Fatty acid content of honeycombs depending on the level of technogenic loading on the environment

The content of heavy metals, non-etherified and anionic forms of fatty acids, and also fatty acids of common lipids in honeycomb cells, obtained from hives placed in areas with high, medium and low levels of technogenic loading is investigated. The hives were located at the educational apiary of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z. Gzhytskyj (area with very heavy traffic and industrial activity) and in private beekeeping farms in the town Vynnyky and the village Chyzhyky, Pustomyty district, Lviv region (areas with lower intensity of traffic and industrial activity). Our research has shown that the newly-built honeycomb cells obtained from hives placed in the territory with medium and low levels of technogenic loading contain a lower quantity of such heavy metals as iron, zinc, cuprum, chromium, nickel, lead and cadmium. The total amount of non-etherified fatty acids with even and odd number of carbon atoms in the chain, monounsaturated fatty acids of n-7 and n-9 families and polyunsaturated fatty acids of n-3 and n-6 families was higher in these honeycombs. However, the total concentration of anionic forms of fatty acids with even and odd number of carbon atoms in the chain, monounsaturated fatty acids of n-7 and n-9 families and polyunsaturated fatty acids of n-3 and n-6 families was lower in these honeycombs. Due to the above-mentioned monounsaturated and polyunsaturated fatty acids the amount of fatty acids of common lipids rises. It leads to increase in the antibacterial and antifungal properties of honeycomb cells and reduction of fragility of their walls. The greatest changes in concentration of heavy metals, non-etherified and anionic form of fatty acids, and also fatty acids of common lipids occur in the newly built honeycomb cells in the territory with low technogenic loading on the environment.


Introduction
Intensive development of the chemical industry and agriculture, environmental degradation and pollution by heavy metals causes growth of toxic lesions in humans and animals.One feature of heavy metals is that in small amounts almost all of them are necessary for plants and organisms (Kulbachko et al., 2011;Brygadyrenko andIvanyshyn, 2014, 2015;Tsvetkova et al., 2016).However, when the permissible amount of these metals is exceeded this leads to serious diseases.Some heavy metals (mercury, cadmium, lead) are distinguished by a special feature of toxicity (Vymazal and Březinová, 2016).The development of industry, agriculture, energy and transport, intensive mining all lead to an increase of the concentrations of heavy metals in the air, water, soil, plants and plant pollen (Conti and Botrè, 2001;Wang Haiyan and Arne, 2003;Pashayan, 2006;Vikram Reddy еt al., 2013).Thus, heavy metals are rapidly accumulated in the tissues of honey bees and honeycombs (Bogdanov, 2006;Perugini еt al., 2011).
On the other hand, fatty acids are a vital component of food for honeybees (Saranchuk, 2013).In particular, the fatty acid composition of pollen affects the productive and reproductive characteristics of honeybees (Loidl and Crailsheim, 2001;Tan еt al., 2015).In addition, fatty acids are the basic building blocks of honeycombs (Buchwald еt al., 2009).There they are mostly linked to long-chain alcohols.Fatty acids in bee combs also occur in free (nonetherified) and bound condition with heavy metals (anionic) (Коnоnskyy, 2006).Thus, the fatty acid and mineral composition of honeycombs is an indicator of the ecological environment.
Having considered the given above, theoretical and practical interest lies in the study of heavy metals, anionic and nonetherified fatty acids and fatty common lipid content in honeycombs, depending on the level of environmental impact.The aim of the study was to find out the contents of heavy metals, anionic and nonetherified fatty acids and common fatty lipids in honeycombs, depending on the level of the technogenic load on the environment.

Material and methods
The level of environmental impact was determined by the content of heavy metals (iron, zinc, copper, chromium, nickel, lead, arsenic and cadmium) in the pollen of dandelions (Taraxacum officinale Wigg.).Samples of pollen from dandelions and freshly built honeycombs for laboratory tests were taken in apiaries located in areas with different intensities of traffic and industry.The hives studied were located at the training apiary of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z.Gzhytsky (an area with very heavy traffic and industrial activity) and private beekeeping farms in Vynnyky town and Chyzhykiv village in Pustomiti district Lviv region (areas with lower intensity of traffic and industry).The private beekeeping farms in Vynnyky town and Chyzhykiv village are located at a distance of 2-3 and 5-6 km from the training apiary of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z.Gzhytsky.Moreover, both the training apiary of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z.Gzhytsky and the private beekeeping farms in Vynnyky and Chyzhykiv of Pustomiti district Lviv region are located along the Lviv -Ternopil highway.
In each of the above-mentioned areas samples of the pollen of dandelions and newly built honeycombs were selected, and in each apiary the samples of dandelion pollen and newly built honeycombs were taken from three hives.To identify the species of the dandelion pollen an identification study was performed using the computer programs Lucia (Laboratory Colour Image Analysis) and Pollen Data Bank.
In the selected samples of the bee pollen heavy metals were found and measured.In the selected samples of newly built honeycombs the concentrations of heavy metals, anionic and nonetherified fatty acids and fatty acids of total lipids was measured.The content of heavy metals in the studied biological material was measured on an atomic absorption spectrophotometer C-115 PC (Khavezov and Tsalev, 1983).The concentrations of anionic and nonetherified fatty acids and fatty acids of total lipids in the studied biological material were measured by the gas-liquid chromatography (Rivis and Fedoruk, 2010).
Mathematical processing of research results was conducted statistically using the software package Statistica 6.0.The results of the average values considered statistically significant at P < 0.05, P < 0.01 and P < 0.001 (ANOVA).
We found out that the level of the technologenic impact on the environment affects the overall content of nonetherified forms of fatty acids in honeybee combs.Thus, the total content of nonetherified forms of fatty acids in honeybee combs which were taken from the beehives located in the areas with medium and low levels of technogenic loading is higher than in the honeycombs which were taken from beehives located in the areas with high anthropogenic loading (Table 2).The highest content of nonetherified forms of fatty acids was found in bee combs received from the beehives located in areas of low technogenic loading.
The higher number of nonetherified forms of unsaturated fatty acids in honeycombs obtained from the beehives located in the areas with medium and low technogenic loading, in comparison with the honeycombs obtained from the beehives located in the areas with high anthropogenic impact can be explained mainly due to a higher content of monounsaturated fatty acids families n-7 and n-9 fatty acids and families of n-3 and n-6 in their composition.The ratio of nonetherified forms of polyunsaturated fatty acids of n-3 family to nonetherified form of polyunsaturated fatty acids of n-6 family is lower (Table 2).It is obvious that the above-mentioned processes take place in the wax glands of honey bees.
Nonetherified forms of caprylic, caprin, lauric, oleic, linoleic and linolenic acids demonstrate the most significant antibacterial and antifungal activity (Bogdanov, 2003;Bogdanov еt al., 2003).We observed that the level of the technogenic loading on the environment has a significant impact on the total concentration of nonetherified forms of caprylic, capric, lauric, oleic, linoleic and linolenic acids in honeycombs.Thus, the total content of nonetherified forms of the above-mentioned fatty acids in the honeycombs obtained from the beehives located in areas with medium and low technogenic loading compared to the honeycombs obtained from the beehives located in the areas with high anthropogenic impact increases.
Nonetherified forms of monounsaturated (oleic and palmitooleyic) and especially polyunsaturated (linoleic and linolenic) fatty acids show maximum antibacterial and antifungal activity (Pauguel еt al., 1993;Manning, 2001;Bogdanov еt al., 2003).We found out that the level of the technogenic loading on the environment has a significant impact on the overall content of nonetherified forms of monounsaturated and polyunsaturated fatty acids in the newly built honeycombs.So, the total content of nonetherified forms of monounsaturated and polyunsaturated fatty acids in the honeycombs obtained from the beehives located in the areas with medium and low technological loading in comparison with the honeycombs obtained from the beehives located in the areas with high anthropogenic impact is higher.
Nonetherified forms of long-chain fatty acids have the ability to bind heavy metals, especially divalent.At the same time, nonetherified forms of long-chain fatty acids (18 or more carbon atoms in the chain) have the highest ability to bind heavy metals (Jenkins and Palmquist, 1982;Bogdanov еt al., 2005).Binding nonetherified forms of long-chain fatty acids with heavy metals takes place in the wax glands of honeybees (Saranchuk, 2013).As the result of this process anionic forms of fatty acids are formed.We found out that the level of technogenic loading on the environment affects the total content of anionic forms of fatty acids in newly built honeycombs.Thus, the total content of anionic forms of fatty acids in the newly built honeycombs obtained from the beehives located in areas with medium and low technogenic loading is lower than in the honeycombs obtained from the beehives placed in the areas with a high level of technogenic (Table 3).The lower number of anionic fatty acids in the newly built honeycombs obtained from the beehives located in areas with medium and low technogenic loading in comparison with honeycombs obtained from the beehives located in the areas with high anthropogenic impact is explained by the lower content of saturated fatty acids with even and odd number of carbon atoms in the chain, monounsaturated fatty acids families n-7 and n-9 fatty acids and families of n-3 and n-6 in the composition of those honeycombs.In this case, the ratio of anionic forms of the n-3 family of polyunsaturated fatty acids and anionic forms the n-6 family of polyunsaturated fatty acids is lower (Table 3).
The process of binding nonetherified forms of fatty acids with heavy metals in honeybee wax glands and the formation of anionic forms of fatty acids can be seen reflected in the content of fatty acids of total lipids in honeycombs.The latter are represented mainly by fatty acids that are found in terrestrial connection with long-chain alcohols.
The lower ratio of saturated fatty acids to unsaturated fatty acids of total lipids in the newly built honeycombs obtained from the beehives located in the areas with medium and low technogenic loading in comparison with the honeycombs obtained from the beehives located in the areas with high anthropogenic impact may indicate a decrease in the fragility of their walls.
Antibacterial and antifungal activity is inherent to many fatty acids -caprylic, capric, lauric, oleic, linoleic and linolenic (Lee еt al., 2001;Bogdanov еt al., 2003).Thus, the content of the above-mentioned acids of total lipids the newly built honeycombs obtained from the beehives located in the areas with medium and low technogenic loading compared to the honeycombs obtained from the beehives located in the areas with high anthropogenic impact is higher.The above-mentioned fact may indicate that antibacterial and antifungal activity of the newly built honeycombs is higher in the areas with lower levels of pollution.
The maximum antibacterial and antifungal activity is observed in monounsaturated (oleic and palmitooleic) and, especially, in polyunsaturated (linoleic and linolenic) fatty acids of total lipids (Bogdanov еt al., 2003).We found out that environmental conditions have a significant environmental impact on the overall content of monounsaturated and polyunsaturated fatty acids of total lipids in the newly built honeycombs.Thus, the contents of the above-mentioned fatty acids of total lipids in the honeycombs obtained from the beehives located in the areas with medium and low technogenic loading in comparison with the honeycombs from the beehives placed in the areas with high anthropogenic impact is higher.Long-chain fatty acids (18 or more carbon atoms in the chain) in the honeycombs can bind heavy mineral elements, especially divalent (Jenkins and Palmquist, 1982;Suárez еt al., 1996;Bogdanov еt al., 2005).We observed that the ecological conditions of the environment influence the content of the above-mentioned fatty acids of total lipids in the newly built honeycombs.Therefore, the content of long-chain fatty acids of total lipids in the honeycombs obtained from the beehives located in the areas with medium and low technogenic loading compared to the honeycombs obtained from the beehives placed in the areas with high anthropogenic impact is higher.
As a result, the newly built honeycombs obtained from the beehives located in the areas with middle-and low technogenic loading contain fewer heavy metals.At the same time, in the honeycombs obtained from the beehives located in the areas with medium and low technogenic loading the content of anionic forms of fatty acids is lower, but the content of nonetherified acids is higher.These combs also contain a higher amount of fatty acids of total lipids.The newly built honeycombs in the areas with low technogenic loading undergo the largest changes in concentration of heavy metals, nonetherified and anionic forms of fatty acids and fatty acids of total lipids.

Conclusions
The newly built honeycombs obtained from the beehives located in the areas with middle-and low technogenic loading contain smaller amounts of iron, zinc, copper, chromium, nickel, lead and cadmium.
The newly built honeycombs obtained from the beehives located in the areas with medium and low technogenic loading havea lower content of anionic forms of saturated fatty acids with even and odd number of carbon atoms in the chain, monounsaturated fatty acids of n-7 and n-9 families and polyunsaturated fatty acids of n-3 and n-6 families.
The newly built honeycombs obtained from the beehives located in the areas with medium and low technogenic loading contain more nonetherified forms of monounsaturated fatty acids of n-7 and n-9 families, and polyunsaturated fatty acids of n-3 and n-6 families.Due to the above-mentioned monounsaturated and polyunsaturated fatty acids the number of fatty acids of total lipids in them is higher.
The largest changes in concentration of heavy metals, nonetherified and anionic forms of fatty acids and fatty acids of total lipids take place in the newly built honeycombs in the areas with low technogenic loading.
Prospects for further research.It is necessary to study the content of heavy metals and fatty acids in bee pollen and ambrosia.