Health Risk Assessment of Heavy Metals for Egyptian Population via Consumption of Poultry Edibles

| Exposure to heavy metals through poultry consumption may lead to health risks in Egypt, especially in areas with expanding industrial and agricultural activities. Lead (Pb), Cadmium (cd), Aluminum (Al) and Nickel (Ni) concentrations were determined in poultry muscle and liver using ZEEnit 700P Atomic Absorption Spectrophotometer with Graphite Furness. Mean concentrations of Pb and Cd in poultry muscle and liver exceeded the maximum tolerable limit set by the European communities. However, Al and Ni concentrations were below the guideline limits. According to the Egyptian standardization, only 33% and 66% of liver and muscle samples from Assiut exceeded the limit, respectively. The estimated weekly and daily intakes in the examined samples were below the FAO/WHO Guidelines limits. Meanwhile, Target Hazard Quotients (THQ) and Hazard Index (HI) were calculated to estimate the human health risk of Egyptian population. In this study, THQ and HI were more than 1 for Cd when compared with their reference dose (RfD) in poultry edibles from both Assiut and Qena. The study emphasizes the potential public health risk of Cd contamination from poultry consumption to the local inhabitants in Egypt.

. Lead, cadmium, aluminum and nickel were selected, in this study, because of their potential for human exposure and increased health risk.Lead and cadmium are among the main toxic and abundant metals which accumulate in food chain (Dermirezen and Uruç, 2006) and easily absorbed from atmospheric air and from the di gestive tract (Krejpcio and Trojanowska, 2000).Exposure to lead may cause kidney and nervous system problems as well as inhibit heme synthesis (Berny et al., 1994).However, cadmium is known to be an endocrine disturbing substance and may lead to the development of prostate and breast cancer as well as kidney and skeletal damage in humans (Saha and Zaman, 2012;Nordberg et al., 2002).
Food is unquestionably the main source of aluminium intake.Exposure is unavoidable because of the wide use of this element in day-to-day life and in industry.For many years, aluminium was not considered a health threat because of its relatively low bioavailability.In 1965, animal experiments suggested a possible connection between aluminium and Alzheimer's disease; the findings of Alfrey were the first to establish a connection between neurologic diseases of dialysis patients (Alfrey et al., 1976).In addition, Aluminum is well known as a neurotoxicant and has been shown to have deleterious effects on skeletal and hematopoietic systems of humans (Domingo, 1995).Nickel is a known hematotoxic, immunotoxic, hepatotoxic, pulmotoxic and nephrotoxic agent.Nickel confirms lethal if it surpasses the allowed amount in edibles (Nriagu and Pacyna, 1988).
The World Health Organization (WHO) reported that about a quarter of the diseases facing mankind today occured due to prolonged exposure to environmental pollution (UNEP, 2008).The number of Egyptian population with kidney failure, due to heavy metal exposure, is about 35000 and the incidence rose scary as it exceeding 4% at a rate of 500 patients per million populations (Abdalla, 2012).In addition, the prevalence of renal failure, liver cirrhosis, and chronic anaemia diseases was markedly increased among the Egyptian population in the last few years due to environmental pollution with high exposure to metal poisoning (Salem et al., 2000).
Poultry is a more efficient feed converter and has a shorter production cycle than red meat animals.Responding to increased demand, Egypt's poultry meat production has trended upward over the last 40 years.Rising demand for poultry meat pushed poultry to 44.3 percent of total domestic production (Taha, 2003).Relatively low and competitive prices as well as dietary and nutritional properties, compared to other meats, are main factors explaining poultry meat's attractiveness.The increasing demand of food safety has accelerated research regarding the risk associated with food consumption contaminated by heavy metals (Mansour et al., 2009).Estimation of the potential risks to human health through the target hazard quotient method (THQ) associated with intake of heavy metals via consumption of poultry is a must for Egypt ian population safety.Although the THQ -based risk assessment method does not provide a quantitative estimate of the probability of an exposed population experiencing an adverse health effect, it does provide an indication of the risk level associated with pollutant exposure.This method of risk estimation has recently been used by many researchers and has been shown to be valid and useful (Akoto et al., 2014;Saha and Zaman, 2012;Wang et al., 2005;Chien et al., 2002).In Egypt, chicken muscle and liver are a major source of animal protein to the population and are widely consumed.Consequently, The Egyptian population face challenges related to food quality and safety.According to our best knowledge health risk assessment of heavy metals for Egyptian population via consumption of poultry edibles is not reported.Additionally, there is no available data about Al levels contaminating poultry muscles or liver.The present work aimed to determine the concentration of heavy metals (Pb, Cd, Al and Ni) in the liver and muscle of poultry collected from Assiut and Qena in Egypt.Although muscle tended to accumulate low concentrations of metals, it is important to compare their levels to the known guideline safe levels because muscle constitutes the greatest mass of the poultry that is consumed.It is also necessary to assess the potential health risk of heavy metals to local residents by poultry edibles consumption.All laboratory equipments and containers were washed with Nitric acid 10% solution prior to each use to ensure cleaning.About 5-6 gm of each tissue sample was cut into small pieces and dried in hot air oven at 80°C until reaching a constant weight.The dried tissues were homogenized and grinded to a powder.One gram of each dry sample was weighed, transferred into screw caped glass bottle and 3 ml of nitric acid high grade (68%) (Merck KGaA, 64271 Darmstadt, Germany) was added and the mixture was left overnight at room temperature.Digestion was completed in a water bath at 90°C for about 1-2 hour until the tissue completely dissolved and the solution become clear (Al-Weher, 2008).The digests was allowed to cool, filtered through Whatman (Ashless no.42) filter paper, then transferred to 25ml volumetric flask and made up to mark with deionized water.Metal analysis (Pb, Cd, Al, and Ni) was carried out in the Central Laboratory of the Faculty of Veterinary Medicine; Assiut University using a ZEEnit 700P Atomic Absorption Spectrophotometer with Graphite Furness Unite (AASG).

saMple analysis
Aqueous standard stock solutions were prepared for Pb, Cd, Al, and Ni using appropriate salts (Merck KGaA, 64271 Darmstadt, Germany).Working calibration standards of Pb, Cd, Al, and Ni were prepared by serial dilutions of concentrated stock solutions of 1000 mg/L.Four working standards were prepared in triplicate for each metal by serial dilution of the stock solution.These standards and blank solution were aspirated into AASG as described by the manufacturers to obtain the absorbance of each of the samples.A calibration curve for the absorbance versus concentration of the standard metal concentrations was prepared for each metal from which calibration graph for each of the metals in the sample was determined as described by Nnaji et al. (2007).

Quality Control and reCovery deterMination
The detection limit for each metal was calculated as double the standard deviation of a series of measurements of a solution, the concentration of which is dis-tinctly detectable above, but close to blank absorbance measurement (USEPA, 1983).In addition, a recovery study of the total analytical procedure was carried out for metals in selected muscle samples by spiking analysed samples with different concentrations of aliquots of metal standards (a multi-element standard solution) and then reanalysed the samples.All determinations were replicated three times.In order to determine the reliability of instruments, a blank and known standard were run after every 10 samples.Acceptable recoveries for the metals were 104.3% for Pb; 89.8% for Cd; 85.3% for Al and 107.1% for Ni.Heavy metal concentrations are expressed on a dry tissue basis and given as µg/gm dry weight (µg/g d wt).Calculated moisture content of the muscle and liver samples were 73.6%± 261 and 74.7± 2.7%, respectively.

deterMination of target hazard Quotients (thQ)
Health risk to Egyptian population from poultry intake was characterized by Target Hazard Quotient (THQ).This is the ratio between the exposure and the reference doses (RfD).Rfd represents reference oral dose that is an estimation of the daily exposure of a contaminant to which the human population may be continually exposed over a lifetime without an appreciable risk of harmful effects (Akoto et al., 2014).RfD value for Pb, Cd, Ni and Al is 0.004, 0.001, 0.02 and 1(µg/g bw/day) respectively (USEPA, 2006).The population will pose no risk if the ratio is less than 1 and if the ratio is equal or greater than 1 then population will experience health risk.The following equation is used (Chien et al., 2002): Where THQ is the target hazard quotient; EF is exposure frequency (365 days/year); ED is the exposure duration (70 years, average lifetime); FIR is the food ingestion rate (g/day); C is the heavy metal concentration in poultry (µg/g); RfD is the oral reference dose (mg/kg/ day); BW is the average adult body weight (70 kg); and AT is the averaging exposure time (365 days/ year × number of exposure years, assuming 70 years).

hazard index (hi)
To evaluate the potential risk to human health through more than one heavy metal, the hazard index (HI) has been developed (USEPA, 1989).The hazard index is Where Σ HQ is the summation of hazard quotients of metals and HQ Pb; HQ Cd; HQ Al and HQ Ni are the hazard quotients for lead, cadmium, aluminium and nickel, respectively.It is assumed that magnitude of adverse effect will be proportional to the sum of multiple metal Exposures.The hazard index for the toxic element Pb, Cd, Al and Ni has been calculated in the present study.When the hazard index exceeds 1.0, there is concern for potential health effect (Huang et al., 2008).

statistiCal analysis
The data was statistically analysed using SPSS version 19 statistical package programs.A one-way analysis of variance (ANOVA) was performed.Differences in mean values were accepted as being statistically significant if P< 0.05.The means were separated using Tukey's test.The statistical significance of the correlation was reported at both P ≤ 0.01 and P ≤ 0.05levels.

RESULTS
Heavy metals (Pb, Cd, Al, and Ni) were estimated in Liver and muscle samples from Assiut and Qena broiler farms.Figure 1 (a & b) shows the mean concentration and standard deviation of the analysed metals.
Irrespective of the poultry source, the range in µg/g (dry weight) obtained for the heavy metals in liver samples ana lyzed were as follows: Al (13.6 ±5.9), Ni (4.1 ±2.8), Pb (2.75 ± 1) and Cd (0.66 ± 0.8).However, heavy metal concentrations (µg/g dry weight) from muscle samples (breast or leg muscle) analyzed were as follows: Al (9.27 ± 2.8), Ni (4.78 ± 3.3), Pb (2.8 ± 0.9) and Cd (1.6 ± 1.5).The mean concentration of Al in liver samples collected from both Qena and Assiut broiler farms was significantly higher (P<0.05)than the level of the other examined metals as observed in figure 1 (a).Additionally, it was clear that the Al concentration in liver samples collected from Qena (16.44 µg/g d wt) was significantly higher (P<0.05)than the concentration in liver samples collected from Assiut (8.44 µg/g d wt).On the other hand, Cd level in liver samples from Qena was 0.24 µg/g d wt and from Assiut was 1.41 µg/g d wt, which indicate that Cd levels were significantly lower than the levels of Pb, Al and Ni in both Assiut and Qena.
Data about the levels of Pb, Cd, Al, and Ni in muscle samples collected from Assiut and Qena broiler farms was presented in Figure 1(b).Among the tested metals, mean concentration of Al (10.1 µg/g d wt) was significantly higher (p<0.01) in muscle samples collected from Qena than the other examined elements.While in muscle samples collected from Assiut the mean concentration of Ni was the highest (7.9 µg/g d wt) followed by that of Al (7.74 µg/g d wt).However, in muscle samples from both Assiut and Qena   of liver and muscle samples from Assiut exceeded the Egyptian limit, respectively.The concentrations of Pb and Cd from liver and muscle samples in both Qena and Assiut exceeded the permissible limits of 0.2 µg Pb/g wet weight and 0.05 µg Cd/g wet weight stipulated by the European Communities (EC, 2001), except liver samples in Qena, only 73% was higher than the standard guideline limit for cadmium.The permissible limit of Ni is 4µg/g (FNB, 2010).Our results revealed that both liver and muscle samples collected from Qena and Assiut showed nickel level below the permissible limit.(Yilmaz, 2003).
According to Allen-Gil and Martynov (1995) the low levels of binding proteins in the fish muscle may account for their low concentrations of heavy metals.Distribution of pollutants among the various organs within an organism is not uniform but rather they accumulate in specific target organs (Terra et al., 2007).The amount of an element, which accumulates in the organs, depends on the interval of exposure, the quantity of ingested element, as well as animal age and breed (Massányi et al., 2000).However, element toxicity is affected by the route and form of ingestion as well as by the interaction between essential and toxic elements (Skalická et al., 2008).

lead and CadMiuM
Pb and Cd are classified among the most toxic heavy metals which have no known biochemical benefits to animals and humans (Akoto et al., 2014).The wide spread industrial production of perfumes, batteries, oils and fats, cement-making, quarrying (especially limestone), and brick-making, as well as, agricultural discharges, sewage effluents, high ways or motor boat traffic, mine and smelting operations, consider main sources of lead in the environment.Discarded manufactured materials to air, soil, water, and food have led to widespread human and animal intoxication (Humphreys, 1991;Mahaffey, 1977).The predominant sources exposure to Cd are through lubricating oils, diesel oil, fertilizers and rubber car tires as well as, the use of fertilizers and sludge containing Cd (Skalická et al., 2002).With inhalation and ingestion as exposure routes, animals in the environment have the potential to act as sentinels for lead and cadmium contamination of water, air, and soil (Ceruti et al., 2002;Bischoff et al., 2010).
It was obvious that there is a great variation between guideline limits from Egypt and the international limits.Levels of Cd and Pb were under the permissible limits of the Egyptian Organization Standardization (EOS, 1993) except for Cd in Assiut.However, Cd and Pb have concentrations greatly above the safe limits recommended by EC.This is of interest considering that Cd and Pb are toxic and their accumulation may lead to serious health issues.The absorption of heavy metals varies to certain extent depending on various factors.Diet, rich in carbohydrates but poor in proteins, calcium and phosphorus accelerates absorption of lead into blood (Sanderson and Bellrose, 1986).Moreover, Pb ingested during period of fasting gets absorbed to a much greater extent than lead ingested with food (WHO, 1992).Clinical signs of acute Pb poisoning in chickens include muscle weakness, ataxia, loss of appetite, marked weight loss, and eventual drop in egg production and/or severe anemia (Salisbury et al., 1958).Chronic exposures may also result in degeneration of motor nerves in the spinal cord and axonal loss in peripheral nerves, along with muscle atrophy and myodegeneration (Kaufer, 1979).
Even trace levels of lead in the diet (1.0 mg/kg) can result in growth retardation (Bakalli et al., 1995).Moreover, ingested Pb resulted in elevated blood levels and deposition in bone, soft tissues and eggs (Trampel et al., 2003).In addition, the accumulation of trace amounts over time in humans, of particular concern is the potential for children to be exposed throw acute neurotoxic effects or abnormal neurodevelopment in chronic lead exposure (Canfield et al., 2003).Excess Pb is known to reduce the cognitive development and intellectual performance in children and to increase blood pressure and cardiovascular disease incidence in adults (EC, 2001).
Cadmium is an environmental contaminant unique among metals because of its diverse toxic effects, extremely protracted biological half-life, low rate of excretion from the body and predominant storage in soft tissue (Beňová et al., 2007).Tissue Cd concentrations in animals are closely related to Cd levels in feedstuffs and the duration of Cd load (Bokori et al., 1995).Absorption and accumulation of Cd in tissue seems to be determined by a wide range of factors: nutritional and vitamin status, such as iron status, age and sex (Torra et al., 1995;Flanagan et al., 1978).Cd Food is the most important source of cadmium exposure in the general non-smoking population in most countries (WHO, 1992).Cd exposure may cause kidney damage including a tubular dysfunction, evidenced by an increased excretion of low molecular weight proteins or enzymes.Skeletal damage, first reported from Japan in the 1950s, called the itai-itai (a combination of osteomalacia and osteoporosis) which may occur in relatively low cadmium exposure, evidenced by low bone mineral density (osteoporosis) and fractures (Staessen et al., 1999;Alfven et al., 2000;Nordberg et al., 2002).Animal experiments have suggested that cadmium may be a risk factor for cardiovascular disease ( Jarup et al., 1998;Nishijo et al., 1995)

aluMinuM
Al is the third most abundant elements in the earth crust and biological system probably evolved in the presence of appreciable concentrations (El-Rahman, 2003).Exposure is unavoidable because of the wide use of this element in day-to-day life and in indus- try (Bohrer et al., 2008).Al is used to make beverage cans, pots and pans, airplanes, siding and roofing, and foil.Aluminum compounds are used in many diverse and important industrial applications such as alums (aluminum sulfate) in water-treatment and alumina in abrasives and furnace linings.Widespread uses of the metal have stimulated considerable interest in their toxicity.Contaminated food is unquestionably the main source of Al intake (FAO/WHO, 2011).
Al has been shown to have deleterious effects on the central nervous, skeletal, and hematopoietic systems of humans (Domingo, 1995).The neurotoxicity of Al to patients with chronic renal disease is well established (Daugirdas, 1994), and its presence in the bloodstream leads to Al accumulation in bone and brain causing an encephalopathy called dementia dialytica.Several studies have pointed to Al as an exogen factor responsible for developed anemia signs both in experimental animals (Zaman et al., 1993;Fulton and Jeffery, 1994) and patients on hemodialysis (Parkinson et al., 1981).Al exposure increased delta-aminolevulinic acid dehydratase (delta-ALA-D) activity in bone marrow which promotes alterations on erythrocyte parameters in rats, probably as a consequence of alterations in the iron status (Farinaa et al., 2005).Al is well known as a neurotoxicant resulted from accumulated Al in brain and altered amino acid neurotransmitters, and its accumulation has been suggested to be an associated phenomenon in various neurological disorders as dementia, senile dementia, and Alzheimer disease (El-Rahman, 2003).Increased Al exposure can be compensated for by excretion via intestines and normal, healthy kidneys.Kidney insufficiency was shown to result in increased Al concentrations in the kidneys of dialysis patients (Krüger et al., 1984).In a more recent study, Al is also discussed as an endocrine disruptor in female Nile Tilapia (Oreochromis niloticus) (Correia et al., 2010).
Within the literature of specialty, we have not found any reference values of the concentration of Al in the tissues and organs taken from poultry, only from fish and plants.Müller et al. (1998) determined Al content of 128 items out of 12 food categories in Germany.Al was detected in Fish as well as meat and meat products at concentration of 3.2 and 5.4 µg/g fresh weight, respectively.In Spain, The more elevated Al concentrations were detected in food with a greater content of spices and aromatic herbs, pasta, certain vegetables and mushroom (López et al., 2002).
The permissible Al dose for an adult is quite high (60 mg/ day) (WHO, 1989).There is no information about maximum Al levels in poultry samples in Egyptian standards.Meanwhile, the concentrations of Al in different poultry parts in our study were below the WHO limit., 2011).The estimated weekly intake (EWI) values were calculated by assuming that a 70 kg individual will consume 100 g and 20 g of poultry muscle and liver serving/day, respectively.Thus, the EWI values of trace elements by an adult (mg/kg body weight) consuming poultry muscle and/ or liver were calculated using the averages posted in figure1 (i.e., EWI in mg/kg body weight = Mean concentration of element (µg /g wet weight) multiplied by the amount of poultry consumed/week (kg) and divided by the average weight of an individual (70 kg) (Table 1).
Hence, the total dietary exposure levels of heavy metals determined in this study were compared with PTWIs by the FAO/WHO ( JECFA, 2004 and2011) to assess potential health risks faced by consumers.The result from Table 1 suggests that the EWI of Pb, Cd, Al, and Ni by a 70 kg adult consuming 820 g of poultry edibles/week in Assiut and Qena were all below the respective PTWIs recommended standard guidelines ( JECFA, 2004 and2011).

health threat froM ConsuMing poultry in egypt
Figure 2 (a & b) shows the estimated Target Hazard Quotients (THQ) for individual metals and Hazard Index (HI) from consumption of poultry in Assiut and Qena in Egypt.The THQ of Cd through the consumption of poultry muscle and edibles were more than 1, indicating that there is a potential significant health risk associated with the consumption of poultry from Assiut.Although Cd has the lowest concentrations among the studied metals,but its health risk was highest.This may be due to lower reference dose in diet, which consequently increased the THQ.The THQ of Pb, Al and Ni is generally less than 1, suggesting that people would not experience significant health risks from the intake of individual metals through poultry consumption.However, Pb may consider one of the health risk contributors, accounted for 60% and 62% of the THQ from poultry edibles in Assiut and Qena, respectively.The potential health risk of Al was the lowest, which may be ascribed to its higher oral reference dose (Figure 2 a).
Health risk index (HI) for Cd was more than 1 for poultry muscle and edibles in Assiut as well as edibles in Qena and among them the highest value was found in edibles in Assiut (2.7) indicating high potential risk to the Egyptian consumers.When the hazard index exceeds 1.0, there is concern for potential health effect (Huang et al., 2008) as exposure to more than one contaminant may produce an additive effect on the consumer.

CONCLUSIONS
Different concentrations of heavy metals were present in poultry edibles at the studied locations in Egypt.
The estimated daily and weekly intake for Pb, Cd, Al, and Ni were well below the established PTWI/PTDI values recommended by FAO/WHO guidelines.Heavy metal levels were below the Egyptian standardization limits in all samples except for Cd.On the other hand, Cd and Pb levels exceeded the permissible limits stipulated by the European Communities nearly on all poultry samples.In addition, Cd has a THQ more than one for poultry edibles suggesting a health threat from Cd and Pb to the Egyptian population in the studied locations in Egypt.

RECOMMENDATIONS
There is a need for a continuous monitoring of contamination level of heavy metals especially Pb and Cd since they can accumulate to toxic levels.Cd and Pb contents will be lowered by applying restrictions on the use of cement and limestone-making, fertilizers

saMple
ColleCtion and preparation Forty eight liver and thirty six muscle samples were collected from broiler poultry farms in Assiut and Qena Provinces.Assiut farms are private farms with capacity ranged from 3000 to 4000 birds.While, Qena farms are governmental farms with 15000-20000 Advances in Animal and Veterinary Sciences January 2015 | Volume 3 | Issue 1 | Page 60 birds' capacity.The samples were collected from 35-42 days -old age birds.Collected samples were transported in an ice box to the laboratory then kept at -20°C until analysis.

Figure 1 :
Figure 1: Mean concentration of heavy metals Pb, Cd, Al and Ni expressed as µg/g d wt in (a) liver samples and (b) muscle samples from Assiut and Qena broiler farms.abc: means with different letters are significantly different (p<0.05) the sum of the hazard quotients as described in the following equation: HI = Σ HQ = HQ Pb + HQ Cd + HQ Al + HQ Ni

Figure 2 :Advances
Figure 2: Estimated potential health risks for Pb, Cd, Al and Ni via consumption of poultry by (a) Target hazard quotients (THQ) and (b) Hazard index for the Egyptian population in Assiut and Qena.The population will experience health risk if THQ ratio for individual metal or HI for the sum of metals is equal or greater than 1 EP: edible parts (liver and muscle)the lowest concentration of the analysed metals was recorded for Cd (0.88 µg/g d wt and 2.44 µg/g d wt, respectively).Additionally, Al level was significantly higher (p<0.01) in liver samples (13.6 µg/g d wt) than in muscle samples (9.27 µg/g d wt), however, Cd levels were significantly higher (P<0.01) in muscles samples (1.6 µg/g d wt) than in liver samples (0.66 µg/g d wt).The results obtained in this study were compared with the maximum permissible limits for Pb, Cd and Ni.The Egyptian Organization for Standardization (EOS 1993) suggested 2, 0.5 and 10 ug/g wet weights as guideline limits for Pb, Cd and Ni in food, respectively.The levels in this study were much below those limits from all samples, except for Cd, 33% and 66%

Table 1 :
Estimated weekly and daily metal intakes (µg/week or µg/day w wt) for Egyptian population consuming poultry edibles

DISCUSSION ConCentrations of heavy Metals in poultry edibles and CoMparison with international dietary standard and guidelines
The total dietary exposure levels of heavy metals(Pb, Cd, Al, and Ni)determined in this study were compared with the provisional tolerable weekly intakes (PTWIs) to assess potential health risks faced by consumers as shown in table 1.The PTWI represents permissible human weekly exposure to those contaminants associated with the consumption of the contaminated organs.It can be observed that the estimated weekly intake of the examined metals in liver and muscle samples as well as edible parts (liver and muscle) either from Assiut or Qena was below the PTWI.The data presented in figure2 (a & b)showed that the THQ and HI value for Cd in muscle samples and edible parts collected from Assiut are more than 1.0 and the highest value for HI was found in edible parts from Qena (1.346) and Assiut (2.662) indicating high potential risk to human health.
. The International Agency for Research on Cancer has classified Cd as a human carcinogen (group I), for prostate and kidney cancer, on the basis of sufficient evidence in both humans and experimental animals(IARC, 1993).