Heavy Metals in the Liver, Kidney, Brain, and Muscle: Health Risk Assessment for the Consumption of Edible Parts of Birds from the Chahnimeh Reservoirs Sistan (Iran)

The concentrations of four heavy metals, zinc (Zn), lead (Pb), nickel (Ni), and cadmium (Cd), were determined in the liver, kidney, muscle, and brain of nine species of birds from the Chahnimeh Sistan from Iran to assess the metal levels and the potential risk to birds and to the people who eat them. Significantly higher levels of all metals were found in the brain than in the other tissues of other birds. There were no significant gender differences in heavy metals in all tissue. The levels of Pb, Cd, Ni, and Zn in the liver and kidney varied as a function of feeding habitats; the median levels were significantly higher in invertebrate predators than they were for fish predators and omnivorous species. Short-distance migrant birds had significantly higher median levels of heavy metals in the liver and kidney than long-distance migrant birds. Ni levels in the liver and kidney tissues in 56% of birds were higher than the critical threshold levels for effects in birds. Our data indicate that environmental exposures to metals were higher in the wintering populations of birds in the Chahnimeh of Sistan from Iran than elsewhere. Concentrations of Zn, Pb, and Cd in a small percentage of birds were above toxicity levels. However, 56% of liver and kidney samples for nickel were above toxicity levels. Determining the exposure frequency and daily intake of birds, the hazard quotient for edible tissues (kidney, liver, and muscle) of these birds showed that their consumption may provide health risk to people consuming them.


Introduction
Aquatic environments accumulate pollutants from runoff and atmospheric deposition. While aquatic habitats are dynamic, they have a limited capacity to accept man-made waste without adverse effects on biota. With further technology advancement and the increased development of industries, the volume of waste imported into water areas will likely increase. Heavy metals are pollutants of concern due to their toxicity, persistence, and accumulation in the tissues of living organisms. Generally, the main heavy metals of concern in the environment are from pesticides, chemical fertilizers, electroplating, preparation of paint, coal production, oil combustion, pigments, batteries, photovoltaic cells, greenhouse gas production processes, vehicles, synthetic plastic, extraction from foundry mines, leather product, urban waste incinerators, and industrial waste [1]. Besides heavy metals deriving from different industrial and agricultural sources, rocks and volcanoes are an additional source [2]. The increase of heavy metals in the biotic and abiotic environment is of great concern because of their adverse human health effects [3]. Small quantities of heavy metals such as lead, cadmium, and chromium and high concentrations of essential elements such as copper and zinc, in living tissues, have caused major concerns due to their serious health effects in birds [4].
Birds are well suited for biomonitoring because their biology is well-known, they have a relatively long lifespan (up to a dozen or more years), and they feed on different levels of the food chain, depending on the species. Birds are therefore one of the best indicators for evaluating heavy metals in the environment [4,5]. Birds are exposed to environmental pollutants from direct contact with contaminated water and food. Studies show that heavy metals accumulate in the organs of birds, especially waterfowl and other bird species that depend on rivers and other aquatic habitats to collect their food. High levels can be harmful and toxic to their reproduction and survival [6]. Also, birds are used as an indicator of environmental pollution on local, regional, and global scales [5]. Local species (that feed locally) can be compared with those that migrate in (and therefore represent contamination over a larger geographical area) [7].
The process of bioaccumulation of heavy metals in birds is very complex and influenced by many factors, including climate, geographical conditions, physicochemical differences, and the mobility and bioavailability of metals [8]. Behavioral factors such as migration, foraging methods, grit collection, and position in the food chain influence exposure as well [9][10][11]. Metals are absorbed in the body, enter the blood circulation, and then exhibit different levels in tissues in relation to reaction to lipids, solubility, and transport in different specific cells [9]. Distribution and concentration of metals in various organs and tissues are influenced by various host characteristics, such as body nutritional status, weight, size, sex, homeostatic mechanisms of genetics, and interaction with nutrients or micronutrients [9,10,12].
Because of the key role the liver and kidney play in detoxification processes, heavy metals such as cadmium (Cd), lead (Pb), nickel (Ni), and mercury (Hg) have been studied most extensively because of their toxicity [13,14]. The levels of Pb are examined in the bone or brain because of their accumulation over a lifetime and the effect they have on the nervous system [15,16]. In recent years, human activities that increased the levels of heavy metals, such as intense agriculture, leakage of contaminated water to groundwater sources, drainage, and hunting, have posed a serious threat to wildlife [17].
Increased anthropogenic pollution has resulted in increased levels of organic matter, nutrients, and heavy metals in water, sediment [18][19][20][21], and fishes [22,23] from Chahnimeh, Iran. Some of the pollutants coming from agricultural and industrial activities in Iran and Afghanistan have run off into the Helmand River, which supplies water to the Hamoun International Wetland and to human-used wells [22]. The amount of heavy metal contamination in birds in this area has not been studied.
The objective of this study is to assess heavy metal levels in birds wintering in the Chahnimeh reservoirs of the Sistan region in eastern Iran. We determined the levels of Cd, Pb, Ni, and Zn in the brain, liver, kidney, and muscle from nine species of birds in Chahnimeh, in the Sistan region in Eastern Iran. We examined metal differences as a function of migration, sex, species, and feeding habits using the liver, kidney, brain, and muscle samples. We also compared the levels to those published in the literature and examined the risk of metals for endangered species of waterfowl in the Chahnimeh of Sistan. These birds were given to us for studies after the Environmental Protection Agency removed them from fishermen who had hunted them illegally. Although sample sizes per species are low, this represents the first metal data of its kind from this region and provides the first risk assessment for humans eating these birds.

Analytical Procedure
Birds were thawed, and liver, kidney, brain, and pectoral muscle tissues were collected. Samples (1-3 g wet weight) were placed into 150 mL Erlenmeyer flasks; 10 mL 65% HNO 3 (Suprapure, Merck, Darmstadt, Germany) was added to the Erlenmeyer flasks and was slowly digested overnight after 5 mL HClO 4 ; 70% was added to each sample (Suprapure, Merck, Darmstadt, Germany) [24]. For digestion, we used a hot plate (sand bath) at the first step at 200 °C, for about 6 h or until the solutions were clear after cooling. In the second step, each sample was transferred to polyethylene bottles, and deionized water was added until the sample equaled 25 mL. In each set of eight samples, one control sample was prepared and analyzed. Then the solution was filtered using a 0.45-µm nitrocellulose membrane filter. A Shimadzu AA 680 flame atomic absorption spectrophotometer was used for determining the concentrations of heavy metals. The detection limits for Cd, Pb, Ni, and Zn were 0.09, 0.04, 0.06, and 0.09 µg/g respectively. Also, the obtained recoveries for Cd, Pb, Ni, and Zn averaged 88%, 90%, 95, and 105% respectively.

Quality Control
Procedural blanks and certified reference material (CRMs, e.g., DOLT-2 (fish liver) and DORM-2) (fish muscles) were included in each sample batch. To determine the detection limit of heavy metals in a sample, blank samples were injected three times for analysis, and the result was 3 times the standard deviation of the procedural blanks (0.08, 0.05, 0.07, and 0.1 μg/g dw in Cd, Ni, Pb, and Zn respectively). The precision and accuracy of the applied analytical method were determined based on CRMs, e.g., DOLT-2 and DORM-2, heavy metal in sample. The results of our CRMs measurements were a good estimate of the real values. In each sample batch, procedural blanks and certified reference material DOLT-2 and DORM-2 were included. For each matrix, the analyses of three blank samples and of three reagent blanks were performed. To estimate the accuracy and precision of the chemical analysis, sample blanks, standard blanks, and three analytical duplicates with the concentration of 1.2 μg/g were injected, and their mean and its 95% confidence interval were calculated. Quantification was based on multi-level calibration on the concentrations of 0.1, 0.5, 3, 15, 50, and 100 µg/g; and then the standard calibration curve was drawn with 99% accuracy. Two certified reference materials (DOLT-2 and DORM-2 from National Research Council Canada, Institute for National) were included for QA/QC to check digestion efficiency and measurement accuracy. The certified values for the reference materials amounted to Zn = 87 ± 2.5, Pb = 0.24 ± 0.3, Cd = 21.8 ± 5, and Ni = 1.3 ± 0.12, and the certified values for the used material amounted to Zn = 88 ± 60, Pb = 0.23 ± 0.4, Cd = 21.58 ± 3, and Ni = 1.2 ± 0.13 (6 replications for 0.8 g sample with the recovery between 88 and 105%). The method's accuracy, understood as the degree of compatibility of results of multiple analyses of the same sample, reached up to 8% (relative standard deviation RSD). All concentrations are expressed in µg/g of dw.

Statistical Analysis
For data analysis, we used SPSS (version 20.0). The data were tested for normality using a Kolmogorov-Smirnov test.
To determine normal distribution and homogeneity of variance of heavy metals levels in the tissue samples, we used the Kolmogorov-Smirnov test, and data were not normal. To normalize our data, we use log-transformation (log 10 ), and, after normalizing all data, we used parametric statistics. To test differences in total heavy metal level of samples among groups, we performed a one-way ANOVA, and then the Duncan's post hoc test for differences in level between areas was used. Spearman's rank correlation coefficients were used to test for correlation among various heavy metals from birds. A P value < 0.05 indicated statistical significance.

Risk Assessment
To assess the health effects and compare them with standards, we converted g/dry weight to g/wet weight. The dry weight/ wet weight ratio was assumed to be approximately 0.3 for all species [25,26]. In this study, the target hazard quotient (THQ) was computed according to the guidelines of the US Environmental Protection Agency, and the level of absorption of heavy metals was considered equal to the absorption of ingestion (assuming that cooking does not affect the level of metals) (USEPA 1989). Furthermore, because of the lack of an oral reference dose (RfDo) for Pb, the value is specified as the permissible tolerable daily intake (PTDI) suggested by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) 2013).
In this study, we calculated the THQ from the following equation: When the target hazard quotient (THQ) is > 1, systemic effect may occur, and in fact, the THQ is the ratio between exposure and reference dose [27]. The reference dose (RfDo) (µg/g/day) is an estimate with uncertainty of the daily exposure of human populations, including sensitive subgroups, without an appreciable risk of deleterious effects during a lifetime. The RfDo values used in this study were 0.001, 0.02, 0.004, and 0.3 for Cd, Ni, Pb, and Zn respectively. The exposure frequency (EF) in this study is about 182.5, the exposure duration (ED) is 72 years, the meal size (MS) is about 95 g [28] and 20 g for kidney and liver [29]. C is the metal concentration (µg/g w.wt) [30,31]. The body weight (BW) is 70 kg [32] and EF × ED = AT (average time): [33] Also, we calculated the estimated daily intake (EDI) and estimated weekly intake (EWI) based on daily and weekly consumption of birds (including liver and kidney muscle).
The estimated daily intake and estimated weekly intake were calculated as follows:

Total Heavy Metal Concentrations in the Liver, Kidney, and Brain and Muscles in Wild Birds from Iran
The levels of heavy metals in the brain, liver, kidney, and pectoral muscle are shown in Tables 1 and 2. The highest median toxic concentrations were of Ni, followed by Pb and Cd; the kidney and liver had the highest levels of Ni. The brain had the highest concentration of Pb (2.7 µg/g dw). For Zn (an essential element), the levels were the highest in the brain (34.50 µg/g dw), followed by the kidney (21.30 µg/g dw), liver (7.30 µg/g dw), and muscle (7 µg/g dw). Studies have shown that there is a homeostatic regulation of the intracellular essential metals in birds [34][35][36][37][38][39].
We considered < 6 µg/g dw Pb in the liver and/or kidney to be indicative of "background" Pb exposure; individuals were considered to be "Pb exposed" when concentrations exceeded 6 µg/g dw in the liver or kidney and were "Pb poisoned" when kidney levels exceeded 20 µg/g dw or when liver levels exceeded 30 µg/g dw [52]. Birds such as shovelers (Anas clypeata), greylag geese (Anser anser), snow geese, brant geese (Branta bernicla), mallards, and black ducks from Northern California, the USA [53], Canada (gosling) [54], four wetland in Spain [55], and northern Idaho, USA [56], had levels of Pb in the livers higher than the threshold level of threat exposure to Pb in the livers. But birds in the Kanibarazan wetland [43], Miyankaleh, and Gomishan wetlands [45] from Iran; Eastern Poland [47]; Donana National Park, Spain [36]; Illinois River [57]; and Eastern Austria [51] were > 5 µg/g dw, indicating the possibility of Pb toxicity.
Concentrations of Cd > 3 μg/g dw and > 8 μg/g dw in the liver and kidney suggest toxic exposure [59], and levels greater than 40 μg/g dw and 100 μg/g dw in the liver and kidney, respectively, indicate toxicities [60]. In this study, except for one black-winged stilt, Cd concentrations of livers were far below the estimated toxic threshold; Cd concentration in one moorhen and one marsh sandpiper were far below the toxicity level [59]. In birds from Iran, the mean cadmium concentrations were 0.43-3.94 µg/g dw in the liver and 0.47-7.47 µg/g dw in the kidney. The concentrations of Cd in liver were (1) similar to those found in birds from Ebro Delta, Spain [55]; Lake Biwa and Mie Izum coast, Japan [61]; and the Chesapeake Bay, USA [35]; (2) were much lower than those observed from Pacific northwest Canada [34], Chaun, Northeast Siberia, Russia [49]; and (3) were much higher than those observed from Zator and Milicz, Poland [62], Mississippi flyway [63], Eastern Poland [47], and an Illinois river [57]. The concentration Cd in kidney was similar to those found in birds from Donana National Park, Spain [36], and Illinois river, USA [57], and were lower than the Zator and Milicz, Poland [62]; Chaun Northeast Siberia, Russia [49]; and Pacific Northwest Canada [34] and were higher than Lake Biwa and Mie Izum coast, Japan [61]; a wetland in Northwestern Poland [58]; Kanibarazan wetland, Iran [43]; and Gomishan and Miyankaleh, Iran [45].
According to studies, Ni concentrations > 10 μg/g dw in the kidney and > 3 μg/g dw in the liver are toxic in wild birds [64]. In this study, 56% of Ni concentrations in the liver and 56% of Ni concentrations in the kidneys were higher than the toxicity level. In birds, Ni concentrations in the liver and kidney are seldom studied. Concentrations of Ni in livers of birds in this study were higher than those from Connecticut, USA [65]; Gdansk Bay, Poland [66]; San Francisco Bay, USA [67]; Jamaica Bay, USA [68]; Wrangel Island, Russia (Hui 1998); and Florida Lake from South Africa [69]. Concentrations of Ni in the kidney of birds in this study were higher than those from Southwest Atlantic coast, France [2]; Gdansk Bay of the Baltic Sea, Poland [70]; and a wetland in Northwestern Poland [58]. Table 1 The concentrations of trace metals (µg/g dw) in the brain and liver, kidney, and brain and muscle of waterfowl from the Chahnimeh of Sistan  In birds, Pb concentrations in the brain >5 µg/g dw are indicative of poisoning [15], and concentrations >16 µg/g dw indicate an advanced state of exposure in birds [71]. In this study, none of the levels of Pb in the brains was higher than the toxic limit threshold.

Variation Among Organs
In this study, the levels of heavy metals in muscle tissue were lower than in other tissues, and our results agree with other studies that reported that muscle tissue was not an active tissue for accumulating these heavy metals. Also, in this study, the brains of birds had the highest concentration of metals, except for Ni (P < 0.05). The level of metal a body absorbs and accumulates depends on the level of exposure, the chemical form of an element, the interaction with other elements, and physiological factors of the bird species (Gochfeld and Burger 1987). The accumulation of pollutants in the internal organs of their bodies is affected by the contaminant level of the food and water ingested. Although the liver and kidney are sites of detoxification, they reflect long-term bioaccumulation [5], while the muscle and brain are sites of accumulation but not of detoxification [72].
If birds are exposed to high concentrations of Pb and Cd, these elements will be accumulating in high concentrations in the brains of these birds, such as in white-tailed eagle and scavenging gulls. Brain tissue levels are related to dietary contamination [70,73]. Relatively low (up to 0.4 ppm wet wt) lead (Pb), but not cadmium (Cd), levels were recorded in the brain of pelagic seabirds [74,75]. Redknobbed coots (Fulica cristata) from industrialized and polluted regions of South Africa had Pb levels in the brain that increased to 25 ppm dw -2 and 4 times as much as in the kidneys and liver [69]. These studies on the accumulation of heavy metals in the brain of birds should be further compared to other studies of birds, both the same and other species. Different adaptations of birds to the environment, as well as the reaction and function of the brain against different contaminants, can be one of the factors affecting the absorption of contaminants in birds' brains. There are very few studies of the levels of heavy metals in the brain tissue of birds. Compared to other studies, the level of heavy metals in brain tissues in this study was higher than other studies from other parts of the world, including Zator and Milicz, Poland [62]; a wetland in Northwestern Poland [58]; Gdansk Bay Baltic Sea, Poland [70]; Nilgiris, Tamil Nadu, India [76]; a lagoon of Marano, Italy [77]; BjØrØya and Jan Mayen Artic [78]; and Pomeranian Bay, Poland [79].
The highest Ni levels were found in the kidneys, the liver and muscles showed slightly lower levels, and the lowest levels were found in the brain (Figures 1 and 2). A significant difference was observed in Ni levels between the kidney and the liver, brain, and muscles (P < 0.05).

Relationship Between Metal Levels, Feeding Habits, and Migration Status
The most important factors that affect the concentration of metals among different species are diet and feeding habits [80]. Diet varies between different bird species depending on the foraging strategies and diet preferences. One of the key pathways for metals to enter the body of birds is through food, water, and by eating sediment, lead shot, and grit (nonfood items). The direct consumption of soil contaminated with metals is a major cause of increased contamination in their bodies, even if the contaminant levels in plants or their prey has not increased [11].
In our study, birds were divided into four groups, invertebrate predator, fish predator, fish and crab predator, and omnivore to examine the effects of type of food on metal levels, using published data [80,81]. In the fourth group, we had only the Eurasian spoonbill (n = 2), so it was excluded from the statistical tests. Diet type had a significant effect on the levels of Zn, Pb, Cd, and Ni in the kidney and liver, with invertebrate species having higher concentrations than fish predators and omnivores (P < 0.05). There were no statistically significant differences for brain and muscle levels for any of the metals examined.
In a study in Shadegan wetland from Iran on mercury pollution in three species of waders, black-winged stilt had higher levels of mercury in the feathers, liver, kidneys, and muscles than other birds in the study [82]. The reason for the increase in mercury in this bird compared to other birds was that its long legs allowed access to deeper water and stilts could hunt larger prey than invertebrates. Similarly, other authors found higher heavy metal levels in the larger species that had access to deeper sections of the water and could hunt larger prey [83]. In the present study, the reason for the increase in metals in the various organs of blackwinged stilt, marsh sandpiper, and northern lapwing was that they fed on agricultural lands irrigated by farmers (and thus were exposed to contaminants in the water). We, and others [81], suggest that these species feed more on agricultural lands than do other species, remaining on the water for several days, rather than on the shores of the Chahnimeh from Sistan. Perhaps the use of chemical fertilizers and pesticides in agricultural lands has increased the exposure of birds to metals. This difference in metal concentration is most likely due to metal biogeochemical behavior, diet, and accidental ingestion of fine soil and sediment particle. However, it is impossible to separate soil selection/soil digestion from diet. Certainly, these two exposure pathways are very effective in concentrating these metals because other metals are correlated with accidental ingestion of fine soil and sediment particle [84]. In our study of heavy metals, birds that are invertebrate predators compared to birds that are predators Table 2 The concentrations of trace metals (µg/g dw) in the brain and liver, kidney, and brain and muscle of waterfowl from the Chahnimeh of Sistan and effect habitat birds * Significant difference between the concentrations of Zn, Pb, Cd, and Ni in the tissues of the liver and kidney of invertebrate predator with omnivores and fish predator (P < 0. at higher trophic levels had higher concentrations of heavy metals in the liver [85][86][87]. Birds of Chahnimeh reservoirs were divided into 2 groups of long-distance migrants, and local migrants that only go to the northern rivers and wetlands of Iran and do not leave Iran. It is noteworthy that there were differences in metal levels between the internal organs of the kidney and liver for all four elements studied, but there was no statistically significant difference between the two groups of birds for brain and muscle tissue ( Table 3). The birds in the southern wetlands  Heavy Metals in the Liver, Kidney, Brain, and Muscle: Health Risk Assessment for the Consumption… from Iran migrate to northern wetlands in the provinces Gilan and Mazandarn in the southern Caspian Sea to avoid the hot summer months in south and southeast Iran [81,88]. Heavy metal levels are high in this region of Iran, Caspian Sea, in fishes, macroalga, sediment, and water [89][90][91][92]. High levels of heavy metals in the south Caspian Sea might explain the high level of these heavy metals in local migrants.
Lower median concentrations of heavy metals (Cd, Pb, Ni, and Zn) in the liver and kidney were detected in the long-distance migrant birds than in the local migrants (P < 0.05) (Figure 1). Low usage of heavy metals and pesticides in breeding regions birds (Siberia or Eastern Europe) [88] that have migrated out of Iran might explain lower heavy metals in these birds.

Correlations Among Heavy Metals
All four elements in this study were positively correlated with each other within organs (P > 0.001, r > 0.603), but none of the elements was positively correlated with the other elements among tissue. This shows that the pathways and sources of entry for the elements studied are similar, but the pathways for accumulation of these elements and the reactions of different organs of the body to these elements are very different. A positive correlation between levels of Zn and Cd in the body of birds may protect them from the effects of increasing Cd in the body [38,48]. Positive correlations of Pb or Cd with other elements in tissues have been reported in birds from Korea [93,94], Cory's shearwater (Calonectris diomedea), and black-backed gulls (Larus fuscus) from England [95]; seabirds from Chaun, northeast Siberia, Russia [49]; and feral pigeons (Columba livia) from Korea [61].

Health Risk Thresholds
One of the non-essential element in the body is Pb that can cause neurotoxicity, nephrotoxicity, and other health effects [96]. Both the Spanish legislation and Australian National Health and Medical Research Council (ANHMRC) proposed 2.0 µg/g ww as the maximum permitted level of Pb in food [97,98]. The median level of Pb in muscle tissue in 6 species of birds (except for Eurasian spoonbill, great crested grebe, and moorhen) was lower than the Spanish legislation and ANHMRC guidelines. The median level of Pb in the liver and kidney of birds was higher than the levels allowed in the Spanish legislation and ANHMRC guidelines (except for cormorant); Eurasian spoonbill also had higher level in the kidney than these guideline (Fig. 3). The action level for human health is 1.7 µg/g ww Pb [99] (Fig. 3). In contrast to these maximum permitted levels for Pb, the Institute of Turkish Standards for Food (ITSF) and the European Commission (EC) introduced the permissible threshold level of 0.1 and Table 3 The concentrations of trace metals (µg/g dw) in the brain and liver, kidney, and brain and muscle of waterfowl from the Chahnimeh of Sistan and effect migration water fowl * Significant difference between the concentrations of Zn, Pb, Cd, and Ni in the tissues of the liver and kidney of long-distance migrants and local migration (P < 0.05)  0.5 µg/g ww, respectively [100,101]. The median level in flesh muscle, liver, and kidney of all birds in this study was clearly higher than these guidelines, and according to these two guidelines, the health of the people of this region is endangered by consuming the muscle and especially the liver of these birds. The maximum permitted Cd level of the ANHMRC, USFDA, and Western Australian authorities was 2, 3.7, and 5.5 µg/g ww, respectively. In our study, none of the birds exceeded this median level of Cd in muscle, but levels of Cd in liver of northern lapwing, moorhen, marsh sandpiper, and black-winged stilt were higher than the threshold levels suggested by the ANHMRC, USFDA, and Western Australian authorities [97,98]. Cadmium levels in the kidney were higher than the ANHMRC threshold in all birds except the cormorant and the Eurasian spoonbill. Also, the great crested grebe, with a Cd level of 4 µg/g ww, was higher than both the ANHMRC and USFDA guidance, and the rest of the birds were higher than all three guideline ANHMRC, USFDA and Western Australian authorities (Fig. 3). In contrast to these maximum permitted levels, the Spanish legislation and EC threshold are 1 and 0.05 µg/g ww, respectively [97,98]. In this study, the levels of Cd in the muscle, liver, and kidney of all birds were greater than these thresholds.

Liver
The permissible limit of Ni in food by the US Food and Drug Administration is 10 µg/g ww [99]. According to this guideline, the levels of Ni in the muscle, liver, and kidney of all birds, except cormorant, were higher than the permissible limit. The permissible limit of the FAO Ni is 13 µg/g ww in food [102], and the levels of Ni in muscle of birds were lower than this limit, except for the liver in black-winged stilt, marsh sandpiper, moorhen, and northern lapwing (13 µg/g ww), and the levels in the kidney of all birds (except cormorant and Eurasian spoonbill) were higher than the FAO guideline (Fig. 3). The Food and Nutrition Board (FNB) [103] introduced the permissible limit of Ni as 4 µg/g ww. Accordingly, the levels of all muscle, liver, and kidney in all birds in the present study were higher than this limit, and the consumption of edible parts of all birds poses a threat for the heath of people in this region.
The ANHMRC and WHO introduced an acceptable limit of 1000 µg/g ww for Zn in food [104,105]. The level of Zn in the muscle, liver, and kidney in all birds of Zabol Chahnimeh reservoirs were below this toxic threshold [97] [106] (Fig. 3).

Health Risk from Consuming Birds in Chahnimeh Reservoir
In our study, the HQ for any of the metals in the muscle for most of birds was < 1, but the ∑HQ was > 1 in moorhen birds ( Table 4). The HQ of Pb in liver was > 1, but in the other birds it wasn't; the ∑HQ was not higher than 1 for any other birds (Table 4). In the edible parts, the level of HQ was high, and except cormorant, in other birds, the level ∑HQ was between 1.24 and 4, which was due to the high level of HQ in the kidneys and muscle of birds in this region (Fig. 4). The ∑HQ of each metal we examined was > 1, suggesting that people would experience health risks from consumption of birds from the Chahnimeh reservoirs (Fig. 4). On the other hand, values of the ∑HQ index for total exposure were > 1 for birds, indicating that the estimated exposure is a major health concern. Studies in the wetlands of northern Iran showed that the pochard is not suitable for consumption. [107].

Estimated Human Daily and Weekly Toxic Elements Intake from Birds
Different metals in different concentrations have different effects on organisms, and some metals can show toxic effects even in low concentrations [29]. In this study, we   [108]. Also, the PTWI according to the guidelines of FAO/WHO ( 2011) is 35 and 7000 µg/kg body weight/week for nickel and zinc, equaling 2450 and 490,000 µg/week for a 70 kg person, respectively [109].
According to Table 4, none of the bird organs had levels of Zn, Pb, Cd, and Ni that were higher than the level of PTWI70. In this study, the EWI of Pb in edible parts of birds B, MS, M, N, and E was higher than PTWI, and this is due to the high level of EWI in the liver of these birds, while the level of lead in the muscle tissue of all birds was within the allowable range for PTDI, PTWI, and PTWI 70.
For Cd, the level EWI in the edible parts was higher than PTWI in all birds, and the EDI level in edible parts birds G, B, MS, M, and N was higher than the PTWT, which is due to the high level of EWI and EDI in the muscle and kidneys of these birds (Table 4).
In this study of Ni, the level EWI in muscle and edible parts was higher than PTWI in all birds, and the level of EWI was higher in B, MS, M, and N of PTDI. The EDI in the birds B, N, M, and MS was higher than the level of PTWI. Except C and E, in all birds, the EWI in kidney was higher of the PTWI, and also the level of Ni in the liver of birds B, Ms, N, and M level of EWI was higher of the PTWI ( Table 4). The level of EDI of Ni in edible parts B, MS, N, and M was higher than the PTWI, and also the level of Ni in kidney M and MS was higher than the PTWI, and finally, the data indicate that the level of EWI in edible parts B, MS, M, and N was higher than the PTDI ( Table 4). The results of this study show that people in this area should not use "edible" parts of the birds examined, and the use of wild birds as daily and weekly food is a serious threat to the inhabitants of this area. This is contrary to the results obtained for birds in the wetlands of northern Iran, where EDI and EWI were within the permissible range and did not pose a threat to the people of the region [107,110].

Conclusion
In this study, the levels of Cd, Pb, Ni, and Zn were investigated in birds of Chahnimeh of Sistan from Iran. The level of all heavy metals (except nickel) in the brains of birds was higher than the levels in other tissues. Differences in metal levels as a function of feeding habitat and migration were  Fig. 4 Estimated potential health risks for Zn, Pb, Cd, and Ni via consumption of the liver, kidney, and muscles collected (edible parts). Hazard quotients (HQ) and ∑ HQ = HQ Pb + HQ Cd + HQ Ni + HQ Zn observed only in the kidney and liver tissues of birds. The levels of heavy metals in some birds were higher than the effect level threshold; 56% of the liver and kidney samples of these birds were above the threat level. The results of this study show that birds in Chahnimeh of Sistan pose a risk to humans from heavy metal contamination. The data show that human consumption (using EDI, EWI, and HQ) of the edible tissues of birds is not suitable: people of the region should avoid eating the edible tissues of wild birds and should particularly avoid eating kidney and liver tissue.