Determination of Fe, Mn, Cu, Zn and Cd after Heat Treatment in some vegetables and Green Bean (Phaseolus vulgaris L.) grown in the Middle Black Sea Region

Background: In this study, Fe, Mn, Cu, Zn and Cd were determined after heat treatment of spinach (Spinacia oleracea L.), leek (Allium porrum L.), green bean (Phaseolus vulgaris L.), lamb’s-quarters (Chenopodium album L.) and mad parsley (Oenanthe pimpinelloides L.) grown in Samsun the city, in the Middle Black Sea region. In this way, it has been aimed to contribute to the subject whether boiled liquors are consumable or should be thrown away. Methods: In our study, before heat treatment, vegetables and green bean (Phaseolus vulgaris L.) analyzed as raws material and after heat treatment separately as pomace and pot liquor. Chopped vegetables are washed, dried and then HNO3 and HClO4 are added and them wet ashing method was using. By taking fi ve samples from each vegetable and fruit, the concentration of Fe, Mn, Cu, Zn were determined with Flame AAS, but Cd was determined with voltammetry. This process was also applied to vegetable and green bean pomaces. The content of the heavy metals in pot liqour was determined by standard addition method. Results: The highest metal ion was iron in spinach (135.1, 181.6 mg kg-1) on the other hand the lowest metal content was cadmium in leek (0.007, 0.001 mg kg-1) in raw samples and pomace respectively. In pot liqour the highest metal concentration belonged to iron with a concentration of 22.9 mg kg-1 in spinach (Spinacia oleracea L.) and the lowest metal concentration belonged to cadmium with a concentration of 0.001 mg kg-1 in mad parsley (Oenanthe pimpinelloides L.). Conclusion: As a result of our experiments, although the industrial areas emit some heavy metals to the environment, the heavy metal content of studied vegetables and green bean (Phaseolus vulgaris L.) were not exceeded the literature and WHO values. Especially pot liquors of mad parsley (Chenopodium album L.), green bean (Phaseolus vulgaris L.) and lamb’s-quarters (Chenopodium album L.) must be thrown away. On the other hand, boiled pot liqour of spinach (Spinacia oleracea L.) and leek (Allium porrum L.) can be consumed. Research Article Determination of Fe, Mn, Cu, Zn and Cd after Heat Treatment in some vegetables and Green Bean (Phaseolus vulgaris L.) grown in the Middle Black Sea Region Seker Fatma Aygun*, Aysegul Yondemir Ince and Behice Yavuz Erdogan Sciences & Arts Faculty, Department of chemistry Kampus-Samsun-Turkey Dates: Received: 06 July, 2017; Accepted: 31 July, 2017; Published: 01 August, 2017 *Corresponding author: S Fatma Aygun, Sciences & Arts Faculty, Department of chemistry, KampusSamsunTurkey, Fax: (90) 362 4576081; E-mail:


Introduction
A human body needs minerals, vitamins and proteins in order to live in a healthy way. One of the routes of entry of metals into the living organisms is through plants. Vegetables and fruits naturally contain the necessary vitamins and minerals but at the same time they may contain toxic substances which can be carried in various ways from such as soil, air and water, those contaminated during industrial processing [1,2]. Plants contain minerals such as calcium, iron, manganese and other micronutrients. From these micronutrients copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) are not only constitutive elements with specifi c functions in plant growth, photosynthesis and respiration [3]. Also these elements are essential for living organisms at low concentrations. However, the high concentrations of even the essential metals are toxic and cause various metabolic abnormalities [1]. Today, heavy metals form a large part of the environmental pollution that threatens our health and have a toxic and carcinogenic effects.
Thus, vegetables can contain some toxic elements in addition to essential substances [4]. to high prevalence of upper gastrointestinal cancer cases [2]. The affected levels of vegetables due to heavy metallic pollution depend on factors such as traffi c congestion, the wind, the distance to the road and the rain.
Samsun has an important place in the residential and manufacturing areas and industries in the Black Sea province shows continuous improvement. This is also why our city pollution and industrialization threatens human health and thus food resources are also polluted.
Heavy metals are determined by various methods in the air, water, cigarette fi lter, cigarette smoke, soil and food. Many methods have been used for the determination of very low concentrations heavy metals, especially fl ame AAS, Graphite Furnace AAS, ICP, ICP-MS or voltammetry [5][6][7][8][9]. The most used of these are graphite furnace and fl ame atomic absorption spectrometry (GFAAS and FAAS) for the determination of trace heavy metal ions (Zn, Fe, Mn, Cu, etc.) due to low cost, easy usage, high sensitivity and short analyzing time [10][11]. Very low concentrations trace metals are also determined with voltammetry within various samples due to simplicity and low cost.
In the previous studies metals were determined in raw vegetables and fruits [12][13][14]. It is known that certain parts of the food stuff interwine to the pot liqour at high-temperatures. However, the portion of metal content transferred to the pot liqour is unknown. In this study, it has been aimed to contribute to the subject of whether the boiled liqours of the plants are to be or not which is a confusing subject for people.
In this study, the determination of Fe, Mn, Cu, Zn and Cd contents after heat treatment of spinach (Spinacia oleracea L.), leek (Allium porrum L.), green bean (Phaseolus vulgaris L.), lamb's-quarters (Chenopodium album L.) and mad parsley (Oenanthe pimpinelloides L.) grown in Samsun, in the Middle Black Sea region was aimed. FAAS was used to determine Fe, Mn, Cu and Zn. We preferred voltammetry for Cd quantifi cation because of its low concentration.

Preparation of the sample
Vegetables and green bean were studied as: raw samples, heat treated samples as pomace. Also the pot liquor of samples were analysed.
All samples obtained from different villages were washed with tap water, than deionized water to clean their damaged, dried leaves and dust, dirt, possible parasites and their eggs on them. Afterwards, samples were divided into small pieces with a plastic knife and dried in the oven at 90 o C for 24 h [15]. This process was also applied to heat treated vegetables and green bean. The concentrations of Cu, Mn, Fe, Zn in raw vegetable and green bean samples were determined by fl ame atomic absorption spectrometer (FAAS) after digestion by wet-ashing method. An aliquot of 1 g sample was digested in Kjeldahl fl ask. The wet-ashing method was applied as: a 50 mL mixture of acids HNO 3 : HClO 4 in the ratio of 8:2 (v/v) was added onto the raw vegetable sample and the fl ask was covered with a watch glass and stored at room temperature for an overnight.

Reagents
All reagents are in high purity and the metal salts used in the preparation of stock solution (1000 mg L -1 ) were supplied from Merck.

Standard solutions
The standard solutions were prepared by the diluting of stock solutions of metal salts (1000 mg L-1).

Apparatus and analysis conditions
In this study, UNICAM 929 Flame Atomic Absorption Gamry Reference 600 voltammeter was used to analyze the Cd content of the vegetable and lady's fi nger samples. The anodic stripping voltammetric experimental conditions were selected as: initial potential -1.0V, fi nal potential 0.0 V, pulse size 25 mV, frequency 25 Hz, accumulation time 60 s while using a hanging mercury drop electrode as working electrode. The supporting electrolyte was 0.5 M acetate solution at pH 4.5. Cadmium gave a single peak at -0.64 V versus Ag/AgCl. Quantifi cation of Cd was performed by using standard addition method. Limit of detection and limit of quantifi cation for Cd were calculated from the calibration curve.
For defi ning the limit of detection the for digestion process and analytical measurement with FAAS sample-free (blank) solutions were analayzed. The mean and the standard deviation of absorbance for 10 blank data were calculated. Three times of the standard deviation of blank was summed with the average of blank absorbance.
Ten times more of the standard deviation obtained in the determination of the detection limit was added to the average of blank absorbance, and Mn, Zn, Cu, Fe concentration corresponding to this value was calculated as the limit of determination.

Statistical analysis
For statistical analysis, one-way analysis of variance (ANOVA) test was applied to metal concentrations in raw vegetables and green bean, pomace and pot liquor with confi dence at level 95 %. Levene test was used to determine the homogeneity of data. The differences between groups were determined with the Duncan test. These analyses were performed using the program SPSS 15.0. The experiments were repeated 5 times in each plant with fi ve different plants and the total number of experiments was 25.

Results and Discussion
Plant samples used in the study were prepared by analytical pretreatment with wet ashing and their heavy metal contents were determined using fl ame atomic absorption spectrophotometer.
The accuracy of the method was determined by recovery studies. Two different vegetable samples were spiked with the satandart solutions inculuding Mn, Zn, Cu, Fe metals. Lamb'squarters and leek vegetable samples were used for recovery studies. Lamb's-quarters except iron are in the range of 90 -112%, because of this reason the method can be accepted as an accurate method. The iron recovery percentage was found to be 136. However, in the blank analysis, the limit of determination of iron was found to be 5.37 mg kg -. 1 . In this case the percent recovery is calculated as 123%. The recovery of metals values in the leek are in 86-119%. Spiked vegetables samples were determined with good recoveries. The limit of detection (LOD) and quantifi cation (LOQ) and recovery values for the analyzed metals are given in table 1,2. In our study, in raw samples the highest and the lowest metal concentrations were found in spinach the highest metal concentration belonged to iron (135.1 mg kg -1 ) and the lowest metal concentration belonged to cadmium (0.006 mg kg -1 ). Similarly, after heat treatment, the highest and lowest concentrations of metal belonged to iron and cadmium in spinach 181.6 mg kg -1 , 0.007 mg kg -1 , respectively (Table 3). In      (Table 7).
Iron is an important element for living things. It is used as crude content in steel industry and iron oxides are used in paint industry as pigments [16]. In this study, iron levels in green Copper is one of the essential elements for feeding and health of people. But if taken in abundance, copper can cause some health problems [24]. It is used widely in electricity, electronic industry and mining enrichment. Copper is left to the environment by means of drain water [16]. In the study, in raw vegetables, copper levels changed from 2.9 mg kg -1 to 5.2 mg kg -1 . It was observed that the lowest copper level was in Allium porrum L. and the highest copper level was in Chenopodium album L. We have been thinking that the reason why the concentration level of copper was high in Spinacia oleracea L.     and Chenopodium album L. probably resulted from the fact that the fi elds on which these plants grew were near the factories.

Metals Raw lamb's-quarters Lamb's-quarters pomace Lamb's-quarters pot liquor
The copper concentrations were found to be lower in our study in comparison to a study conducted in Kayseri (Turkey) [25] reported as 22.19 -76.5 mg kg -1 for urban and rural areas. Also Yusuf et al reported 25.08 -56.84 mg kg -1 copper in plants in a study carried out in Nigeria which had higher values than our study [15]. On the other hand, our data except for vine leaves (0.01 -5.67 mg kg -1 ) was found to be compatible with the study carried out in Manisa (Turkey) by Bagdatlioglu et al. [19]. In heated vegetables the copper levels were between 3.  [19]. However, Zn level reported as 136 mg kg -1 in the spinach carried out by Smolen and Sady (2012) [28] in Krakov (Poland) was found to be higher than the our values.
In heat-processed vegetables the zinc level changes from 4.8 mg kg -1 to 43.2 mg kg -1 and the lowest zinc concentration was in Allium porrum L. and the highest zinc concentration is in Spinacia oleracea L. In pot liquor zinc concentration was in the change of 5.9 mg kg -1 to17.6 mg kg -1 . After the boiling process the lowest concentration was in Oenanthe pimpinelloides L. and the highest concentration is in Phaseolus vulgaris L. According to the World Health Organization (WHO), the maximum Zn concentration should be 60 mg kg -1 [16]. In all the vegetables and the level of Zn was found to be lower than upper limits.
There is a statistically signifi cant difference in the levels of zinc vulgaris L. (green bean) should be thrown away but also the juice of green beans should not be eaten at all. However, if green bean is consumed higher than 250 g in a day, the limit of WHO exceed. WHO defi ned the maximum permissible Cu and Zn concentrations in vegetables as 40 mg kg -1 and 60 mg kg -1 , respectively. As for leeks, it can be suggested to be eaten raw because metals determined are below the limit value of WHO and with the boiling process the amount of metals are found less. But the amount necessary for the human body can be taken better by eating raw leek. As for Oenanthe pimpinelloies L., metal concentration increases by boiling. We're of the opinion that this plant, too, should be eaten raw without being cooked as salad like some people do. With regard to Chenopodium album L., it is suitable to be eaten by throwing away the boiled juice.
In Black Sea Region, frequntly, vegetables are consumed as fried after boiling and separating from the pot liquor. In this study, heavy metal contents of vegetables as raw and boiled were determined. Besides the heavy metals were also investigated in pot liquors. To our knowledge, there is no publishes data concerning the ratio of metals passing to the pot liquor from boiling vegetables. In our study its planned to enlighten whether pot liquor should be removed or not, which causes confusion in public.

Conclusion
In conclusion, heavy metals in vegetables analysed in our study are determined to be lower than limit values of WHO. When compared with other vegetables grown in the world, our results are in the range of literature values. When compared with the studies made in Turkey, usually the data of this study have been found higher. For this reason, our municipalities have to inspect factories in places where the iron concentration is high and check the water purifi cation and also the waste water treatment plants. Besides it is a must that the natural gas systems. And it is suggested that our people should throw away the boiled vegetable juice then consume as fried.