A total of 394 seafood samples were collected from selected major fish landing ports of Fisheries Development Authority of Malaysia (Lembaga Kemajuan Ikan Malaysia, LKIM) and wholesale markets in Peninsular Malaysia. The sampling was conducted from June to December 2009. This paper reported results for nine heavy metals concentrations, which consisted of 296 numbers of samples included 46 species of marine fish. The selection of fish was based on the most popular/consumed seafood by the local population as described elsewhere [6]. Six major fish landing complexes of LKIM and five wholesale wet markets throughout Peninsular Malaysia were selected as the sampling location. The selected sampling locations are the main fish landing port where fishes were distributed throughout the Peninsular Malaysia, which is represented the whole population consumption of fish (Figure 1). The complete methodology of the sample calculation and collection has been described by Ahmad et al., 2015a [6].

All single elements stock solution (selenium, cadmium, lead, copper, zinc, antimony, tin, chromium and manganese) and mixed internal standard solution (indium, yttrium, holmium, and scandium) with the concentration of 10 mg/L were delivered by Perkin Elmer^® and certified for purity and concentration. A mixed working standard with the concentration of 1 mg/L was prepared by pipetting 5 mL of each stock solution into a 50 mL volumetric flask and diluted up to 50 mL with 0.2% nitric acid (HNO₃ Suprapur^®, Merck). This standard solution was used for calibration. All the laboratory apparatus were decontaminated by soaking in 10% v/v HNO₃ for 24 hours before analysis. Ultra-pure water with 18.2 M-ohm was used for the preparation of all solution and reagents.

The dried fish samples were weighted for 0.5 g and digested with a mixture of 5 mL concentrated nitric acid and 2 mL of hydrogen peroxide in a microwave digestion system (Multiwave 3000, Anton Paar). The power profile for the digestion process was as follows: During the first phase, the power of the digestion system was set at 600 W, followed by 5 minutes ramping and holding, respectively. At the second phase, the power was increased to 1400 W followed by 5 minutes ramping and 10 minutes holding time. Finally, in the third phase, the power was turned to zero withholding time of 15 minutes. After the digestion process, samples were filtered through a 0.45 μm acid resistant membrane. The solution was transferred into a 25 ml volumetric flask and diluted with ultrapure water. The analytical reagent blanks also prepared in the same manner but without the dried fish samples [6] [28].

The heavy metals were analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) ELAN 9000 (Perkin Elmer, Sciex, Canada) equipped with a Meinhard concentric quartz nebuliser, cyclonic spray chamber, nickel sampler, nickel skimmer cones and an autosampler (Perkin Elmer). The calibration curves were established using a mixed working standard solution containing 1 mg/L (Se, Cd, Pb, Cu, Zn, Sb, Sn, Cr and Mn) as described before by subsequent dilution into concentrations of 10, 40, 70 and 100 μg/L of each element, with 20 μg/L of mixed internal standard (In, Y, Ho, and Sc). The calibration curve showed linearity with a correlation coefficient of higher than 0.9995 (r² > 0.9995). Limits of detection were determined for each element by using seven replicate aliquots of the fortified reagent blank (0.2% HNO₃ + 0.05 µg/L of each element). Three times of the standard deviation of the aliquots readings were calculated to determine the detection limit and the sample analyses were conducted triplicate to ensure accuracy of the samples. The concentrations of heavy metals were converted to wet basis values in order to compare with PTWI. The formula as follows; wet weight concentration = dry wet concentration × (100/100 moisture percentage) [6]. The moisture content of the fish was calculated based on the study by Tee et al., 1997 [29] and Nurnadia et al., 2011 [30].

The precision of the method was evaluated by adding 20 µg/L of the standard solution of multi-elements (Se, Cd, Pb, Cu, Zn, Sb, Sn, Cr and Mn) to the fish sample. The concentration of the sample before and after the addition was measured. The recovery values of these elements were between acceptable ranges, which were within 85% to 115%. The standard reference samples (NIST SRM® 1946-Lake Superior Fish Tissue) were analyzed to ensure quality control of this analysis. The average recovery of reference standards was 90.7%.

To prevent human health impairment by heavy metals originating from dietary sources, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) assigned a PTWI [31] for total Cd, Pb, Cu, Zn and Sn that are 0.007, 0.025, 3.5, 7.0 and 14.0 mg/kg b.wt./week respectively [20] [21]. According to FAO 2009, fish consumption for an average age of Malaysian per capita is 160 g/day/person [32] [33]. The average body weight for the Malaysian population is 62 kg [34]. In order to estimate the risk of exposure from consuming fish, Estimated Weekly Intake (EWI) of elements of fish was calculated as follows [20] :

EWI = concentration of metals ( mg / kg , wet weight ) × weekly consumption body weight ( kg ) (1)

Risk assessment is a tool to estimate the probability of health effects due to exposure to the hazard, which in this study is the exposure through consumption of fish. USEPA developed the oral reference dose (RfDs) for Se as 5E⁻³ mg/kg/day; Cd as 1E⁻³ mg/kg/day; Zn as 3E⁻¹ mg/kg/day; Sb as 4E⁻⁴ mg/kg/day; and Mn as 1.4E⁻¹ mg/kg/day (Risk Information System (IRIS), USEPA). There is no established reference of dose for Pb, Cu, Sn and Cr under IRIS USEPA. The health risks assessment for non-carcinogen metals were calculated based on the following equation [22] [35] [36] :

HQ = EF × ED × FIR × C RfD × BW × AT × 10 − 3 (2)

where HQ is chemical-specific Hazard Quotient; EF is the exposure frequency (350 days/year); ED is the exposure duration (30 years); FIR is the fish ingestion rate (160 g/day/person); C is the metal concentration in the muscle of fishes (mg/kg wet weight); RfD is the oral reference dose (IRIS, USEPA); BW is the average adult body weight (62 kg) and AT is the average exposure time for non-carcinogen (365 days/year × number of exposure years, assuming 30 years).

A summation of the hazard quotients for all chemicals to which an individual is exposed was used to calculate the hazard index as described in following equation [35] [36] :

HI = HQ Se + HQ Cd + HQ Zn + HQ Sb + HQ Mn (3)

where HI is the hazard index; HQ_Se is the target hazard quotient for Se intake; HQ_Cd is the target hazard quotient for Cd intake; HQ_Zn is the target hazard quotient for Zn intake; HQ_Sb is the target hazard quotient for Sb intake; HQ_Mn is the target hazard quotient for Mn intake. If the HQ and HI values were greater than 1, then it is implied that there is a potential non-carcinogenic health risk related to the studied metals.

Before statistical analysis, the data was cleaned and checked for discrepancies. The non-parametric technique was chosen for analysis as the data was not normally distributed because of the presence of outliers. The median was calculated using SPSS (version 11.5 for Windows, 2002, SPSS Inc.). The median was calculated from triplicate analysis of each sample and interquartile range was calculated to show dispersion of dataset. The statistical significance of difference was assessed using Mann-Whitney’s (MW) test for two groups and Kruskal-Wallis’s (KW) test for three groups or more. The level of significance was designated as p < 0.05 (5%).

A total of 296 marine fish samples were analyzed in this study. The samples included 177 samples of pelagic fish, which were classified into two families; Carangidae (80 samples) and Scrombidae (97 samples). There were 119 samples of demersal fish samples which were categorized into five families; Lutjanidae (24 samples), Latidae (14 samples), Dasyatidae (25 samples), Sciaenidae (25 samples), and Nemipteridae (31 samples). The size of fish from collected samples ranged between 12.0 cm (Selaroides leptolepis) to 142.0 cm (Himantura uarnak), the smallest weighed between 0.02 kg and 0.29 kg and the largest weighed between 2.26 kg and 3.04 kg (Table 1).

Heavy metals median concentration of nine elements (Se, Cd, Pb, Cu, Zn, Sb, Sn, Cr and Mn) from marine fish samples were summarized in Table 2. Results were expressed in wet weight (w/w) basis for comparison later with PTWI. Overall, the heavy metals accumulation in marine fish samples was found to be dominated by Zn followed by Sn, Se, Cu, Mn, Cr, Pb, Cd and Sb which ranged between 15.9612 mg/kg (Zn) to 0.0002 mg/kg (Sb) wet weight. Among the heavy metals studied, Zn showed the highest level of accumulation and Decapterus macrosoma showed the highest concentration of Zn (15.9612 mg/kg) whereas Otolithoides biauritus showed the lowest concentration of Zn (2.3000 mg/kg). Sb showed the lowest level of accumulation, which was ranged between 0.0048 mg/kg (Decapterus macrosoma) and 0.0002 mg/kg (Selar crumenopthalmus, Otolithoides biauritus, Nemipterus tambuloides). Otolithoides biauritus demonstrated the lowest concentration for all heavy metal except for Pb. The highest concentration of Cd (0.0786 mg/kg), Zn (15.9612 mg/kg) and Sb (0.0048 mg/kg) were found to be in Decapterus macrosoma samples. Sample Himantura gerrardi demonstrated the highest concentration of Pb (0.057 mg/kg) while sample Nemipterus tambuloides showed the highest concentration of Sn (5.717 mg/kg). The highest concentration of Se was found in Thunnus tonggol (1.221 mg/kg) while the highest concentration of Cu was found in Megalaspis cordyla (1.614 mg/kg). Nemipterus nemurus showed the highest concentration of Cr (0.411 mg/kg) while Dasyatis zugei showed the highest concentration of Mn (1.542 mg/kg).

IQR: Interquartile range. ND: Not detected.

The estimated weekly intake (EWI) was presented in Table 3. The results were expressed as per unit body weight per week (ug/kg b.wt/week). Our results estimated that EWI for Cd consumption from Peninsular Malaysia ranged between 0.01 to 1.42 ug/kg b.wt/week. The estimated EWI of Cd in the study was far below the established PTWI of FAO/WHO JECFA (7 ug/kg b.wt/week). The calculated EWI of Pb from consumption of various fish species was between 0.03 and 1.02 ug/kg b.wt/week which was lower than PTWI guideline for Pb (25 ug/kg b.wt/week). The estimated EWI Cu ranged between 0.69 and 27.65 ug/kg b.wt/week, Zn ranging from 41.55 to 288.3 ug/kg b.wt/week and Sn were between 5.12 and 103.27 ug/kg b.wt/week. The results showed that the calculated EWI was below than established PTWI of Cu (3500 ug/kg b.wt/week), Zn (7000 ug/kg b.wt/week) and Sn (14,000 ug/kg b.wt/week). Therefore consumption of studied fishes was not considered to pose adverse effects to consumer based on FAO/WHO JECFA guidelines.

PTWI values for Cd, Pb, Cu, Zn and Sn are 7, 25, 3500, 7000 and 14,000 ug/kg b.wt./week (FAO/WHO, 2004; 2011).

Table 4 showed the estimated hazard quotient (HQ) and hazard index (HI) for the consumption of fish for each species. Overall results showed the HQ and HI values were less than one indicated that exposure to the studied metals through ingestion is unlikely to result in adverse health effects. There was a significant discrepancy of HQ among different metals. HQ values of Se were the biggest ranging between 0.0365 (Otolithoides biauritus) and 0.6041 (Thunnus tonggol). Decapterus macrosoma demonstrated the highest HI value (0.8930) compare to other species while Otolithoides biauritus showed the lowest value of HI (0.0593).

HQ - hazard quotient; HI - hazard index.

Significant variation of heavy metals concentrations, HQ and HI values for different factors were presented in Table 5. The median concentration of Cu, Se, Cd and Zn in pelagic fish were significantly higher compared to demersal fish (p < 0.05). Pb and Mn levels were significantly higher in herbivorous compared to the omnivorous and carnivorous (Pb, p = 0.001; Mn, p < 0.05). While Zn level was significantly higher (p = 0.014) in omnivorous compared to other feeding habits. Among family group, Pb and Mn levels were found to be higher in Dasyatidae compared to other family groups. Level of Se was higher in Scrombidae; Cu, Cd and Zn levels were higher in Carangidae; Sn level was higher in Sciaenidae compared to other family groups. Meanwhile, Cr and Sb levels showed no

*Significant different (p < 0.05).

significant difference between the family group. No significant differences were shown for heavy metals level between local and imported fish (p > 0.05). The coastal region showed significant differences for Cr, Se and Sb levels (p < 0.05). The highest concentrations of Cr were found on the south coast, Se in east coast while Sb in the west coast. The concentration of Cu, Sn, Se and Cd showed significant different (p < 0.05) between fish landing complexes and wholesale wet market. The levels of Cu, Sn, Se and Cd. Sn and Cd were found to be higher in Kuantan compared to other sampling location. Cu levels were higher from Selayang, and Se levels were higher from Chendering compared to other locations. Only Cd level was found significantly different (p = 0.025) for smaller fish compared to bigger fish.

Study on heavy metal in fish has been reported in many studies in Malaysia. However, most of the study focused on the specific location, certain heavy metal and particular fish species. Up to our knowledge, this is first publishing in Malaysia with a high number of samples, representing each state throughout Peninsular Malaysia, and covered nine elements of heavy metals. The results indicated that Zn was the most abundant metal in the fish samples. Even though Zn is essential for human, excessive amount could cause a severe health problem. It is quite interesting to discuss on this topic regarding its source, safe daily intake and health risk of zinc exposure through consumption of fish. The data also showed that there were interspecific differences in heavy metals level for all metals. This paper discussed the differences based on several factors, i.e., habitats, feeding habits, family, origin, coastal area, sampling points, sampling location and body length of fish. Each of these factors is discussed below.

Results of this study demonstrated Zn is the most accumulated metals in fish sample coincided with results reported in several studies conducted in Malaysia [9] [25] [26] [27] [37], which conducted the study in 6 coastal areas in Peninsular Malaysia found that Zn concentration in fish muscle relatively high among the studied elements (26.8 ug/g dry wt.). The study conducted by Bashir et al., 2013 [9] reported Zn was the highest metal concentration in two fish species, namely Arius thalassinus and Johnius belangeri, ranged between 30.21 to 13.12 ug/g dry weights. Fathi et al., 2013 [37] did a study in the eastern coast of Malaysia, recorded the highest mean concentration of Zn in Arius thalassinus (35.4 ug/g dry wt.), and the lowest mean concentration of Zn was in Megalaspis cordyla (17.54 ug/g dry wt.). Zn concentration in Ong et al., 2016 [27] study was lower compared to our study, ranged between 11.172 ug/g dry wt. (Thunnus sp.) to 5.861 ug/g dry wt. (Nemipterus sp.). While Kamaruzzaman et al., 2010 [26] study showed Zn range between 12 ug/g dry wt. (Selaroides leptolepis) to 25 ug/g dry wt. (Rastrelliger kanagurta) nearly equivalent to our study. The results indicated that bioaccumulation of Zn from water to muscle of fish is higher compared to other metals [27]. The source of Zn in natural water mostly from rock weathering process or human activities such as wastes water discharges from industrial. Even though Zn is an essential element for the human, higher concentration of Zn could cause health problems such as skin annoyances, stomach cramps, anaemia, vomiting and nausea. High levels of Zn could also cause damage to the pancreas and disturb the protein metabolism, and cause arteriosclerosis [38]. This study showed that Decapterus macrosoma recorded the highest concentration of Zn (63.013 ug/g dry wt.) and Cd (0.310 ug/g dry wt.). However, the levels were lower compared to a study conducted by Khalaf et al., 2012 [3] (Zn 94.57 ug/g, Cd 2.32 ug/g dry wt.). Agusa et al., 2005 [25], reported the much lower result of Zn (29.1 ug/g dry wt.) and Cd (0.162 ug/g dry wt.) in Decapterus macrosoma sample. Elements such as Pb, Cd, and Cr were subject to many studies because of their toxicity [25] [39]. Pb is other known significant contaminants in the environment and occurs naturally in soils, sediments and hydrosphere. Pb is also widely used in industries which may contribute to pollution in the environment [7]. Cd levels in this study were significantly high in Kuantan area. Kuantan is an urban area and situated near to petrochemical industries, which might contribute to Cd pollution in the coastal area [40]. Burning of fossil fuels and municipal waste are known to be the largest sources of Cd release to the environment [38].

Generally, the EWIs obtained from this study did not exceed the standard PTWIs recommended by JEFCA for Cd, Pb, Cu, Zn and Sn. Nevertheless, the EWI results from our study were higher compared to study conducted by Zaza et al., 2015 [21] in Italy, where EWI for Cd and Pb were 0.33 ug/kg/week and 0.49 ug/kg/week respectively. The study by Peycheva et al., 2016 [20] in Bulgaria reported that the EWI of Cu were ranged between 0.480 to 1.279 ug/kg/week, which is about 25% lower than our study. While the EWI of Zn demonstrates significantly lower than our study (7.334 - 15.983 ug/kg/week). The calculated HQ and HI in this study demonstrated that HQ values were lower than 1, which implied that fish consumption from Peninsular Malaysia has low non-cancer risk towards the human. Similar to the findings by Storelli et al., 2008 [2] showed HQ values lower than 1 for Cd. However, the values ranges were much lower compared to our study (HQ; 0.01 to 0.04). The study by Peycheva et al., 2016 [20] also demonstrated that their HQ values for Zn (0.0005 to 0.0010) were lower compared to our study. Although the values of HQ and HI were much lesser than 1, the consumer should consider the daily intake of fish to ensure safe consumption of fish.