was taken from Semporna in Sabah, showed that zinc, iron and copper were present in this species. The concentrations of these three metals were below 3.5 g/100g (Mansoor et al., 2012). From the study, it can be concludes that sea conditions and environments affect the concentration of essential metals in seaweed. In other research, to determine the composition of seaweed by using different types of seedling production, the micropropagated Kappaphycus alvarezii yielded significantly higher concentration of cobalt, copper, manganese and zinc compared to farm-propagated Kappaphycus alvarezii (Suhaimi et al., 2014). Fayaz et al., (2005) stated that Kappaphycus alvarezii species is a good source of essential metals, containing 0.033% of iron and 0.016% of zinc. The molybdenum is also present in Kappaphycus alvarezii about 0.04 milligrams (Suresh, 2014) and about 1.56 ppm of cobalt found in this species (Rajasulochana et al., 2012).
4.5.2 Ulva reticulate species
Ulva reticulata species from Pattani was reported to contain high level in manganese and iron and also small amount of zinc and copper. The concentration of manganese, iron, zinc and copper are 48.1 mg/100g, 174.8 mg/100g, 3.3 mg/100g and 600 μg/100g respectively (Pattama & Anong, 2006). Awang et al. (2008) reported the range amount of iron is 6.5 mg to 11 mg per 100g, 2 mg to 7 mg per 100g of zinc, less than 0.55 mg/100g of copper and less than 3 mg/100g of chromium present in this species.
4.5.3 Gracilaria changgi species
Gracilaria changgi species was reported to contain high level of zinc, iron, and copper with the concentration of 13.8 mg/100g, 95.6 mg/100g and 0.8 mg/100g respectively (Norziah & Chio, 2000). The result showed that this species has high amount of iron when compared to other vegetables reported by Tee et al. (1988). The comparison of iron content in Gracilaria changgi species and other vegetables are shown in table 3.
Table 3: Concentration of elements (mg/100 g wet weight) present in G. Changgi (Norziah & Chio, 2000) and in some vegetables (Tee et al.,1988).

Foodstuffs

Iron, Fe

G. changgi

95.6

Green bean s prouts

1.7

Soya beans, white

6.0

Peas

1.9

Red spinach

4.0

Carrots

0.8

Broccoli

0.7

From the study, it shows that seaweed is the best source of iron compared to other land vegetables due to its metabolic system which it can absorb elements directly from the seawater.
4.5.4 Caulerpa lentillifera species
Iron, zinc and copper were present in Caulerpa lentillifera species which is taken from Semporna in Sabah with the concentration of 21.37 mg/100g, 3.51 mg/100g and 0.11 mg/100g respectively (Suhaila et al., 2009). Duduku et al. (2008) determined the amount of 6.45 mg/100g of iron, 3.53 mg/100g of zinc, and less than 1mg/100g of copper and chromium found in this species. The sample was also taken from Sabah. The study about the composition in Caulerpa lentillifera species showed that iron, manganese, copper and zinc were present with concentration of 9.3 mg/100g, 7.9 mg/100g, 2200 μg/100g and 2.6 mg/100g respectively (Pattama & Anong, 2006).
4.5.5 Sargassum polycystum species
A research has been made that used sample of Sargassum polycystum species from Seribu Island in Jakarta showed the concentration of 0.002 mg/g of copper, 0.004 mg/g of zinc, 0.277 of iron and 0.010 of chromium were present (Joko, 2006). In other research that used sample from Kota Kinabalu, the present of iron, zinc and copper were also reported in small amounts with the concentration of 68.2 mg/100g, 2.15 mg/100 g, and 0.03 mg/100g respectively whereas other essential trace metals were not determined (Suhaila et al., 2009).
4.6 Methods used to analyze the essential trace metals in seaweed
Atomic spectroscopy techniques viz., flame atomic absorption spectroscopy (FAAS), graphite furnace atomic absorption spectroscopy (GFAAS), inductively coupled plasma optical emission spectroscopy (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) are widely used to analyze trace element. Nageswara (2013) used FAAS method to determine the trace element in seaweed, the instrument was calibrated with standard solutions and a hollow cathode lamp was used for detection of lead, cadmium, copper, nickel, iron, cobalt, chromium, manganese, zinc, platinum and palladium. In other research, Awang et al. (2008) used GFAAS method whereas Rodenas et al. (2009) used ICP-MS method and Suhaimi et al. (2014) and Suresh et al. (2014) used ICP-OES method.
This research uses ICP-MS method. Inductively coupled plasma mass spectrometer is suitable for the determination of trace metals with pretreatment and laser ablation device. This method has high sample throughput and the sensitivity is extremely high. It has lower detection limits and the ionization is efficient. In order to get the concentration of essential trace metals in seaweed, the elements are placed in solution by acid digestion. This solution is then nebulized into spray chamber and then carried by argon gas into a torch. ICP is argon plasma and can reach temperatures of 10,000 Kelvin. This allows the atomization of the trace metals in seaweed to complete and reduced the interferences of potential chemical (PerkinElmer, 23 October 2014). The positive ions in the plasma are focused down a quadrupole mass spectrometer. This quadrupole mass spectrometer rapidly detects the mass range. By getting the mass spectrum of the plasma, information about the trace metals can be obtained.