A total of 30 specimens of each bivalve species including green-lipped mussel (P. viridis), Asiatic hard clam (M. meretrix), Pacific oyster (C. gigas) and marsh clam (P. expansa) were acquired from fishermen who harvest different species of bivalves in the Marudu Bay (Figure 1). The specimens were transported to the laboratory in polystyrene box containing ice cubes. The specimens were then dissected by using acid washed stainless steel knife. The whole soft tissues were kept in zip-lock bag then stored in −80˚C ultralow freezer (U570-86, New Brunswick). Prior to further analyses, the specimens were oven-dried at 105˚C for 24 hours [9] . Then grounded to powder using mortar and pestle, and kept in zip-lock bags until analysis.

Sample digestion was carried out in hot block digester (Tecator Digestor Auto 8, FOSS) according to the method described by [10] . Prior to digestion, all glassware were first rinsed with 1 N nitric acid. Then, 0.5 g of powdered sample was weighed using electronic balance (Pecisa 404A) and placed in digesting tubes. 15 ml of 65% nitric acid (Merck) and 2.5 ml of 70% - 72% perchloric acid (Merck) were added in the tubes. The tubes were heated at 140˚C for 10 minutes in hot block digester. Then, 5 ml of 37% hydrochloric acid (AR Grade, Qrec) was added in the tubes. Subsequently, the temperature was raised to 200˚C and digestion process proceeded for 3 hours. When digestion completed, the tubes were left to cool at room temperature before adding 1.25 ml of 65% nitric acid and 10 ml of double distilled water. The solution was filtered using Whatman filter paper (No. 1001-110) and top-up to 35 ml with double distilled water. Finally, the filtrates were kept in 20 ml glass vials and stored at 4˚C until heavy metal analysis.

Heavy metal content in the bivalve samples was determined by using the Inductively Couples Plasma-Optical Emission Spectroscopy (ICP-OES), Perkin Elmer Optima 5300DV. Quality Control Standard 21 (Perkin Elmer Pure) was used as Standard Reference Material (SRM1646a) for instrument recovery. Triplicate readings were obtained for every sample. Assessment of measurement quality and precision was achieved by running blank samples and certified standard materials Dogfish liver DOLT-5 (National Research Council Canada) alongside with the bivalve samples.

Health risk assessment was carried out by calculating the Hazard Quotient (HQ) according to [4] and [11] (Table 1). Health risk assessment of the toxicants was interpreted based on the values of HQ and Total Hazard Index (THI) [12] .

HQ = ( EF ) ( ED ) ( IR ) ( C ) ( RFD ) ( WAB ) ( TA )

The THI is the sum of HQ of respective heavy metal in the sample. Where, HQ_n is the hazard quotient for metal n in the sample.

THI = ∑ HQ = HQ 1 + HQ 2 + HQ 3 + ⋯ + HQ n

Values of HQ and THI below 1.0 indicate no risk of adverse health effect and the greater the value, the greater the risk level of the heavy metal intoxication [12] .

To compare the heavy metal contents in different bivalves, metal pollution index was calculated by using the following formula [15] .

MPI = ( Cf 1 , Cf 2 , ⋯ , Cf n ) 1 / n

where Cf_n is the concentration of the metal n in the sample. The MPI index value was adapted from [2] as shown in Table 2.

The SPSS Windows Statistical Package (version 22.0) was used for all statistical analyses. All data were subjected to one-way ANOVA with all variables tested for normality and homogeneity of variances. Significance level was set at p <

0.05. One-way ANOVA was performed and followed by the Tukey multiple comparison test (Tukey HSD) to make specific contrast of heavy metals content in the bivalves. A multivariate analysis (Principal component analysis) was performed to highlight the relationship between heavy metals. Varimax rotation was performed to help interpret the principal components.

The heavy metals contents in the four bivalve species collected from Marudu Bay are as shown in Table 3. It was noted that the As and Mn contents in all species of bivalves exceeded the permissible limits. The same was noticed to Cu, Pb and Zn in Pacific oyster and Zn in marsh clam (Table 3).

Hazard Quotient (HQ) exceeding 1.0 is considered threatening human health [1] . In this study, HQ for Fe andLi was not calculated because its oral reference dose and permissible limits have not been specified by any international standard providers. The calculated HQ of heavy metals in the four bivalve species is given in Table 4. Based on the HQ cut-point, it showed that all four bivalves can promote health risk due to As intoxication as the HQ values were greater than 1.0.The cumulative effect of HQ of the respective heavy metals was indicated by the value of THI. It was found that the THI values for all species of bivalves were greater than 1.0. The THI value, in decreasing order, of the respective bivalve begins with Pacific oyster (5.07) follows by green-lipped mussel (4.40), marsh clam (1.86) and Asiatic hard clam (1.60).

Table 5 shows the MPI of bivalves obtained from Marudu Bay. The highest MPI was obtained from Pacific oyster (15.03) followed by green-lipped mussel (8.31), marsh clam (6.86) and Asiatic hard clam (5.79).

The results of principal component analysis (PCA) of the heavy metals content

Note: b.d.l = below detection limit, Rfd = oral reference dose (USEPA IRIS, 2006), n.a = value not available, 1) MFR (1985), 2) EU (2002), 3) FAO/WHO (1984).

Note: n.a = value not available. Values of HQ greater than 1.0 are highlighted in bold indicating presence of adverse health effect associated with respective heavy metal.

in the four bivalve species are illustrated in Table 6. It showed that heavy metals reduced into four factors with the total variation explained of 35.1%, 18.2%, 17.2% and 10.8% for Factor 1, Factor 2, Factor 3 and Factor 4, respectively. Strong positive correlations were observed between 1) metals As, Cd, Cu, Pb, V and Zn with Factor 1; 2) Fe and Mn with Factor 2; 3) Cd and Li with Factor 3 and 4) Ni with Factor 4. On the other hand, opposite correlations were noticed between a) Fe and Mn with Factor 1; b) As, Cd and Li with Factor 2; c) Cu, Ni, Pb and Zn with Factor 3, and d) Cu, Mn and Zn with Factor 4.

Bivalves are known to have the ability to accumulate great level of heavy metals in their body [16] . This makes them good candidates for bio-indicator to assess the level of metal pollutants in an aquatic system. However, the contents of heavy metals in bivalves are affected by numerous factors. It was noted that intrinsic factors such as species, age, size, metabolic demand, spawning process [17] [18] [19] [20] and extrinsic factors such as type of heavy metals, location and various environmental factors [1] [17] [20] can affect the heavy metal content in bivalves. Many human activities (Table 7 and Table 8) and natural sources such as rocks weathering, runoff of mineralized rocks and hydrological conditions are known to contribute to heavy metals load in the aquatic environment [18] . In the present study, we observed that surface runoff [21] , domestic wastewater [22] and boating activities [23] [24] may contribute to the heavy metals deposition in the bay and caused the As and Mn contents in the bivalves to exceed the permissible limits (Table 3).

Although the As and Mn contents in all analyzed species exceeding the permissible limits, only the HQ value of As was greater than 1.0, which indicates presence of adverse health effects due to As intoxication. The HQ value is not only attributed to the extent of heavy metal content in the food, but also influenced by the exposure frequency as well as the ingestion rate of the contaminated food [25] . Furthermore, the differences in oral reference dose of the respective metal may also affect the HQ value. Upon consumption, the bio-accumulated heavy metals in bivalves body can eventually transferred to human [26] [27] . Inhalation and dermal contact are other recognized routes of heavy metal exposure [28] in human. As reviewed elsewhere [29] [30] [31] [32] , heavy metals and

metalloids have been reported to cause disturbances at cellular level especially in 1) promoting oxidative stress due to the initiation of reactive oxygen species (ROS), 2) causing DNA damage and impair its repair mechanisms, 3) hindering normal function of cell membrane and the assimilation of nutrient and 4) promoting malfunction of proteins. Moreover, most metals are known to form covalent bonds with carbon and resulting in metal-organic compounds which then interfering the normal functions of various organ systems [33] .

Metal Pollution Index (MPI) is used to identify the extent of heavy metal pollution [48] either in non-biota or biota. According to [25] , variation in MPI values noted in different species of organisms is attributed to the differences in feeding habits. However, variation in MPI can also be influenced by season [48] .

In the present study, it was shown that Pacific oyster exposed to medium contamination while the other species were categorized under low contamination. Previous study has reported that, oyster C. virginica, was unable to eliminate accumulated metals from their soft tissue even after depuration [49] . This condition is attributed to the presence of metallothionein, a cysteine-rich protein that has high affinity to heavy metals [50] which accumulate inside the cells in a non-toxic form [49] . Metallothioneins are responsible in the homeostasis of essential metals (Zn and Cu), detoxification of toxic metals (especially Cd) as well in scavenging free radicals [51] . They were known to be rapidly induced in the liver by a wide range of metals, drugs and inflammatory mediators [52] .

Principal Component Analysis (PCA) was applied in order to highlight the relationship among the heavy metals. Metal elements belong to the same groups are usually considered to be originated from the same source [53] . In the present study, As, Cd, Cu, Pb, V and Zn which are dominant variables on Factor 1, are likely to be originated from fertilizers, pesticides and herbicides [5] [6] [41] [42] . These substances are known to be used extensively in oil palm plantation. Leaching of heavy metals from contaminated soil may also occur during surface runoff. Moreover, despite various possible sources of Fe and Mn, both metals could have been originated from the same source of weathering process or from the domestic wastewater discharge. This was evident in current study where Mn and Fe were grouped together under Factor 2. Cd and Li were dominant variables on Factor 3. These elements can be naturally found in rocks minerals, thus weathering process as well as the sand dredging activities which have been reported to have taken place in some rivers flowing into the Marudu Bay [41] may contribute to such finding. Last but not least, oil residue from fishing boats, residues from forest burning during land clearing for agricultural purposes within and in the vicinity areas of study site could have been responsible for the dominance of Ni in Factor 4.

Water is crucial for the longevity of aquatic organisms. Despite its importance, it receives various environmental threats originating from agricultural activities, effluents from industries and households, and others [62] [63] . Upon the discharge of these wastes, heavy metals and other contaminants are loaded into the aquatic environment. Consequently, these heavy metals will be accumulated in the marine organisms, especially in bivalves following their exposure. This situation can harm human because those metals and other contaminants can be transferred to human through food chain.

Heavy metal contamination can be overcome or removed by mean of bioremediation [64] . It involves the use of living organisms such as algae, bacteria or plants to convert or reduce heavy metals into less toxic forms. The application of microbial bio-sorption to remove or recover heavy metals from aquatic environment has been reviewed by [65] . Risk of heavy metal intoxication and infection of various pathogens arising from the consumption of bivalves can also be reduced by mean of depuration [66] . Depuration is a process whereby bivalves are immersed in clean water environment for a certain period of time to allow purging of biological contaminants and physical impurities from them. El-Gamal [67] noticed that Zn, Pb, Ni, Mn, Cu, Cr and Cd can be reduced by 44%, 23%, 25%, 17%, 61%, 41% and 75% in clam, Paphia undulata after immersion in clean artificial seawater for 24 hours, respectively. The same author also showed that depuration process reduced the presence of Vibrio sp. (75%), Shigella sp. (31%), Escherichia coli (68%) and Salmonella sp. (36%) in the clam. Subjecting the newly caught bivalves to depuration process for more than 24 hours in clean water environment before consumption will help reduce health risk from contaminants including heavy metals.

Besides, strict enforcement of environmental law and policies in the bivalve natural habitat also help keep them away from being exposed to heavy metal contaminants. In Malaysia, the Ministry of Natural Resources and Environment has recognized the important of public participation in the effort to mitigate environmental pollution by providing an online complaint service for public to report any environmental polluting activities occurring in their surroundings [68] .