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Determination of proximate composition, selected essential and heavy metals in sesame seeds (Sesamum indicum L.) from the Ethiopian markets and assessment of the associated health risks

In this study, the proximate composition, levels of essential and heavy metals; and health risk assessment were evaluated in sesame seed (Sesamum indicum L.) samples purchased randomly from local markets of three study sites in Ethiopia. A 0.15 g of dried and homogenized samples were digested using 6 mL of HNO3 (69–72%) and 3 mL of HClO4 (70%) at 120 °C for 3 h. The levels of proximate composition and essential metals in the sesame samples were determined by the Association of Official Agricultural Chemists (AOAC) and Flame Atomic Absorption Spectrometer (FAAS), respectively. The moisture content, crude fiber, ash content, fat, crude proteins and carbohydrate of sesame samples ranged between 3.15% and 5.52%, 4.21–4.40%, 3.10–4.75%, 55.75–56.9%, 22.65–23.39% and 8.34–8.80%, respectively. The accuracy of the procedure was validated by percent recovery of samples for all metals varied from 85.60% to 102.00%. The mean levels of the metals (mg/kg) were found in the range of 37.20–70.10 (Na), 315.1–338.00 (K), 209.00–216.50 (Mg), 328.00–341.30 (Ca), 12.60–12.90 (Fe), 4.90–5.00 (Cu) and 8.30–8.70 (Zn) mg/kg. The THQ values of heavy metals were found < 1, indicating that the consumption of sesame may not cause health risks to local consumers. The values of HI ranged from 1.5 × 10−3 to 1.8 × 10−3 in the study areas and may not have a possible adverse health effect on the adult population from the consumption of sesame seeds.


Introduction
Sesame (Sesamum indicum L.), commonly known as Beniseed, is a flowering plant in the genus Sesamum. It is wide spread in tropical regions and is one of the oldest known oil seed-producing crops [1]. It is cultivated in several countries such as India, Sudan, China, Ethiopia and Burma, which are considered the major producers (60% of its total world production) [2][3][4].
Sesame is often grown as a single crop. However, sesame and other oilseeds are sometimes grown intercropped and/or in borders with sorghum or teff and other grain crops in North Shewa and South Wollo, central Ethiopia mainly to diversify production as well as add value to the income of farmers [5]. It is a multi-functional seed used as a food supplement in addition to spices, insecticides, medicines, soap, green manure and ornaments.
In Ethiopia, sesame is the most powerful economic source of agricultural products. Because of the excellent quality of the edible oil it produces, it is often called as queen of the oilseed crops. Sesame varieties showed significant differences in some physical properties, proximate mineral, anti-nutritional (phytic acid), and antioxidant compositions. Its product quality depends on suitable agro-ecological areas, making its diversified products. It grows in an altitude of lower than 2000 m above sea level and is a well-established crop in Amhara, Tigray, Benshangul Gumz and Oromia regions, Ethiopia [5].
Heavy metals such as chromium, copper, iron, manganese and zinc are essential for humans and they may play an important role in many biochemical processes [6]. Some metals like Fe, Zn, Cu, Cr, Co and Mn are essential for the metabolism of the human body. Some other metals like Pb, Cd, Ni and Hg are toxic in certain concentrations. Fe, Cu, Zn, Na, Ca and Mg are essential metals, but in excess can be toxic [7]. Thus, food safety issues and their subsequent problems are serious public and environmental concerns. Shelf stability and heavy metal concentration have a physiological impact in the human body. If the heavy metal concentration is above the optimum value, it will lead to toxification and if it is below the optimum value, it will lead to deficiency disease.
Toxic metals are given more attention due to their toxicity and mutagenic effects even at very low concentrations (Quadra et al., 2019; [8]). Preventing food items from heavy metal contamination is, therefore, one of the important aspects of food quality assurance. Accordingly, the content of essential and heavy metals in sesame is of great interest to consumers [9]. Seeds of sesame are considered as major source for solving the problem of deficiency of micronutrients in modern-day nutrition.
Sesame is not only rich source of oil but also a potential source of carbohydrates, proteins, fats, fibers, and vitamin B1 [9]. Sesame seeds are also an excellent source of minerals such as phosphorus, iron, magnesium, calcium, manganese, copper and zinc. In addition to these important nutrients, its seeds contain two unique substances, sesamin and sesamolin. Both substances belong to a group of particularly useful dietary fibers, the lignans and have a cholesterol-lowering effect in humans, prevent high blood pressure, degeneration of vessels, antioxidant activity and increase the vitamin E supply in animals [9,10]. Sesame seeds are used in the making of tahini (sesame butter) and halva and for the preparation of rolls, crackers, cakes and pastry products in commercial bakeries [11]. Therefore, the objective of this study was to determine the proximate composition; levels of essential and heavy metals of sesame seeds from three districts of the North Shewa Zone and local markets in Ethiopia; and assessment of the associated health risk.

Reagents and chemicals
During the study, 69-72% HNO 3 and 70% HClO 4 were used for the digestion of sesame seed samples. 40% Boric acid was used for total nitrogen content analysis in the sample. A 1000 ppm of the metals (Fe, K, Zn, Cu, Ca, Mg and Na) were used for the preparation of a series of working standard solutions to calibrate the instrument. Selenium and potassium sulfate was used as a catalyst for the determination of nitrogen, whereas lanthanum oxide was used for the determination of magnesium and calcium. A 50 ℃ boiling point of petroleum ether was used as a solvent for the determination of crude fat. Distilled water was used throughout the experiments for sample preparation, diluting the solutions and rinsing apparatus before and during analysis. Chemicals and reagents used in this study were all analytical grade.

Description of the study area
Moretna Jeru, Merhabete and Mida Woremo were study areas found in the North Shewa Zone, Amhara Regional State, Ethiopia. The study sites were selected because they are potential production areas and sesame seeds are commonly consumed in these areas. Moretna Jeru located between the Jamma river and the districts of Shewa Meda. The district has a longitude and latitude of 10 • 10 ′ 0 ′′ N, and 39 • 0 ′ 0 ′′ E, respectively. Merhabete is bordered on the south by Ensaro, on the west by the Oromia Region, on the north by Mida Woremo, on the east by Menz Keya Gabriel and on the southeast by Moretna Jiru. It is located at longitude 10 • 15 ′ 0 ′′ N and latitude 38 • 45 ′ 0 ′′ E. Mida Woremo is one of study sites located at longitude and latitude of 10 • 20 ′ 0 ′′ N and 38 • 50 ′ 0 ′′ E, respectively.

Sampling and sample preparation
The sesame seed (Sesamum indicum L.) samples were purchased from three different local markets in the districts of North Shewa Zones, Ethiopia. About 25 g of sesame seed samples, were randomly purchased from farmers of each sampling site at the local market. The sesame seed samples, marketed from the same origin, were pooled together to obtain a representative composite samples. The composite subsamples were cleaned of stones, sand, soil and other particles; washed, sun-dried and ground into a fine powder using mortar and pestle; homogenized and sieved with a 5 mm sieve. Finally, the fine powdered packed with polyethylene plastic bags and stored in the fridge, until they were taken for digestion and analysis. Data for the estimation of daily intake have been collected from the three districts of the study areas. A total of 180 adult people (90 men and 90 women separately) from each sampling sites were interviewed for daily consumption of sesame. The mean values of each interviewee were considered to estimate the daily intake of sesame in the local sampling sites.

Proximate composition analysis of sesame seed samples
The proximate compositions of the sesame samples were estimated as described in previous reports [11,12] with simple modifications.

Determination of ash content
A 0.5 g samples were dried in the moisture extraction oven until constant weights were obtained. Then, the samples were transferred into the muffle furnace (FUSEF1AH, R000047 UK) with a pair of tongs and ashed at 500 • C for 12 h until white ash was obtained. The samples were removed from the furnace and cooled in a desiccator and weighed. The percentage of total ash content of sesame seeds was calculated using Eq. 1.

Determination of moisture content
Standard official methods of analysis of the AOAC were used for moisture content determination [12]. 2 g of each sample were weighed into clean, dried, and pre-weighed crucibles. The sesame seed samples were dried in the moisture extraction oven at 100 • C for 3 h. The dried samples were then removed from the oven, cooled in a desiccator and reweighed. The samples were again put back into the oven and dried until a constant weight was obtained. This analysis was carried out in triplicate and the average value was recorded as moisture content. In this regard, the weight of the empty dried dish was taken as W 1 ; the weight of 2 g samples with the dry dish was taken as W 2 and the sample was dried at 100 • C for 3 h and taken as W 3 . The percentage moisture content was calculated by Eq. 2: Where, W 1 = Initial weight of empty crucible, W 2 = Weight of crucible + sample before drying, and W 3 = Final weight of crucible + sample after drying.

Determination of fiber content
2 g of samples were weighed; 200 mL of sulfuric acid was added to the boiling mixture and heated for 30 min, and filtered. After the residue was washed three times with hot water, 100 mL of 2% NaOH was added to the boiling mixture and heated for 30 min, and filtered. Then, the filterate washed carefully three times with hot water until it was free from the acid. The sample was dried under suction and transferred to an oven at 105 • C overnight, and finally weighed. The residue was ashed in a muffle furnace at 550 • C for 3 h till a light grey ash was formed, then weighed to a constant, and the total crude fiber percent was calculated by the Eq. 3: Where: W 1 is the weight of sample before ignition, W 2 is weight of sample after ignition and W 3 is the original sample weight.

Determination of crude protein
The Kjeldahl method was used to determine the crude protein. A 5 g of sesame sample was weighed with electrical balance and placed in digestion flask. A 2 g of Kjeldahl catalyst and 200 mL H 2 SO 4 were added and boiled until the solution cooled and became clear. 60 mL H 2 O and 6 drops of the mixed indicator were added and heated until all N 2 was distilled. Finally, the solution was titrated with NaOH and the crude protein content calculated by Eq. 4 [13]: Where A is the volume (mL) of 0.25 N HCl used for sample titration, B is the volume (mL) of 0.25 N HCl used in blank titration, N is the normality of HCl, W is the weigh (g) of sample, 14.007 is the atomic weight of nitrogen, 6.25 is the protein-nitrogen conversation factor.

Determination of fat content
A 4 g of sesame sample were weighed (w 1) and taken into Soxhlet; 250 mL of petroleum ether was added and heated at 85 • C until the solvent is completely evaporated and assigned as w 2 . Then, the bottle containing the sample was cooled in desiccators and reweighed (w 3 ) then the fat content was calculated by Eqn5: Where: W 1 is the weight of empty bottle, W 2 is the weight of bottle and oil, W 3 is the weight of sample.

Determination of carbohydrate
The percentage of carbohydrates in sesame seed was calculated by using the following formula with the same amount of seed sample (Eq. 6);

Mineral analysis of sesame seeds
In the current study, sesame seed samples were washed with distilled water and then dried in an oven. The dried sesame seed samples were ground with an electrical grinder and made into powder. The dried and powdered sesame seed samples were digested in a microwave digester (Top Wave Analytical Jena). The solvent was separated from crude fat by a rota evaporator (RE200). The crude fat was extracted by a Soxhlet extractor (RS2071).
Microwave-assisted digestion with mixtures of nitric acid and perchloric acid technique was used for the dissolution of sesame samples. This digestion technique in closed vessels under pressure has gained popularity as a simple and fast dissolution technique that minimizes acid consumption, the risk of sample contamination and the loss of volatile elements [14]. A 0.15 g of dried and homogenized sesame seed samples were transferred into a digestion tube with a 9 mL mixture of 69-72% HNO 3 and 70% HClO 4 (2:1 v/v) and digested. The solution was allowed to cool for 3 h at 120 • C. Lanthanum oxide was added as a catalyst and distilled water was added to dissolve the precipitate formed on cooling [15]. Then, the solution was transferred to a 50 mL volumetric flask and diluted with deionized water and filtered with Whitman filter paper #42 and the digested samples were stored in the refrigerator until the content of all metals in the sample solutions was determined by FAAS equipped with deuterium arc background correctors and hollow cathode lamps with air-acetylene flame. The operating parameters for the determination of metals used in FAAS measurements were given in Supplementary data Table 1.

Instrument calibration
The concentration of metals in the sample solution was determined using calibration curves. As a result, several working standard solutions from 10 mg/L were prepared, the range of which includes the unknown concentration of the metal. Wavelengths, standard working concentrations, the regression equation, and the value of the correlation coefficients are summarized in Table 1. The correlation coefficients (R 2 ) of the calibration curves were found in the range of 0.9967-0.9993, suggesting that there was a very high correlation between concentration and absorbance.

Validation of the analytical method
The parameters: accuracy, precision, sensitivity, limit of detection (LOD) and limit of quantification (LOQ) were used for method validation [7,16].
The LOD and LOQ values of the investigated metals with FAAS were found to be 0.080 and 0.266 mg/L for Na; 0.034 and 0.113 mg/L for K; 0.015 and 0.050 mg/L for Mg; 0.0028 and 0.009 mg/L for Ca; 0.0009 and 0.003 mg/L for Fe; 0.0032 and 0.010 mg/L for Cu and 0.0045 and 0.015 mg/L for Zn, respectively (Table 1), which showed that the method was valid to the determination of the investigated metals in sesame seeds samples.
The accuracy of the method was evaluated by spiking 0.15 g of sesame seeds from Moretna Jeru with 6.75 µL, 27.75 µL, 33.75 µL, 18 µL, 0.75 µL, 0.525 µL and 1.2 µL of 1000 mg/L of Na, K, Ca, Mg, Zn, Cu and Fe, respectively. Spiked samples were digested using the optimized digestion method and analyzed in triplicate under similar conditions to non-spiked samples. The results of recovery analysis are shown in Table 2 and the percentage recoveries lied within the range of 85.71 ± 3.80%− 108.25 ± 2.85%, which were within the acceptable range of recoveries (80-120%) [17].

Health risk assessment of heavy metals in sesame seeds consumption
The human health risk of ingestion of sesame seeds contaminated with heavy metals (Cu, Zn, and Fe) was assessed based on metal estimated daily intake (EDI), target hazard quotient (THQ) and hazard index (HI).
The EDI values of metals were calculated to estimate the daily exposure to metals in the body system for a given body weight of a consumer and calculated according to Eq. 7 [18]: Where C metal (mg/kg) is the mean concentration of heavy metals in sesame, IR (ingestion rate) is the average daily consumption of sesame (gram/day person), and BW is the average body weight (kg). The average daily intakes of sesame seeds calculated from collected data were 70 and 60 mg/person/day for males and females, respectively. The average body weights of adults were considered 67.0 kg for males and 62.3 kg for females [19] for this study. The THQ values of metals were calculated based on Eq. 8) [20]: The reference dose values (RfD) of Cu, Zn and Fe are 0.04, 0.30 and 0.70 mg/kg/day, respectively [21]. A THQ value greater than 1 indicates that the population poses a health risk associated with the consumption of metal-contaminated food crops [18].
The hazard index (HI) helps to assess the overall non-cancer risk to human health from more than one heavy metal. It is the sum of the hazard quotient (THQ) of all heavy metals as described in the following equation: The population is not at risk if the HI score is less than 1, and if the HI score is equal to or greater than 1, then the population is at risk [18].

Statistical analysis
Experiments were carried out in triplicate for each sample and the results are reported as the mean ± standard deviation. One-way analysis of variance (ANOVA), was performed using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) software for Windows, where p < 0.05 was considered statistically significant.

Proximate composition analysis
The results obtained by this study showed some variations in sesame seeds' proximate composition among the sesame samples ( Table 3). The moisture contents of sesame seed samples from three districts of the North Shewa Zone were between 3.15% and 3.52% and had no significant (p > 0.05) differences between the samples. The results of moisture contents of sesame seeds in the present study were slightly lower than the results of the study reported (4.16-4.62%) by [22] and (6.21 ± 2.41%), by Christian Ebere, [11]. However, the moisture content of sesame reported by Nweke et al., [23] was in the range of 0.22-3.5%, which is slightly lower than the present study. The moisture content for sesame seeds has profiled the study areas in the order of Merhabete > Mida Woremo > Moretna Jeru.
Ash content showed a significant difference (p < 0.05) between sesame samples from Merhabete with Moretna Jeru and Mida Woremo but no significant difference (p > 005) between Moretna Jeru and Mida Woremo. The ash content of sesame was obtained in the range from 3.10% (Mida Woremo) to 4.75% (Merhabete) as shown in Table 3, which agrees with the results reported by [23] (1.44-5.93%) and [24] (4.77%). Ash content values reported by [22] were in the range of 3.67-5.39% for Turkish sesame seeds, and by [12] ranged from (4.8-4.01%), which are similar to the result of this study. However, the ash content value of the sesame reported by Mbaebie et al., [25] (8.94 ± 0.96%) was higher than the present study.
The percentage of crude fiber was obtained in the range of 4.21%− 4.30%. The results were comparable with the results in the three study areas as shown in Table 3. The result had also no significant differences (p > 0.05) between samples. The values for the crude fiber were lower than the results (6.60 ± 4.39%) reported by Mbaebie et al., [25] and (12.01%) by Iliya, [24]. However, higher than the result (1.30%− 3.30%) reported by Dimitrov et al., [26].
There is a significant (p < 0.05) difference in the protein content between the samples of Merhabete from Moretna Jeru and Mida Woremo. Moretna Jeru and Mida Woremo had no significant differences (p > 0.05) in the protein content. The results shown in Table 3 of the crude protein content obtained in the range of 22.65%− 23.39% showed good agreements between the samples. The crude protein content of sesame reported by [24,25] (4.32% and 16.63 ± 11.96%), respectively, were lower than the present study.
As the study revealed, the Moretna Jeru sesame seed has a higher carbohydrate and fat composition as compared with Merhabete and Mida Woremo sesame seeds ( Table 3). The carbohydrate content of the sesame samples analyzed in this study was obtained to be 8.34-8.80%, which showed nearly similar results reported by [27,28] i.e., 10.64% and 9.40 ± 0.02%, respectively. However, the results reported by Comfort et al. [29] and Dravie et al., [30] were 40.08% and 18.44%, respectively, which were higher than the present study. However, this value is below the value given by FAO/WHO RDA (55%), so a sesame seed diet must be consumed in conjunction with other food with much higher carbohydrate content to meet the daily carbohydrate requirement from the diet. Carbohydrates not only represent the most accessible source of energy for our body, but also play an important role in many physiological processes [27]. The fat content of the sesame samples was found in the range of 55.60%− 56.90% and as shown in Table 3, there were strong agreement with the value reported by [30] (i. e., 56.56%). The fat content from Moretan Jeru was significantly (p < 0.05) different from the results obtained from Merhabete and Midaw Woremo, however, the results from Merhabete and Mida Woremo has no significant difference (p > 0.05).

Concentrations of metals in sesame seed samples
The mean concentrations of Na, K, Mg, Ca, Fe, Cu and Zn in sesame seed composite samples, cultivated from the three study areas of the North Shewa Zones, Ethiopia, were presented in Table 4. In this study, the concentrations of Na. K, Mg, Ca, Fe, Cu, and Zn in sesame seed samples were ranged from 37.2 ± 2.6-70.1 ± 1.1, 315.1 ± 4.5-318.0 ± 1.5, 209.0 ± 3.7-216.5 ± 0.95, 328.0 ± 0.56-341.3 ± 1.21, 12.6 ± 2.3-12.9 ± 1.4, 4.9 ± 0.65-5.0 ± 1.3 and 8.3 ± 3.12-8.7 ± 0.34 mg/kg, respectively. The data obtained for the elemental concentrations of sesame seeds can also help to decide on the dose or amount of consumption as herbal remedies made from this crop [11]. Sodium is an essential mineral that regulates blood volume, blood pressure, osmotic equilibrium and pH. The minimum physiological requirement for sodium is estimated to range from about 120 mg/day in new born to 500 mg/day over the age of 10. Excess sodium has direct adverse effects on target organs, beyond the increased risk of hypertension (HTN) [31]. The amount of sodium needed to maintain homeostasis in adults is exceedingly low (< 500 mg) compared to the average intake of most Americans (> 3200 mg) [32]. The concentration of sodium obtained in sesame seed obtained from three study sites is below the limit of detection of the applied method. Data in Table 4 revealed that Na was detected in all sesame samples at a concentration of 70.1 ± 1.1, 49.5 ± 0.5 and 37.2 ± 2.6 mg/kg from Moretna Jeru, Merhabete and Mida Woremo, respectively. The concentration of Na in all samples showed statistically significant differences (p < 0.05). The concentration of Na reported in Nigeria (0.54 ± 0.03 mg/kg) [28] was lower than the present study. However, the concentrations of Na, in all the study areas, were much lower than reported by Yeasmin [33] (422.0 ± 0.60 mg/kg) in Bangladesh.
An adequate intake of potassium was 3400 mg/day for adult males and 2600 mg/day for adult females. The average recommended intake by Americans is 2591 ± 9 mg/day [34]. In the present study, the concentration of K was 319.8 ± 1.5, 338.0 ± 1.5, and 315.1 ± 4.5 mg/kg obtained from Moretna Jeru, Merhabete and Mida Woremo, respectively ( Table 4). The concentration of K from Merhabete was significantly different (p < 0.05), however, the results obtained from Moretna Jeru and Mida Woremo were not significantly different (p > 0.05). According to Bolaños et al. [35] and Yeasmin [33] reports, the concentration of K in the present study was significantly lower than reported in Argentina (5845.53 ± 77.61 mg/kg), however, its values were significantly higher than reported by Bangladesh (1203.0 ± 0.29 mg/kg).
The recommended daily intake of Mg is 400-420 mg/day for men and 310-320 mg/day for women and the mean intake of Mg was (412.68 mg/day) [36]. The concentration of Mg, in the present study, was below the optimum limit. The highest Mg concentration of 216.5 ± 0.95 mg/kg was recorded in Merhabete, while the lowest concentration of 209.0 ± 3.7 mg/kg was found in Mida Woremo.The level of Mg obtained from Mida Woremo was significantly different (p < 0.05) from the results found in Moretna Jeru and Merhabete, whereas there are no significant differences (p > 0.05) between Merhabete and Moretna Jeru ( Table 4). The Mg concentrations found in this study were significantly lower than the values 3743.60 ± 49.42 mg/kg and 996.0 ± 0.51 mg/kg reported by Bolaños et al., [35] and Yeasmin [33] for Argentina and Bangladesh sesame seeds, respectively.
Calcium is an important element in the body. It plays an essential role in the health of bone as well as in regulating blood pressure [37]. The recommended upper limit for calcium is 2500 mg/day for adults aging 19-50. For those 51 and older, the limit is 2000 mg/day [36]. The obtained calcium concentration in this study was below the tolerable limit. The highest mean concentration obtained for Ca was 341.3 ± 1.21 mg/kg from Moretna Jeru ( Table 4). The order of mean concentration of Ca is Moretna Jeru > Mida Woremo > Merhabete. The concentration of Ca obtained from Moretna Jeru was significantly different (p < 0.05) from Merhabete and Mida Woremo sites, but the values were statistically similar (p > 0.05) between Merhabete and Mida Woremo ( Table 4). The Ca content of this study was lower than values of other study in Ethiopia reported by Zebib et al. [38] with values of 1172.8-12257.1 mg/kg. Similarly, the concentration of Ca in this study was lower than the results reported in Argentina, 10463.47 ± 152.76 mg/kg [35] and in Bangladesh, 2510.0 ± 0.78 mg/kg [33].
Fe is an essential element for metabolism in the human body acting as a catalyst. In the absence of enzymatic catalysis, most biochemical reactions are so slow that they would not occur under the mild conditions of temperature and pressure that are compatible with life [3]. Iron is an essential trace element for hemoglobin formation [29]. Fe content obtained from sesame samples in this study ranged from 12.6 ± 2.3-12.9 ± 1.4 mg/kg, which is below the recommended safety limits for consumption (80-110 mg/L) [3]. Therefore, sesame seeds that contain Fe can be consumed without any health problems by the local community. In this study, the results of the One-way ANOVA analysis also showed that there were no significant differences (p > 0.05) between the three sites. According to the research conducted in other countries, the concentration of Fe detected is lower than the results found in the present study, 0.349 mg/kg in Iran [7], and 0.1251-1.9815 mg/kg in Iraq [2], whereas the level of Fe reported in other studies in Ethiopia found 39.1 mg/kg [3], 102.4-107.5 mg/kg [38] was higher than the present study. The concentration of Fe reported by Yeasmin, [33] in Bangladesh sesame seed (29.0 ± 0.20 mg/kg) and previously reported by Hu and Zhou [4] in China (61.9-86.8 mg/kg) [4] were higher than the results of the present study.
Copper is essential for their normal biological activities. It is therefore necessary that humans consume a certain amount of copper to maintain their health [3]. Copper is also involved in hemoglobin synthesis, connective tissue metabolism, bone development, tryptophan synthesis and iron transport. Consuming too much can cause oxidative stress and damage the immune system [39]. The mean concentrations of Cu in e sesame seeds were 5.0 ± 1.3, 4.9 ± 0.45 and 4.9 ± 0.65 mg/kg from Moretna Jeru, Merhabete and Mida Woremo, respectively. The concentrations of Cu in sesame seed samples from the three study sites were statistically similar (p > 0.05). The concentration of Cu reported by Abu-Almaaly [2] in Iraq was ranged from 0.1988 to 1.1002 mg/kg; Eghbaljoo-Gharehgheshlaghi et al. [7], in Iran with a value of 0.35 ± 0.017 mg/kg and Bolaños et al. [35] in Argentina 0.30 ± 0.01 mg/kg were lower than the present study. The concentration of Cu reported by Hu and Zhou [4] in China ranged from 16.3 to 30.1 mg/kg was higher than the present study.
In the human body, zinc is essential for the proper functioning of the immune system. It facilitates division of cells, the growth of cells, wound healing and carbohydrate catabolism [3]. The mean concentrations of Zn in sesame seed samples were 8.3 ± 3.12, 8.7 ± 0.34 and 8.4 ± 0.47 mg/kg, respectively, as presented in Table 4. One-way analysis of variance (ANOVA) was tested to check the data homogeneity and validatity of the results. The results of ANOVA and least significant difference (LSD) statistical tests showed statistically similar values of Zn in the three sites (p > 0.05). The results of study researched by Eghbaljoo-Gharehgheshlaghi et al. [7] showed that the mean level of Zn (1.707 ± 0.022 mg/kg) in Iran was lower than the present study. The level of Zn, in this study, was also significantly lower than reported by Bolanos et al.  The similarities and variations in essential and heavy metal levels in sesame seed samples in the present study and studies in Ethiopia and other countries might be attributed to different varieties, growth environments, data pretreatments and analytical methods. Several factors might also contribute to variations in soil metal levels, including industrialization and the pollution of the biosphere, fertilizer use, pesticide treatment and others.
Fe, Cu, and Zn are known components of proteins, enzymes and redox systems for humans. The deficiency or excessive intake of Fe, Cu, and Zn in humans can lead to several disorders or diseases [4]. Heavy metals can play an important role in the physiological and biological functions of the human body when it is below tolerable limits. The recommended dietary allowance (RDA) of Zn, Fe and Cu in the USA for adults is 8-11, 8-18 and 0.9 mg/day, respectively [40]. These metals are physiologically important metals while their concentration is on the permitted level.
The average daily Fe intake from foods and supplements is 13.7-15.1 mg/day for children aged 2-11 years; 16.3 mg/day in children and teens aged 12-19 years; 19.3-20.5 mg/day in men and 17.0-18.9 mg/day in women older than 19 years [41]. In the current study, the concentrations of Fe were found to be within the tolerable limit (Table 4).
For cupper, the recommended daily intake (RDI) is 11 mg/day for adult men and 8 mg/day for adult women. Pregnant and breast feeding women should consume 11 and 12 mg/day Cu, respectively [42]. Cu is an essential element, used in a variety of biochemical processes. However, at higher levels, it is toxic and affects soft tissues particularly the blood and kidneys [39]. Similarly, the recommended dietary allowance (RDA) of Zn for adults above 19 years is 11 mg/day for men and 8 mg/day for women. Pregnancy and lactation require slightly more than 11 mg/day and 12 mg/day, respectively [40]. The tolerable upper intake level is the maximum daily intake unlikely to cause harmful effects on health. The concentration of copper and zinc is below this tolerable limit.

Risk assessment of heavy metals
Estimated daily intake (EDI), Target hazard quotient (THQ) and Hazard index (HI) values of heavy metals via consumption of sesame seeds were given in Table 5. According to the Environmental protection agency (EPA), human health risk assessment is defined as the process of estimating the nature and likelihood of adverse health effects in people exposed to chemicals in contaminated environmental media now or in the future. The heavy metal risk score is calculated using parameters such as estimated daily intake (EDI), Target hazard quotient (THQ), and Hazard index (HI). These parameters for risk assessment are caused by any chemical contaminants with prolonged exposure [21]. They not only depend on the amount of pollutant ingested but are also related to the frequency and duration of exposure, average body weight and the reference oral dose (RfD) [43]. The values of THQ must not exceed 1; if the values exceed 1, it is an implication of causing potential non-carcinogenic risks to the exposed population [43,44]. It should also be noted that THQ is not a risk measure but reflects the level of concern [43]. According to Comfort et al. [29] report, the EDI values of Cu, Zn and Fe were 1.51 × 10 − 3 , 1.88 and 5.39 × 10 − 3 mg/day, respectively, which were higher than the present study as shown in Table 5.
The uptake of heavy metals through food chain is related to the concentration of heavy metals in food and the amount of food consumed [20]. The possible health risk of sesame seed consumption was evaluated. The HQ and HI values are related to all heavy metals in the sesame samples were less than one, which indicates no potential health risks in sesame consumption, which is consistent with the results reported by [20]. Therefore, based on the THQ and HI indices, consumption of the studied sesame is not harmful due to the presence of Cu, Zn and Fe. The THQ and HI values of Cu from Moertna Jeru for females were slightly higher than the other samples ( Table 5). The THQ values of Zn and Fe for females from Moretna Jeru and Mida Woremo showed similar results. The lowest HI values of metals in sesame samples were obtained from Mida Woremo for females. The THQ and HI values for males were slightly higher than for females.

Conclusion
This study investigated the proximate compositions and levels of essential (Na, K, Ca, Mg) and heavy (Zn, Cu, and Fe) metals in sesame seeds grown in the North Shewa Zone by AOAC and FAAS techniques, respectively. The proximate and essential elemental components have revealed the presence of several useful elements in the human body. Low LODs, good linearity (R 2 > 0.996) and recoveries in the range of 85.60-102.00% were achieved for all metals tested. The developed analytical method was successfully applied to the analysis of proximate composition and heavy metals in sesame seed samples originating from districts of the North Shewa Zone. The concentrations of all essential metals were found to be lower than the permissible limit recommended by FAO/WHO. From health risk perspective, the THQ level of Zn, Cu and Fe in sesame seeds did not contribute to the non-cancer causing risk. The Hazard index (HI) values showed that the consumption of sesame has no potential negative effects on human health.

CRediT authorship contribution statement
Atnafu Guadie: Conceived the idea, designed the experiments and performed the experiments. Tamene Beshaw, Kindnew Demssie: Performed the experiments, analyzed and interpreted the data; wrote the paper. Molla Tefera: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Funder information
The laboratory experiments were conducted at Wolkite University and we acknowledged the University for helping us in the completion of this research. However, the research was not financially supported. Body weight for an adult = 67.0 for males and 62.3 kg for females, respectively Average daily consumption of sesame = 70 and 60 mg/person/day for males and females, respectively

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
All data used are included and acknowledged in the manuscript.

Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.toxrep.2022.09.009.