The Use of Natural Bee Products as Bioindicators of Environmental Pollution - The Detection of Heavy Metals

Introduction

Bees and their products have been suggested as biological indicators of environmental pollution in many studies. The pollen powder collected by honey bees from flowers is known as honey bee pollen ¹. Pollen is stored in the chambers of the hives by honeybees.  Bee pollen is a ball or pellet of pollen collected in the outdoors by worker honeybees and utilized as the hive’s major food supply. All of the additional nutrients required for honeybee development and growth are found in pollen. It is a wide-ranging plant product. Bees use it as a raw material to make bee bread. Pollen colours ranged from bright yellow to dark brown. Pollen grains can be as large as 2.5–250 m in diameter and vary in shape, colour, size, and weight depending on the plant species. Proteins, amino acids, carbohydrates, lipids, fatty acids, phenolic compounds, enzymes, and coenzymes, as well as vitamins and bio elements, were discovered in pollen powder ². Study on Pollen is important as it provides a “real” picture of the atmosphere; however, it is difficult to provide clear ideas about pollen and pollution interactions because multiple components are found mixed in various types of pollutants in different regions of the world, and finding a single experimental device for pollen-pollutant detection is currently difficult ³. Pollen powder from various floristic components has a composition of fifty percent total carbohydrates, 2-16 percent polysaccharides and dietary fibers, 6-28 percent proteins, 4-8 percent lipids, and 6 percent free amino acids ⁴. Because of the high concentrations of flavonoids and other phenolic components in bee pollen powder, it possesses anti-inflammatory, antiviral, antioxidant, antifungal, antibacterial, and immune-stimulating characteristics [1] ^5-6. Selected heavy metals that have been studied, or Potentially Toxic Elements (PTE), lead (Pb), cadmium (Cd), copper (Cu), zinc (Zn), chromium (Cr) is noticed in foods, and generally, their concentrations are higher than the approved limit ^7-8. The Food and Agriculture Organization (FAO) of Macedonia, as well as the World Health Organization (WHO), have both registered and have made the legal norms (Official Gazette of RM-No.102 / 2013) in favours of it ⁹. A high concentration of heavy metals can reason health problems. Pollen powder is one of the bee products that can be used as a bioindicator ¹⁰ because its final product contains a collection of several factors, soil, water, plants, air, etc. ^10-13

Experimental Methods

For experimentation purposes, we have ensured the following steps.

Sampling

Sampling of pollen

The pollen powder samples were collected from the bee apiaries from four different locations of the Pelagonija region, and conserved in the vacuum-packed bags at the temperature (<-40C). All samples are collected in the same season [from May 2020 to October 2020]. Half gram (0.5 gram) samples are taken in macerated in a porcelain plate for the test, and then added the HNO3 69%, MERCK Trace Pure (7ml+2ml H₂O₂), and Alkaloid. Left the sample mixture in the oven (CEM Mars 5 Digestion Oven) for 24 hours. The solutions are filtrated by (Munktell Filter – Qualitative Filter Papers) and put into plastic laboratory pumpkins of 25ml for the experiment ¹⁴.

Sampling of soil

From the same locality, soil samples were also collected for the detection of Potentially Toxic Elements (PTE), as well as an overview of the flora from the same regions.

The Region Selected for sampling

The region selected for the collection of experimental samples is ecologically polluted due to the different established industries. The region situated geographically at the latitudes of 40°50″ and 42°20″ N and longitudes of 20°27′ and 23°05″ E, and further, we subdivided it into two groups: two lowland areas near to river areas around the village of  Novaci (41°2′34.49″ N, 21°27′35.81″ E, 600 m a.s.l.), the village of  Makovo (41°7′4.91″ N, 21°36′31.6″ E, 620 m a.s.l.) and two mountain areas or highland areas named the village of Rapeš (41°6′9.73″ N, 21°38′50.18″ E, 802 m a.s.l.) and the village of Orle (41°8′53.82″ N, 21°36′48.28″ E,780 m a.s.l.).

Figure 1: The geographical map of the Republic of Macedonia (taken from Google Maps). Click here to View figure

The landmarks are such as the red spot is for the village Novaci and the white spot is for village Makovo (both are lowland areas) and village Orle is a black spot whereas village Rapeš blue (both are highland areas).

Instrumentation

The Potentially Toxic Elements (PTE) were examined by the application of atomic emission spectrometry with inductively coupled plasma, ICP-AES (VARIAN spectra AA240) applied an ultrasonic nebulizer CETAC (ICP/U-5000AT+) for better results and sensitivity. The Rosh–Drying oven DZLG-9023A, Soxhlet apparatus (Sigma-Aldrich Z556173), Kjel Digester K-449 and Buchi Scrubber K-415, Büchi Kjeldahl and Sistem Kjel Flex K-360, HPLC -МКС EN 14463:2010, and electronic balance were used the sample analysis.

Phytosociology

In the phytosociological literature, the maximum and permissible limit scale is used that is proposed by Braun Blanquet. This scale contains not only numerical grades but also descriptive, but it does not have a mathematical data processing application.

Experiments

The samples analysis was carried out by an accreditedlaboratory and test methods. The concentrations of the Potentially Toxic Elements (PTE) such as lead (Pb), cadmium (Cd), copper (Cu), zinc (Zn), chromium (Cr), were examined in the pollen samples, soil, and the agricultural products of the region with help of the instruments discussed above.

For the Physical and chemical composition of beepollen samples, 1 grof bee pollen is taken at 105⁰C, and firstly examined the ash content by using Rosh –Drying oven DZLG-9023A. For the test of the lipid concentration, two-gram (2g) pollen samples are taken at (>78°C) for 5 hours and determined with the help of Soxhlet apparatus (Sigma-Aldrich Z556173) automated method applied and using diethyl ether (80 ml) as a solvent. The concentration of nitrogen components was determent with the classic method by Kjeldal method, taking 1g of sample for the test. A KjelDigester K-449 and Buchi Scrubber K-415 are used for homogenization, and digestion a Büchi Kjeldahl and Sistem KjelFlex K-360 is used. For the determination of fructose and glucose in the sample powder HPLC spectrometer is used. The HPLC spectrometer used for the purpose is ISO Standards and МКС EN 14463:2010 by Agilent technologies G7129A/1260.

Data analysis

Data were analysed using packages software SPSS 19.0. Means and Standard deviation, one-way ANOVA test was used, the statistical significance was p< 0.05. The same data analysis package was used for bee products ^15-16.

Results and Discussions

It was assumed that bee powder is an ecologically pure product that can be a bioindicator [14]. of environmental pollution. The physical-chemical composition of bee pollen, the mean composition data of lipid, percentage of heavy metals in food grains and soil, of the target location, may confirm the above assumption. The current study compels us to accept the said assumption that the bee pollen powder can be used as a high degree of the eco-monitoring system with the targeted locations ¹⁷.

The results of phytocoenological structure obtained from the different target locations are presented in Table -1.

Table 1: Phytocoenological findings of the target locations

Sequence number of the recording	1	2	3	4	1							2							3
Locality	v. Novaci	v. Makovo	v. Orle	v. Rapeš	v. Novaci		v. Makovo		v. Orle		v. Rapeš	v. Novaci	v. Makovo		v. Orle		v. Rapeš		v. Novaci	v. Makovo		v. Orle	v. Rapeš
Надморска висина (m)	600	620	780	802	Degree of presence							Mean cover value							Frequency (%)
Exposure	E	E	E	E
Slope (0)	25	25	35	35
Coverage (%)	60	60	40	50
Surface image	100	100	100	100
Geological basis	clayey	clayey	clayey	clayey
*Sequence number of the recording	10	10	10	10
Medicago sativa- alfalfa	3.3	3.4	2.2	1.1	V	IV		II		I		37,5		37,5		17,5		5,0	100		80	40		20
Trifolium pratense- red clover	3.3	3.2	1.1	2.1	V	IV		I		II		37,5		37,5		5,0		17,5	100		80	20		40
Cirsium arvense – creeping thistle	3.2	1.3	1.1	1.1	V	IV		II		I		37,5		17,5		5,0		5,0	100		80	40		20
Clematis vitalba L. – traveller’s joy	3.3	2.3	2.1	1.2	IV	III		II		II		37,5		17,5		17,5		5,0	80		60	40		40
Campanula medium-canterbury bells	3.2	1.2	+	1.1	IV	III		I		II		37,5		17,5		0,1		5,0	80		60	20		40
Salvia nemorosa – balkan clary	2.2	1.2	1.3	3.1	IV	III		II		V		17,5		5,0		5,0		37,5	80		60	40		100
Heliantus annus – sunflower	3.4	1.1	+	+	IV	I		I		I		37,5		5,0		0,1		0,1	80		20	20		20
Medicago sativa – alfalfa	3.3	2.3	1.1	+	V	IV		II		I		37,5		17,5		5,0		0,1	100		80	40		20
Gossypium – cotton	1.2	1.2	+	+	III	II		I		I		5,0		5,0		0,1		0,1	60		40	20		20
Rubusidaeus L. – red raspberry	+	1.1	2.3	2.2	II	II		IV		V		0,1		5,0		17,5		17,5	40		40	80		100
Ulmis campetris L .- elm	1.2	1.1	3.3	3.4	III	III		IV		IV		5,0		5,0		37,5		37,5	60		60	80		80
Corylus colurna L. – hazel	1.1	1.2	3.4	2.3	II	II		IV		IV		5,0		5,0		3,5		17,5	40		40	80		80
Trifolium repenss L. – white clover	3.2	3.4	2.2	+	IV	IV		III		II		37,5		37,5		37,5		0,1	80		80	60		40
Tymus serpyllum – thyme	1.2	+	1.2	+	III	II		IV		V		5,0		0,1		5,0		0,1	60		40	80		100
Salvia verticullata L. – ring sage	1.3	1.3	+	+	III	III		II		II		5,0		5,0		0,1		0,1	60		60	40		40
Borago officinalis L. – borage	1.2	1.1	1.1	+	III	II		III		II		5,0		5,0		5,0		0,1	60		40	60		40
Rubus fruticosus L .- rubus	2.1	1.1	3.1	3.3	II	II		IV		IV		17,5		5,0		37,5		37,5	40		40	80		80
Rubus idaeus L. – raspberry	+	1.1	3.3	3.3	I	II		V		IV		0,1		5,0		37,5		37,5	20		40	100		80
Lotus corniculatus – bird’s-foot trefoil	2.3	1.2	1.1	+	II	II		II		II		17,5		5,0		5,0		0,1	40		40	40		40
Cucurbita pepo – pumpkin	2.4	1.2	r	r	IV	II		I		I		17,5		5,0		0		0	80		60	20		20
Stachys annua L. –wight  basil	1.1	1.3	1.1	1.1	II	III		II		II		5,0		5,0		5,0		5,0	40		60	40		40
Lytrum salicaria L. – purple loosestrife	1.1	1.2	+	+	II	III		I		II		5,0		5,0		0,1		0,1	40		60	20		40
Mentha longifolia – horse mint	2,4	+	1,1	2,3	III	I		II		III		17,5		0,1		5,0		17,5	60		20	40		60
Rosa canina L. – dog-rose	3.3	3.3	3.4	3.4	III	III		IV		V		37,5		37,5		37,5		37,5	60		60	80		100
Robinia pseudoacacia L. – black locust	2.3	2.2	1.1	+	IV	IV		II		I		17,5		17,5		5,0		0,1	80		80	40		20
Lavandula officinalis spp. – lavender	2.1	1.1	3.1	2.1	II	III		IV		III		17,5		5,0		37,5		17,5	40		60	80		60
Humulus lupulus – hops	1.1	1.2	2.4	2.4	II	II		IV		IV		5,0		5,0		17,5		17,5	40		40	80		80
Castanea sativa – sweet chestnut	2.1	2.1	1.1	1.1	IV	IV		I		I		17,5		17,5		5,0		5,0	80		80	20		20
Iris germanica L. – bearded iris	1.1	1.1	1.1	1.1	I	II		III		III		5,0		5,0		5,0		5,0	20		40	60		60
Campanula roduntifolia L. – harebell	+	1.1	1.1	r	I	II		II		I		0,1		5,0		5,0		0	20		40	40		20
Melisa officinalis L.- lemon balm	1.2	1.1	+	+	III	III		I		I		5,0		5,0		0,1		0,1	60		60	20		20
Mentha spicata – diosmos	1.3	1.2	1.3	1.3	II	II		III		III		5,0		5,0		5,0		5,0	40		40	60		60
Prunus domestica L. – plum	+	+	2.3	1.2	I	I		IV		II		0,1		0,1		17,5		5,0	20		20	80		40
Salix alba – white willow	1.1	1.1	1.1	1.1	I	III		I		I		5,0		5,0		5,0		5,0	20		60	20		20
Prunus cerasifera- cherry plum	1.1	r	1.1	+	II	II		III		I		5,0		0		5,0		5,0	40		20	60		20
Polygonium persicaria – redshank	1.3	1.1	r	+	III	II		I		I		5,0		5,0		0		0,1	60		40	20		20
Saturea montana l.- winter savory	1.2	1.1	2.2	2.1	III	II		IV		IV		5,0		5,0		17,5		17,5	60		40	80		80
Solidago virgaurea L. – european goldenrod	1.2	1.3	1.2	+	III	III		II		I		5,0		5,0		5,0		0,1	60		60	40		20
Salvia officinalis L. -common sage	1.3	1.1	1.1	r	IV	V		III		II		5,0		5,0		5,0		0	80		100	60		40
Calluna vulgaris – hull	2.1	2.1	1.2	1.1	III	II		IV		IV		17,5		17,5		5,0		5,0	60		40	80		80
Satureja montana -winter savory	1.1	+	1.1	1.2	II	II		III		III		5,0		0,1		5,0		5,0	40		40	60		60
Castanea sativa – sweet chestnut	r	r	1.1	1.2	I	I		III		II		0		0		5,0		5,0	20		20	60		40
Rubus fruticosus L. – blackberry	1.2	1.1	2.2	3.4	I	II		IV		V		5,0		5,0		17,5		37,5	20		40	80		100
Rubus idaeus L. – red raspberry	1.1	1.2	3.2	3.3	I	II		IV		V		5,0		5,0		37,5		37,5	20		40	80		100
Tymus serpulum – wild thyme	1.1	1.2	3.4	3.4	II	II		IV		V		5,0		5,0		37,5		37,5	40		40	80		100
Juglans nigra L. – black walnut.	+	r	3.3	3.4	I	I		IV		IV		0,1		0		37,5		37,5	20		20	80		80
Hipericum perforatum – perforate St John’s-wort	+	r	1.1	3.3	I	I		II		IV		0,1		0		5,0		37,5	20		20	40		80
Bruns spruce – bruns	1.1	+	+	1.1	I	I		II		III		5,0		0,1		5,0		5,0	20		20	40		60
Pinus silvestris – wight pinus	1.1	+	1.1	2.3	I	I		II		IV		5,0		0,1		5,0		17,5	20		20	40		80
Alnus glutinosa – black alder	+	+	+	1.3	I	I		I		III		0,1		0,1		0,1		5,0	20		20	20		60

Table 2: Physical-chemical composition of the collected bee pollen from the target locations

Component	Novaci	Makovo	Orle	Rapeš
Lipid	3.49±0.67	4.44±1.25	7.31±0.52	5.38±1.14
Protein	18.10±1.61	21.65±0.22	24.47±1.42	26.45±3.02
Fructose	11.73±0.28	12.70±0.61	11.60±0.92	14.53±0.61
Glucose	8.42±0.06	8.49±0.21	12.56±1.12	14.17±1.65
Moisture	22.46±0.09	23.36±0.12	25.34±0.16	26.31±0.31
Ash	2.18±0.04	2.31±0.01	2.35±0.05	2.48±0.06

Note. Data are given as means ± SD (medians); * p< 0.05, ** p < 0.01, *** p < 0.001; c -Novaci, d – Makovo, c- Orle, d -Rapeš

The results show that the finding of Lipids is in order of Orle 7.31 mg/kg> Rapeš, 5.38 mg/kg> Makovo 4.44 mg/kg> and Novaci 3.49 mg/kg. Orle has the highest among all 7.31mg/kg.

Table 3: The concentration of heavy metals Cu, Pb, Cd, Cr, and Zn (mg /kg) in the bee powder samples from the target locations.

Four different Areas are targeted
Element	v.Novaci	v.Makovo	v.Orle	v.Rapeš
Cu	2.01±0,42 (2.04)	1.60±0,24 (1.50)	1.62±0,37 (1.50)	1.85±1,31 (1.62)
Pb	0.78±0.31 (0.72)	1.10±0.80 (1.03)	1.21±0.69 (0.90)	1.07±0.55 (0.82)
Cd	0.5±1.12 (0,60)	0.12±0.40 (0.70)	0.02±0,10 (0.78)	0.03±0.25 (0.64)
Cr	1.14±1.12 (0.78)	0.80±0.40 (0.70)	0.60±0.10 (0.60)	0.55±0.25 (0.64)
Zn	38.50±9,24 (37.58)	34.05±7.70 (32.17)	29.68±4.58 (30.14)	27.84±3,61 (28.33)

Note. Data are given as means ± SD (medians); * p< 0.05, ** p < 0.01, ***  p < 0.001; c – Novaci, d – Makovo, c-  Orle, d – Rapeš.

The Potentially Toxic Elements (PTE) such as lead (Pb), cadmium (Cd), copper (Cu), zinc (Zn), chromium (Cr) are analyzed quantitatively of the soil samples from the four different targeted locations of study ⁸. 

Table 4: The results of the Concentration Potentially Toxic of elements Cu, Pb, Cd, Cr and Zn (mg/kg) of the soil samples from the four different targeted locations.

Element	Area
	Novaci	Makovo	Orle		Rapeš
Cu***	15.41 ± 2.48 c*d*** (16.40)	26.64 ± 1.52 acd*** (26.85)		12.08  ± 1.85 d** (12.10)	8.95  ±  0.74 (9.05)
Pb***	17.15 ± 1.17 cd*** (17.55)	14.01 ± 0.84 cd*** (14.15)		11.62  ± 1.02 d*** (11.45)	8.26  ±  0.71 (8.20)
Cd***	0.25 ± 0.05 (0.27)	0.60 ± 0.09 cd*** (0.58)		0.36  ± 0.09 a* (0.36)	0.42  ±  0.05 a*** (0.41)
Cr***	38.28 ± 0.83 bd***c* (38.45)	34.47 ± 2.14 d*** (33.80)		36.02 ± 1.78 d*** (36.80)	27.67  ± 1.46 (27.80)
Zn***	29.57 ±  2.01 (29.15)	53.26 ± 2.02 ac*** (53.55)		43.52 ± 1.62 a*** (43.40)	72.03 ± 1.55 abc** (72.20)

Note. Data are given as means ± SD (medians); * – p< 0.05, ** – p < 0.01, *** – p < 0.001; c – Novaci, d – Makovo, c – Orle, d – Rapeš

However, in principle, these elements are part of human food, but they should be under the authorized limit. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO), as well as the government of Macedonia (Official Gazette of RM-No.102/2013), have fixed the optimum limit of human food and its product.

According to the regulation of WHO (World Health Organization), the optimum limit of Cu should be 20.0 mg/kg, but the above-obtained results showed a very low concentration of Cu only 2.01 mg/kg.  Even much lower than the results obtained by Harmanescu (2007), according to his findings from different areas are as Agricultural area <4.47, The Industrial area <4.58, and in the desert area <5.34 mg/kg.

The study found the concentration of Cd in bee powder is above the optimum limit in the low land areas, the data shows as in v. Novaci the average concentration of Cd is 0.5mg/kg, and in v. Makovo Cd is 0.12mg/kg.

The Cd concentration in the soil from the different locations, mentioned by the same said author was as in the order of 0.5mg/kg in Novaci > 0.1mg/kg in Makovo > 0.03mg/kg in Rapeš > 0.02mg/kg in Orle. Similar results were presented by Sattler D.A.G, and S. Sandikci Altunatmaz as well.

The amount of Cr was found in Novaci 1.14mg/kg > in Orle 0.80mg/kg> in Makovo 0.60mg/kg > in Rapeš 0.55mg/kg respectively. But as compared to the value found by the Kostić and et.al ⁴ is much lower, is between 0.170 mg/kg to 0.465 mg/kg.

The mean amount of Zn in bee powder was found in order of from minimum to maximum as 27.84 mg/kg in Rapeš, <29.68 mg/kg in Orle < 34.05 mg/kg in Makovo <38.50 mg/kg in Novaci. According to WHO (World Health Organization), the optimum limit of Zn is specified as 60.0 mg/kg. Similar results were reported by Hamza and co-authors of Jordan as well.

The concentration of Pb in bee powder were found very low in all four target locations, the results are found as 1.21mg/kg in Orle > 0.78mg/kg in Novaci > 0.07mg/kg in Rapeš >0.10mg/kg in Makovo.

It has been confirmed from the current study that the concentration of heavy metals in Pollen’s powder is depended on the type of plant, soil, and the local geographical structure.  Pollen powder is one of the bee products that can be used as a bioindicator, which includes a compilation of several stages, soil, water, plants, and air. A higher or lower concentration of the above investigated heavy metals can cause human health problems ^{9, 18-20, 11-13, 21-29}

Conclusions

The focus of the present work is to identify heavy metals like Pb, Cr, Cd, Zn, and Cu in bee pollen from various parts of the Pelagonija region. Bee pollen powder is a substance collected by bees from flowers. Bees and their products have been suggested as biological indicators of environmental pollution in many studies. We can be concluded from the collected data that heavy metal levels are associated with the type of plant, soil, and geographical conditions. The concentrations of Cd and Cr elements were found to be higher than expected. Steps should be taken to protect the ecological environment, the submitted results are evidence of the alarming situation of pollution in the existing region.

Acknowledgment

We gratefully acknowledge the Faculty of Natural Sciences and Mathematics / Institute of Chemistry – Skopje. The authors also acknowledge and special thanks to Faculty of Biotechnical Sciences – Bitola, University St. Kliment Ohridski, Partizanska n.n, Bitola, R.N. Macedonia, who supported in our work.

Conflict of Interest

The author declares that there is no conflict of interest.

Funding Sources

None

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