Due to its multifunctional and complex role in the honey bee colony functioning and health (construction material allowing food storage, brood rearing, thermoregulation, mediation in chemical and mechanical communication, substrate for pathogens, toxins and waste), Apis mellifera beeswax has been widely studied over the last five decades. This is supported by a comprehensive set of scientific reports covering different aspects of beeswax research. In this article, we present an overview of the methods for studying chemical, biological, constructional, and quality aspects of beeswax. We provide a detailed description of the methods for investigating wax scales, comb construction and growth pattern, cell properties, chemical composition of beeswax using different analytical tools, as well as the analytical procedures for provenancing beeswax and beeswax-derived compounds based on the hydrogen isotope ratio (IRMS). Along with classical physico-chemical and sensory analysis, we describe more precise and accurate methods for detection of adulterants in beeswax (GC-MS and FTIR-ATR). Moreover, we present methods for studying the influence of beeswax (comb foundation) adulteration on comb construction. Analytical protocols for determining the pesticide residues using different chromatographic and spectroscopic techniques are also described. As beeswax is an agent of high risk for the transmission of bee diseases, we present methods for detection of pathogens in beeswax. To ensure the reproducibility of experiments and results, we present best practice approaches and detailed protocols for all methods described, as well as their advantages and disadvantages.

S u m m a r yConiferous honeydew honey, mainly Abies alba was characterised. Samples chosen for the study had organoleptic traits characteristic for the variety: greenish, opalescence tone of brown colour, mild, sweet flavour with pleasant, slightly resinous aftertaste and aroma as well as electrical conductivity over 0.95 mS/cm. To define composition and physicochemical parameters of the variety, contents of water and total sugars were determined. In addition various carbohydrates were identified and their contents were assessed as well. These were: fructose, glucose, sucrose, maltose, turanose, trehalose, isomaltose, malezitose. Other examined parameters related to honey quality were: free acidity, pH, the content of 5-hydroxymetylofurfural (HMF), the main amino acid -proline, and the activity of α-amylase enzyme (Diastase Number). The following properties were proven to be characteristic for this variety: high electrical conductivity with the average value of 1.14 mS/cm, ranging from 0.96 to 1.32 mS/cm; content of monosaccharides lower by few percent in relation to other honey varieties (from 58.2 to 67.4 g/100 g; on average 62.0 g/100 g) and a higher content of disaccharides and trisaccharide -melezitose. The presence of this sugar confirms that a considerable part of the honey was produced from honeydew. The average value of melezitose was 3.2 g/100 g, ranging from 0.9 to 5.9 g/100 g. Also, the results of the pH measurements were slightly higher than in other honey varieties (from 4.23 to 4.99; on average 4.63). The colour value in mm Pfund ranged from 74 to 105, with the average of 93.

a b s t r a c t here we describe a method of hydrocarbon (alkanes, alkenes, dienes) identification and quantitative determination of linear saturated hydrocarbons (n-alkanes) in beeswax using gas chromatography with a mass detector technique (gc-ms). beeswax hydrocarbons were isolated using a solid-phase extraction (spe) technique with neutral aluminum oxide (alumina -n, 1000 mg, 6 mL), then were separated on a non-polar gas chromatography column Zb-5ht inferno (20 m×0.18 mm×0.18 µm). qquantitative analysis of n-alkanes was conducted by the method of internal standard with squalane used as the internal standard. the basic parameters of validation (linearity and working range, limit of determination, repeatability and reproducibility, recovery) were determined. for all of the identified compounds, satisfactory (≥0.997) coefficients of correlation in the working ranges of the method (from 0.005 to 5.0 g/100 g) were obtained. the elaborated method was characterized by satisfactory repeatability and within-laboratory reproducibility. the average coefficients of variation for the total n-alkanes did not exceed 2% under conditions of repeatability or 4% under conditions of reproducibility. the recovery for individual n-alkanes was above 94%; for their total content, it was 100.5%. in beeswax originating from apis mellifera, n-alkanes containing from 20 to 35 carbon atoms in their molecules were determined. the total content of these alkanes was between 9.08 g and 10.86 g/100 g (on average, 9.81 g/100 g). additionally, apart from the saturated hydrocarbons, unsaturated hydrocarbons and dienes were identified.

A b s t r a c t To detect beeswax adulteration with hydrocarbons of alien origin (e.g. paraffi n), gas chromatography with mass detector (GC-MS) technique was used. The method has been verifi ed here on beeswax samples with different addition (3, 5, 10, 30, and 50%) of paraffi n and validated under the conditions of repeatability and within -laboratory reproducibility. The addition of paraffi n to beeswax can already be detected on the basis of an analysis of the chromatograms. The intensity of individual alkane peaks increased with the increase of the amount of paraffi n added to the beeswax. This increase was the mostly visible for the alkanes with even numbers of carbon atoms in the molecule: C 24 H 50 , C 26 H 54 , C 28 H 58 , C 30 H 62 , C 32 H 66 , and C 34 H 70 . These observations have also been proven by quantitative analysis performed using the internal standard method. Adding paraffi n to beeswax resulted in an increase in the total contents of n-alkanes as well as individual alkanes, and in particular, of the even-numbered alkanes. The addition of paraffi n to beeswax also resulted in the appearance of alkanes containing over 35 carbon atoms in the molecule, which were not detected in beeswax. The method for determination of beeswax hydrocarbons with the GC-MS technique is characterised by satisfactory repeatability and within-laboratory reproducibility. This method can be used for the detection of beeswax adulteration with hydrocarbons of alien origin (e.g. paraffi n).Keywords: adulteration, beeswax, GC-MS, hydrocarbons, n-alkanes, paraffi n.Research Institute of Horticulture, Apiculture Department, Puławy, Poland INTRODUDCTIONDue to the insuffi cient domestic production of beeswax and the lack of obligatory legal regulations concerning beeswax quality, instances of its adulteration are quite frequent. The addition of alien substances such as paraffi n, decreases beeswax quality as well as the quality of the foundation made of such adulterated beeswax. As a consequence, it may no longer be possible to use this product in beekeeping. It may also not be possible to use such a product in some branches of industry, for example, the cosmetic, pharmaceutical, and food industry. The sensory assessment and physico-chemical characterisation are only approximate and do not allow to detect the adulteration of beeswax unambiguously. Beeswax which has paraffi n added, becomes chinawhite and pliable when kneaded with the fi ngers. What is more, the addition of paraffi n causes the beeswax to become smooth and shiny where rubbed (Curyło, 1983). As we mentioned previously (Waś et al., 2014a), a lot of methods so far used for the determination of hydrocarbons in beeswax and for the detection of beeswax adulteration were based on the physico-chemical parameters. These parameters include: melting point, density, solubility, and saponifi cation, acid and ester numbers, and iodine index (Tulloch, 1973;Serra Bonvehi et al., 1989;Serra Bonvehi, 1990;Vit et al., 1992;Poncini et al., 1993). Some interrelations were found be...

A b s t r a c t The hydrocarbon composition of beeswax secreted by Apis mellifera was characterised. In the studies, analyses were made of virgin beeswax (obtained from light combs, socalled "wild-built combs") that was collected at different dates, and beeswax obtained from dark combs ("brood combs"). A qualitative analysis did not show any differences in the hydrocarbon composition of beeswax originating from light and dark coloured combs. The same hydrocarbons (n-alkanes, alkenes, and dienes) were identified in virgin beeswax and beeswax collected from brood combs. However, the studies showed differences in the content of n-alkanes in the beeswax obtained from light and dark coloured combs. In comparison to the virgin beeswax, the beeswax obtained from dark combs had higher content of the total n-alkanes, higher total contents of even-numbered alkanes and odd-numbered alkanes, and higher contents of certain alkanes. Furthermore, it has been found that the hydrocarbon composition of beeswax did not depend on the collection period.