MATERIALS AND METHODS

Reagents: The reagents with super quality, analytical grade Nitric acid (70%) and hydrogen peroxide (30%) were acquired from Merck (Darmstadt, Germany) along with the stock standard solutions of Al, Cr, Ni, Zn, As, Cd and Pb in concentrations of 1,000 mg L^-1. All the plastic and glassware were cleaned by soaking in dilute HNO₃ (10%) and were rinsed with de-ionized water prior to use.

Apparatus: A Perkin Elmer model Elan 9000 inductively coupled plasma-mass spectrometry (ICP-MS, USA) was used in current study. After calibrating the apparatus with standard solutions derived from commercial materials, it was optimized according to the manufacturing standards the cones and tubes were carefully cleaned to get rid of any possible residues.

Sample collection and preparation: Eggs sample from chicken (Gallus gallus), quail (Coturnix coturnix japonica), guinea fowl (Numida meleagris) and pigeon (Columba livia) were collected from commercial farm located in a rural area in Kajang city(latitude 2°59’N, longitude 101°47’E), Selangor, Malaysia. About 36 eggs (for each species) were chosen randomly during the period (from May to October 2010), all eggs were weighed, chicken egg (40.79±4.01 g), guinea fowl egg (38.55±1.75 g), quail egg (11.38±0.87 g) and pigeon egg (12.16±1.35 g). To analyze the metals in egg content according to method previously described by Burger and Eichhorst (2005) with slight modification, the eggs were washed vigorously with de ionized water, then open carefully the eggshell separated from egg content, the egg content were homogenizes and put it in Petri dishes the samples were drying at 70°C for 24 h the egg content grain to powder by mortar, 0.5 g of the sample soaked in nitric acid 70% and hydrogen peroxide 30% overnight in room temperature, the digested completed in bloke thermostat (150°C) for 4 h until solution were clear.

Table 1:	Analytical results for the Certified Reference Materials (CRM) and its certified values for each metal (μg g-1 dry weight)
The CRM for Al is not available

After cooling the solution was diluted to 50 mL with de ionized water, then were filtrated throw 0.45 μm acid resistant filter paper. The solutions were stored at 4°C for later metal analysis. Concentration of Aluminum (Al), Chromium (Cr), Nickel (Ni), Zinc (Zn) Arsenic(As), Cadmium (Cd) and lead(Pb) Were determined by inductively couple plasma-mass spectrometry (ICP-MS, model Perkin-Elmer Elan 9000 A). Each analysis was carried in duplicate, standard and blank samples were analyzed every 20 sample. All concentration were expressed in μg g^-1 on dry weight basis. The accuracy of the applied analytical method was validated by accurate analysis of Certified Reference Materials (CRM) for lobster hepatopancreas (TORT-2, National Research Council Canada). All the runs were carried out in triplicate. The results obtained on the SRMs are showed in Table 1. The recoveries of all the metals were satisfactory.

Health risk estimation: To estimate the human health risk from consuming metal contaminated eggs, the methodology for estimation of target hazard offers an indication of the risk level due to pollutant exposure, this method was available in US EPA Region 111 Risk based concentration table (USEPA, 2000). It is described by following equation:

where, EF is exposure frequency (from 365 days/year for people who eat egg 7 times a week to 52 days/year for people who eat egg one time a week. ED is the exposure duration (70 years), equivalent to the average lifetime; FIR is the food ingestion rate (egg: 37.2 g/person/day). C is the metal concentration in egg content (μg g^-1), RFD is the oral reference dose (USEPA, 1997, USEPA, 2000); WAB is the average body weight (64 kg, the reference weights for the age categories were derived from several local studies in Malaysia (Lim et al., 2000) and TA is the averaging exposure time for non carcinogens(365 day/yearxED). If HQ>1.00, indicating that there is a potential risk associated with that metal (Khan et al., 2008). The lifetime Cancer Risk (CR) for arsenic was obtained by using the chemical Slope Factor (SF) (USEPA, 1997) and the estimated metal exposure dose mg/kg/day (EED) in the following equation:

Statistical analysis: All calculations were performed using SPSS for Windows (vers. 18.0, SPSS Ltd., Woking, Surrey, UK). The descriptive statistics (mean values, standard deviation) for values of egg content were analyzed by one-way analysis of variance followed by the Tukey honestly significant difference test. Differences were considered significant at the p<0.05 level.

RESULTS

Metal analysis in eggs: Result demonstrated that disparity in metals concentrations in eggs among four bird’s species, among all the heavy metals, Zn concentration was maximum and Cd was minimum in four species eggs, the highest concentration of Zn were occurred in quail eggs, followed by chicken and guinea fowl however, their level does not significantly differ among species. Al concentration was significantly (p<0.05) highest in quail eggs followed by those of chicken and pigeon. Mean while, Cr was occurred in analogous values in chicken and guinea fowl eggs (3.2 and 3.6 μg g^-1), respectively followed by those of guinea fowl and pigeon. The same situation was observed in nickel concentration (Table 2). As and Cd levels in eggs of birds were found in less values compared to those of other metals. For As chicken showed the highest value. While for Cd concentration, quail presented the highest once. As far as, the highest values for Pb were obtained in Quail eggs while the lowest value found in guinea fowl eggs.

Health risk estimation: According to Norimah et al. (2008) that daily consumption of chicken egg for one person in Malaysia about one egg (medium) daily, which equivalent (without eggshell) average (37.2 g/person/ day), we suggest that daily consumption of other birds eggs was in the same rate. Metals (As, Cd and Pb) were choose to estimate the THQ due these metals may inadvertently enter the food chain and pose health risk to human (Zhuang et al., 2009) and Zn because it has high concentration among diagnosed metals in current study. Target hazard quotient and cancer risk of As were presented in Table 3.

Table 2:	Mean concentration of heavy metals (μg g-1 dry weight±SD) in egg content of domestic avian species
Values with different letters within arrow are significantly (p<0.05) differ

Table 3:	Health risk estimate for As ingestion from domestic birds eggs

Table 4:	Health risk estimate for Zn ingestion from domestic birds egg

In order to estimate the human health risk from consuming metal-contaminated egg, we used the reference dose of metals RFD and estimated metal exposure dose EED (mg/kg/day). The THQ values of As in egg of domestic bird were ranged from 0.5 to 0.03 for people who eat eggs 7 times a week, from 0.3 to 0.02 for people who eat eggs 4 times a week and from 0.2 to 0.005 for people who consume eggs one time a week (Table 3). For Zn, TQH values ranged from 0.07 to 0.05 for people who eat eggs 7 times a week, from 0.06-to 0.03 for people who consume eggs 4 times a week and from 0.03 to 0.007 when human eat eggs one time a week (Table 4). For Cd the values of THQ reached from 0.12 to 0.063 for eggs consumed in 7 times a week, from 0.07 to 0.04 in 4 time a week and 0.025-0.008 in one time a week of egg consumption (Table 5). While THQ values of Pb have been ranged from 0.2 to 0.04, 0.1-0.02 and 0.03-0.004 for people who eat egg 7, 4 and 1 time a week respectively (Table 6).

Table 5:	Health risk estimate for Cd ingestion from domestic birds egg

Table 6:	Health risk estimate for Pb ingestion from domestic birds egg

Table 7:	HQ (hazard quotient) values when the consumption of eggs more than one eggs/person/day

Risk to human health from the intake of metal contaminated eggs was characterized using hazard quotient HQ is a ratio of determined dose of a pollutant to the dose level (a reference dose or RfD). If the ratio is less than 1 like in Zn, Cd and Pb, there will not be any noticeable risk (Table 7). Conversely, an exposed population of concern will experience health risks if the dose is equal to or greater than the RfD (Khan et al., 2008).

DISCUSSION

Metals concentrations in avian eggs: Demonstration of trace elements in eggs may be an important instrument for nutritionists and environmentalists. Thus, current study provide a base information of the composition of domestic avian eggs in trace elements, such information was rare before in Malaysia. Present study confirmed that a large variation in metals concentrations among domestic avian eggs, this differences may be resulted from feeding behavior and different ability for birds to ingest soil and grass and the metabolic activities among different bird species (Nisianakis et al., 2009). In this study, diet, geographical location, or environmental pollution could not have contributed to such differences due all birds were fed a common diet and kept at the same range area free from any local pollution. Concentration of Zn and Ni in chicken egg content in present study reached 34.22 and 1.11 μg g^-1, respectively were higher than values previously recorded by Van Overmeire et al. (2006), Meanwhile, Pb and Cd concentration in current study in chicken eggs content get to (0.42, 0.05 μg g^-1, respectively) were less than values reported by Fakayode and Olu-Owolabi (2003). Arsenic burden was found in high concentration in chicken egg content than pigeon egg content, similar observation was also reported by Nisianakis et al. (2009). According to Malaysian Food Act (1983) concentration of Cd, Pb and Zn were lower than the recommended legal limit (1, 2 and 100 μg g^-1, respectively). As concentration in chicken egg was 0.3 μg g^-1 and these results are obviously lower than the permitted limits 2.0 ppm (ANZFA, 2001).

Health risk estimation: Some of the heavy metals such as Zn, Cr, Ni and Cu act as micro-nutrients at lower concentrations, they become toxic at higher concentrations. Small amounts of these elements are common in our environment and diet and are actually necessary for good health but large amounts of any of them may cause acute or chronic toxicity (poisoning). Heavy metal toxicity can result in damaged or reduced mental and central nervous function, lower energy levels and damage to blood composition, lungs, kidneys, liver and other vital organs. Long-term exposure may result in slowly progressing physical, muscular and neurological degenerative processes that mimic Alzheimer's disease, Parkinson's disease, muscular dystrophy and multiple sclerosis. Repeated long-term contact with some metals or their compounds may even cause cancer (IOSHIC, 1999).

The estimated THQ values in present study seem to indicate that consumption of these egg does not implicate an appreciable human health risk for the consumer due THQ values of (As, Zn, Cd and Pb) were less than 1, cancer risk SF for arsenic in this study ranged 10^-4-0^-6 (Table 3) the US EPA considers cancer risk that are below about 1 chance in 1,000,000 (1x10^-6) to be so small as to be negligible and risks above 1x10-4 to be sufficiently large that some sort of remediation is desirable. Excess cancer risks that range between 1x10-6 and 1x10-4 are generally considered to be acceptable (USEPA, 1991). If we suggest that consumption of chicken eggs will be increase from 1 egg to 2 or 3 eggs/day/person. Then values of THQ of Zn, Cd and Pb were still in permission level except for Arsenic value which it value exceed to 1.2 and 1.7 respectively (Table 6) this indicates that human health risk might be of concern in case of increased eggs consumption.

CONCLUSION

We conclude that the concentration of Al, Cr, Ni, Zn, As, Cd and Pb were varied among the egg of domestic bird. The eggs of quail were accumulated high levels of Al, Zn and Pb compared to those in other birds eggs, meanwhile, Cd was occur in less concentration in the egg of chicken, guinea fowl and pigeon . From the standpoint of human health As, Zn, Cd and Pb. THQ values (<1) show a situation without risk to the consumer in the area of study. Further ecotoxicological studies are needed on the eggs of species in different area of Malaysia.

ACKNOWLEDGMENTS

We take this opportunity to thank the Department of School of Environmental Science and Natural Resources, UKM, Malaysia for kindly providing all of the samples collected. The author wish to acknowledge the financial support provided by the Iraqi Ministry of Higher Education, Iraq.