Mineral element and heavy metal ( cadmium , lead and arsenic ) levels of bee pollen in Turkey

In this study, mineral element and heavy metal levels in bee pollen produced in various regions of Turkey (20 samples) and imported bee pollen (4 samples), were determined using an Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) device. Mineral and heavy metal levels of bee pollens produced in Turkey were determined to be within the following ranges (minimum-maximum, µg/gpollen); Cr (chromium): 0.124-1.595, Cu (copper): 3.728-14.994, Fe (iron): 28.603-725.360, Mg (magnesium): 271.107-1278.340, Mn (manganese): 8.151-201.036, Se (selenium): 0.593-5.085, Zn (zinc): 14.832-39.079, B (boron): 0.592-61.33, Ni (nickel): 0.002-1.761, Si (silicon): 0.082-10.500, K (potassium): 992.107-2894.154, Ca (calcium): 491.853-1472.102, P (phosphorus): 795.899-5246.992 and Cd (cadmium): 0.006-0.181, Pb (lead): 0.000-0.479, As (arsenic): 0.006-1.035. As a result of this study, it can be concluded that bee pollens produced in Turkey may be a good source of mineral regarding the trace elements Se, Mn, Fe, Cr and Cu.


Introduction
Bee pollen is a nutrient collected from various flowering plants by bees and stored and brought to the beehive in their back legs (Dagaroglu, 2004).Bees use the pollen as the primary source of nutrition for their nourishment.It is constituted of the bee's oral secretions and some nectar.The main difference between bee pollen and flower pollen is that bee pollen is diluted with nectar, saliva and honey (Campos et al., 2008;De-Melo et al., 2015).Therefore, the mineral level in flower pollen is higher than mineral levels in bee pollen (Stanciu et al., 2011).
Alongside its nutritional value, bee pollen may also contain toxic elements (Bogdanov, 2006).Due to environmental contamination; heavy metals, radioactive isotopes, organic contaminants, pesticides, antibiotics and pathogenic microorganisms may be present in the air, soil, water and plants, and spread to bee pollen via these routes.Among heavy metals, Pb and Cd may have industrial origin as well as being present in the pesticide compound (Bogdanov, 2006;Campos et al., 2008).Arsenic may be present in many environmental sources such as; rocks, soil, water, dust and air.It has been reported that air and water play an important role in As contamination (Morgano et al., 2010).
It has been stated that, bee and bee products may be used as a bioindicator in determining environmental pollution and presence of heavy metals (Nispet et al., 2013).To collect pollen, bees roam an area with an average diameter of 7-13 km and approximately 80-100 flowers.Therefore, bees in the hive interact with approximately one million flowers (Dagaroglu, 2004;Nispet et al., 2013).For optimum pollen quality, pollen must be collected from areas at least 3 km away from the source of contamination (Bogdanov, 2006;Campos et al., 2008).
Turkey has an ecosystem suitable for apiculture.In Turkey, apiculture products are gaining importance due to an increasing demand for natural products as well as a long-standing apiculture

Mineral element and heavy metal (cadmium, lead and arsenic) levels of bee pollen in Turkey
Sema Sandikci ALTUNATMAZ 1 *, Duygu TARHAN 2 , Filiz AKSU 1 , Umıt Bora BARUTÇU 2 , Mehmet Erman DR 3 a tradition.The aim of this study is to determine mineral element and heavy metal (Pb, Cd, As) levels of bee pollens in Turkey.

Sample collection
Bee pollen samples were collected from areas of intensive beekeeping.The samples were dried and prepared, suitable for consumption and their origins were determined via producers and sellers.Element measurements for pre-determined mineral and heavy metals were carried out in a total of 24 bee pollens.20 of these samples were obtained from different regions of Turkey representing; the Black Sea, Central Anatolia, Eastern Anatolia, Marmara, The Mediterranean and Aegean regions.Four imported bee pollens, originating from Canada, China and Santiago and sold in Turkey, were included in the study (Table 1).

Preparation of samples for measurement
Pollen samples were placed into tared tubes, weighed using a precision balance and the weights recorded.2 mL Nitric acid (HND 3 ) was added to pollen samples placed into a heat-resistant graduated tube in a 200 °C drying oven and left to dissolve.The pollen and nitric acid mixture was left to cool at room temperature, after which 1 mL Perchloric acid (HClD 4 ) was added and the mixture was returned to the 200 °C oven.The compound was subjected to wet ashing in the oven, vortexed and distilled water added, totaling its volume to 12 mL.It was vortexed once more and prepared for element analysis in the ICP-DES device.

Element measurement
Suitable wavelengths of 267.716, 324.754, 259.940, 285.213, 257.610, 196.090, 206.200, 189.042, 249.773, 221.647, 251.611, 228.802, 220.353, 766.490, 317.933 and 177.495 nm were used for the analysis of Cr, Cu, Fe, Mg, Mn, Se, Zn, As, B, Ni, Si, Cd, Pb, K, Ca, P elements respectively in the ICP-DES device (Thermo iCAP 6000 series).Samples were subjected to wet ashing and prepared for measurement.Four samples from each pollen type were prepared and mean values of the element analysis results were calculated.The stock solution was prepared from standard solutions containing Cr, Cu, Fe, Mg, Mn, Se, Zn, As, B, Ni, Si, Cd, Pb, K, Ca ve P (Chem-Lab NV) and distilled water was used for the blank solution.The calibration graph was obtained from the ICP-DES device using blank and standard solutions and element concentration measurements of the prepared pollen samples were carried out.The measurements were repeated 4 times and mean values were determined.

Discussion
Within the scope of this research, similarities or varying degrees of differences between mineral levels and the regions from which the bee pollens originated can be seen in all the bee pollens.In 3 bee pollens obtained from plants of different origin (colour differences) in the city of Çanakkale, while some mineral values were very close, differences between the Fe (55.333,168.449),Mn (11.605,54.628) and Ca (493.998,954.493)values were interesting.In this study, it was concluded that similarities as well as differences may be present in mineral levels of bee pollens obtained from the same city.These differences or similarities in mineral levels are related to factors such as; flower varieties from which the bee pollen was collected, climate conditions, geography, environmental conditions, genetic composition of the plant species, agricultural procedures (fertilization and agricultural spraying) and apicultural processes (Bonhevi & Jorda, 1997;Dagaroglu, 2004;Dinkov & Stratev, 2016;Feas et al., 2012;Harmanescu et al., 2007;Nispet et al., 2013).It has been stated that, mineral levels are related to plant type rather than the soil and geographical situation.It has been expressed that, some of the minerals in bee pollen are of nectar origin (Kostic et al., 2015).Turkey has regional (Bursa, Çanakkale etc) Boron reserves.The fact that Boron levels are generally high in bee pollen obtained from regions and surrounding areas where Boron reserves are high suggests that soil type is also important.Changes in mineral levels of bee pollens in relation to many factors may cause difficulties regarding a standard product.It is thought that, the control of human related factors such as, environmental pollution and apicultural procedures, may prevent differences, particularly with respect to heavy metals.In fact, in one study, Dinkov & Stratev (2016) expressed that there were no major differences between regions regarding Cd and Pb levels in bee pollens.It has also been stressed that, by the bee's feeding on heavy metal-containing nutrients, such as sugar, heavy metal contamination may also be present in bee products (Dinkov & Stratev, 2016).
In this study, amounts of mineral elements in one gram are arranged from greatest to smallest as; P, K, Ca, Mg, Fe, Mn, Zn, B, Cu, Se, Si, Cr, Ni.In some studies, the most common mineral element is reported to be K (Campos et al., 2008;Morgano et al., 2012;Szczesna, 2007).Despite changes in amounts depending on the type of mineral element in bee pollen, it has been reported that the mineral content of bee pollen is K 60%, Mg 20%, Na and Ca 10% (Campos et al., 2008).In 27 bee pollens collected in Poland, South Korea and China, mineral elements were reported to be arranged as K>Mg>Na>Ca>Fe>Mn>Zn>Cu (Szczesna, 2007).In another study, this line up was K > P > Ca > Mg > Na > Fe > Mn > Zn > Cu (Morgano, et al., 2012).In 25 bee pollens investigated in a study in Serbia, K: 2462-4236, Ca: 856-2032, Mg: 503-964, Fe: 39.56-141.29,Zn: 28.76-75.92,Mn: 13.52-92.23mg/kg values were determined and the bee pollens were reported to be very rich with respect to Fe and Zn, while most commonly found were K, Ca and Mg (Kostic et al., 2015).When compared to the results of this study, in bee pollens produced in Turkey, Ca, K and Zn values were found to be lower, while Fe, Mg and Mn values were higher.The mineral element P was found to be more common in bee pollens produced in Turkey.
In a study carried out in Poland, South Korea and China (27 bee pollens, mg/kg), the following high values were determined; Poland, Ca: 542-1080 and Cu: 5.6-23.9,China Mn: 13.2-429.8,Mg: 1126-1893, K: 4247-5976, South Korea, Zn: 23.7-60.7 and Fe: 74.3-365.9(Szczesna, 2007).In bee pollens produced in Turkey, while the Ca value is higher, Cu, K, Fe, Mg, Mn and Zn values were found to be lower.In this study, Mn, Mg and K levels in bee pollens from China were seen to be lower than those in the present research (Tables 4, 5).In another study carried out in China, levels of P: 5946, K: 5324, Ca: 2068, Mg: 1449, Fe: 119.3, Mn: 70.23, Zn: 45.10, Cu: 17.35 mg/kg were determined in bee pollens (Yang et al., 2013).In comparison with the mentioned study, the Fe level determined in the present study was seen to be higher, while P, K, Ca, Mg, Mn, Cu and Zn levels were lower, and that the Zn level in bee pollen from China was similar.The Fe level determined in the present study was found to be higher than the 26.1-92.0,3.61-8.41,1.24-2.49,1.07-1.69µg/g values determined by Formicki et al. (2013) in regions of Southern Poland and the Fe: 40.14-88.7 ppm value determined by Harmanescu et al. (2007).In 8 different bee pollens collected from various regions in Romania, Cu: 5.51-12.07ppm, Zn: 33.93-54.35ppm and Mn: 12.78-98.25ppm was determined (Harmanescu et al., 2007).It can be seen that, in the mineral levels in bee pollens produced in Turkey, the Cu value is similar, the Mn value is higher and the Zn value is lower (Table 4).Concentration of the mineral substances were evaluated in terms of mg/kg where values were; Fe: 11.1-80.3,Ca: 252.9-850,Zn: 18.8-81.1,K: 2485-6411, Cu: 4.1-15.7,Mg: 273-815.7,P: 294.7-854.4,Mn: 7.4-8.3and 20 bee pollens were evaluated (Bonhevi & Jorda, 1997).When compared to the present study, Fe, Ca, Cu, Mg, P and Mn values were found to be higher, while Zn and K values were found to be lower (Tables 2-5).
Mineral substance mean values (µg/g pollen ) for bee pollens produced in Turkey were determined to be; K: 1945.870, Ca: 862.435, Mg: 669.769, Fe: 203.165, Cu: 10.418, Mn: 29.33, Zn: 29.153, Se: 2.560, P: 2659.73.In 154 bee pollen samples collected from different regions in Brazil, values were; K: 5089, Ca: 2215, Mg: 1179, Fe: 82, Cu: 9.6, Mn: 64, Zn: 46.7, Se 0.51, P: 4828 mg/kg.It has been reported that K, P, Ca and Mg are major mineral elements in pollen and that Mn and Se are rich sources for Brazilian pollen, while Cu, P and Zn may be a source (Morgano et al., 2012).When mean values were compared, Cu, Fe, Mn and Se values were found to be higher in the present study, while Zn, K, Mg, Ca and P values were seen to be lower.It can be said that, bee pollens produced in Turkey may be a rich source of Se and Mn minerals, while also a source of Ca.In another study carried out in 5 bee pollens in Brazil, mineral substance mean values (mg/100g) were determined as; Ca: 68.9, Fe: 13.4,Cu: 0.87, Cr: 1.0, Mn: 4.0, P: 480, Zn: 3.5.In view of the results obtained in the present study, Ca, Fe and Cu levels were high and Cr, Mn, P and Zn levels were low.According to legislative regulations in Brazil, depending on its mineral content, food is described as "source food" if RDI values >15% and "high content food" if RDI >30%.In the study carried out for bee pollens in Brazil, bee pollen was classed as "high content food" depending on its essential elements Fe, Cr and Mn (Sattler et al., 2016).In the light of the data obtained in this study, bee pollen can be evaluated as "high content food" with respect to Se (46%), Fe (36%), Cr (36%), Mn (32%) and "source food" regarding Cu (28%).
In this study, Ni level was determined to be 0.002-1.761µg/g pollen .In their studies, Morgano et al. (2010) and Harmanescu et al. (2007) determined Ni level to be < 0.10-1.13mg/kg and 0-0.72 ppm, respectively.The results found in the present study were higher than these values, however, lower than the result found by Formicki et al. (2013) (Ni: 3.61-8.41µg/g).According to Brazilian legislation, Maximum Tolerable Limit (MTL) has been determined as 5 mg/kg for Ni.In glucose metabolism, Cr plays a role together with insulin (Harmanescu et al., 2007;Mertz, 1981).According to Polish standards, Cr must not exceed 0.14 mg/kg (Roman, 2007;Roman et al., 2016).In one study, Cr was seen to be the most problematic element and 50% of the samples exceeded the limit value (0.14 mg/kg) (Roman et al., 2016).In the 20 bee pollens produced in Turkey, examined in this study, Cr value was determined to be below the 0.14 mg/kg value in only one sample.In the research carried out in Southwest Poland, Cr was determined as 0.138 mg/kg (Roman et al., 2016).In Brazil, Sattler et al. (2016) determined the recommended daily intake (RDI) for Cr as 35 µg.In this case, the Cr level found in this study supplies 36% of daily requirement (Sattler et al., 2016).For adults, the safe and sufficient Cr value has also been reported as 50-200 µg (Mertz, 1981).Differences in national standards and the lack of international standards cause difficulties in the assessment of bee pollens with respect to minerals.The mineral elements B and Si analysed in this study, had not been widely researched in previous bee pollen studies.
The heavy metal levels (µg/g pollen ) of the analysed bee pollens were; Pb: 0.000-0.479,Cd: 0.006-0.181,As: 0.006-1.035,and Pb: 0.000-0.339,Cd: 0.044-0.081,As: 0.008-1.054 in imported bee pollens.It has been reported that, the heavy metal content in bee pollen should not exceed the amounts of; Cd: 0.03 mg/kg, Pb: 0.5 mg/kg, As: 0.5 mg/kg (Campos et al., 2008).In the light of this criteria, when all bee pollens in this study were assessed, it was observed that; As level had exceeded the limit in 8 bee pollens, Cd level had met these values in only 2 Turkish bee pollens and that Pb value had remained under the limit in all bee pollens.According to Polish standards, the following limits must not be exceeded; Cd: 0.05, Pb: 0.50 and As: 0.20 mg/kg (Roman, 2007;Roman et al., 2016).With regard to this standard, As value exceeded the limit in 13 and Cd value in 15 bee pollens in the present study.
Considering heavy metals can spread with environmental pollution via soil, water and air, environmental steps must be taken to prevent it reaching dangerous levels for health.

Conclusion
This study suggests that bee pollens produced in Turkey may be assessed as a good source of mineral regarding the trace elements Se, Mn, Fe, Cr and Cu, in particular.When compared to other studies, the Zn level was found to be lower.The lack of international limits to determine bee pollen quality, as well as differences in national standards, causes difficulties in assessment.In recent times, bee pollen has become a frontline product due to increased interest and consumer demand for natural products.Both its use in apitherapy and its components renders it important.Considering the fact that bee pollen may be consumed by children, elderly people and pregnant women, both producers and consumers should be cautious with respect to heavy.

Table 1 .
Regional distribution of bee pollen samples.

Table 2 .
Mineral element and heavy metal mean values (µg/g pollen ) of bee pollens produced in Turkey.

Table 3 .
K, Mg, Ca, P mean values (µg/g pollen ) of bee pollens produced in Turkey.

Table 5 .
K, Mg, Ca and P mean values (µg/g pollen ) of imported bee pollens.

Table 4 .
Mineral element and heavy metal mean values (µg/g pollen ) of imported bee pollens.