Beeswax Samples Research Articles

Variations in lead, cadmium, arsenic, and mercury concentrations during honeybee wax processing using casting technology

Beeswax foundations are a necessary material in intensive modern beekeeping. Heavy metals can accumulate in these foundations for decades, as it is a common beekeeping practice to recycle wax. Beeswax samples were analysed using GFAAS for As, Cd, Pb, and Hg concentrations during the production of beeswax foundations using casting technology with a prolonged cooling and sedimentation phase. Significant differences were determined in the concentrations of As, Cd, Pb (p<0.01, all), and Hg (p<0.05) between the three levels of wax in a double-walled steel casting container and comb foundations (CF) during the processing stage. Concentrations (mg kg-1) of the examined metals in comb foundations as the final product ranged as follows: As 0.01-0.88; Cd 1.26-3.55; Pb 82.5-171, and Hg 0.29-1.46. All examined element concentrations demonstrated similar distribution and ratio in different layers, ranging from the lowest concentrations in layers from which wax material is used for comb foundations as the final product, to the highest concentrations in sedimented layer, which represents waste. The obtained results suggest that the described method could effectively eliminate a significant amount of heavy metals from the initial material used for the production of new beeswax foundations.

Efficiency of GC-MS method in detection of beeswax adulterated with paraffin

Abstract The efficiency of the gas chromatography - mass detector (GC-MS) technique for the detection of beeswax adulterated with paraffin, was evaluated. For this purpose, beeswax samples with paraffin additions (3, 5, 10, 30, 50%) were analysed. Since not enough is known about paraffin compositions, and since it is difficult to detect paraffin in beeswax, the aim of our research was also to compare the hydrocarbon composition of different types of paraffin. The analysis showed that the types of paraffin available on the market, differ qualitatively and quantitatively as far as their hydrocarbon compositions are concerned. In all kinds of paraffin, we found homologous series of n-alkanes that were much longer than those in beeswax. In beeswax, the amount of added paraffin that is possible to detect, differs and depends on the kind of paraffin used for adulteration. In this study, the minimum estimated percent that was detected using the GC-MS technique, was 3%. The adulteration is indicated by the presence of hydrocarbons containing over 35 carbon atoms in the molecule, and by the higher contents of n-alkanes (C20H42 - C35H72), in comparison to the concentration of these compounds determined in pure beeswax. We also presented the results of the quality control of commercial beeswax. Based on our results, it can be stated that beeswax adulteration is currently a problem.

Analysis of Physico-Chemical Properties of Beeswax Produced in Bale Natural Forest, South-Eastern Ethiopia

The study was conducted to evaluate physico-chemical properties of beeswax from Bale natural forest Southeastern of Ethiopia in 2014/15. For analysis fifteen (15) crude beeswax samples each of 1 to 3 kg were obtained from three locations considering the different agro-ecologies of Bale and compared against to the national and international standards. The physico-chemical characters evaluated were: melting point, specific gravity, refractive index, volatile matter, acid value, saponification value, ester value, and acid to ester ratio. Although there were some significant variation (P &lt;0.05) between the different agro-ecologies, all samples collected were found in the range of the national and international standard limits. The means results of beeswax sample collected were Melting Point (62.39±0.12), Acid Value (22.33±0.39), Ester Value (75.58±0.84), Ester to Acid Ratio (3.38±0.07), Saponification Value (98.04±0.86), Specific gravity (0.9598±0.001), Refractive Index (1.4426±0.0002) and Volatile Matter (0.3335±0.027). Results obtained in this study, indicate that beeswax obtained from Bale show excellent quality characters according to the national and international standards. Hence, it is advisable to utilize the potentials for export market with better improved technology interventions.

Determination of sulfonamides in beeswax by liquid chromatography coupled to tandem mass spectrometry.

The manuscript presents the development of a new method for the quantification of 16 sulfonamides in beeswax. Different sample preparation techniques were tested and modified to maximise the recovery of the target analytes and minimise the amount of coeluted impurities under conditions that provide reproducible results. The proposed method consisted of melting and dilution of beeswax in a mixture of n-hexane and isopropanol followed by extraction with 2% acetic acid. The extract was cleaned up by solid-phase extraction using strong cation exchange phase. Determination of the sulfonamides was achieved by liquid chromatography coupled to tandem mass spectrometry with the use of a pentafluorophenyl analytical column and applying a gradient elution with acetonitrile and 0.01% acetic acid as mobile phases. The limits of detection and limits of quantification ranged from 1 to 2μg/kg and from 2 to 5μg/kg, respectively. The recoveries varied between 65.2% and 117.8% while coefficient of variation of the method was less than 24.2% under intermediate precision conditions. Finally, the method was applied to the analysis of real samples of beeswax from beekeepers and commercial foundations manufacturers.

A 10 year survey of acaricide residues in beeswax analysed in Italy.

The aim of this work was to provide an overview of the prevalence and level of acaricides in beeswax used in Italy in the past 10 years by analysing 1319 beeswax samples processed by the certified laboratory of the Italian Bee Research Institute. The proportion of samples positive to at least one active ingredient decreased between 2005 and 2009 (from 69 to 32%) and then increased again between 2009 and 2014 (from 32 to 91%). This trend is in agreement with reports from beekeepers that the use of synthetic acaricides decreased in the second half of the past decade and increased after the beginning of the colony losses phenomenon. The active ingredient with the greatest overall proportion of positive samples was coumaphos (49%), followed by fluvalinate (38%) and chlorphenvinphos (25%). The indicator for amitraz, 2,4-dimethylphenylformamide (DMPF), was detected in a very small proportion of samples (6%), while residues of cymiazole were never found. In more than half of the analysed samples, residues of at least one active ingredient were detected. The mean levels of residues of all the considered active ingredients in the positive samples may represent a source of accumulation in beeswax and pose risks to honey bee health. © 2015 Society of Chemical Industry.

Analysis of Intact Glucosinolates in Beeswax by Liquid Chromatography Tandem Mass Spectrometry

A new method was developed to determine 12 intact glucosinolates (glucoiberin, glucoraphanin, glucoerucin, gluconapin, glucotropaeolin, glucobrassicin, gluconasturtiin, glucoalyssin, 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, sinigrin) in beeswax, which is an authorized food additive in the European Union, by means of liquid chromatography tandem mass spectrometry with electrospray ionization. An efficient extraction procedure was proposed (average analyte recoveries were between 88 and 101 %); this involved the dissolution of melted beeswax with hexane, a solvent extraction using a mixture of ethanol and water (80:20, v/v), and a concentration step in a rotary evaporator. Chromatography was performed on a C18 analytical column with a mobile phase of formic acid in water (0.5 %, v/v) and formic acid in acetonitrile (0.5 %, v/v), at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification values were below 7 and 23 μg/kg, respectively. The proposed method was applied to analyze beeswax samples collected from organic apiaries.

Application of Gas Chromatography with the Mass Detector (GC-MS) Technique for Detection of Beeswax Adulteration with Paraffin

Abstract To detect beeswax adulteration with hydrocarbons of alien origin (e.g. paraffin), gas chromatography with mass detector (GC-MS) technique was used. The method has been verified here on beeswax samples with different addition (3, 5, 10, 30, and 50%) of paraffin and validated under the conditions of repeatability and within - laboratory reproducibility. The addition of paraffin 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 paraffin added to the beeswax. This increase was the mostly visible for the alkanes with even numbers of carbon atoms in the molecule: C24H50, C26H54, C28H58, C30H62, C32H66, and C34H70. These observations have also been proven by quantitative analysis performed using the internal standard method. Adding paraffin 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 paraffin 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. paraffin).

Physical characterization of wax/oil crystalline networks.

The objective of this research was to evaluate the physical properties of different types of wax/oil systems. Olive (OO), corn (CO), soybean (SBO), sunflower (SFO), safflower (SAFO), and canola (CAO) oils were mixed with sunflower oil wax (SFOW), paraffin wax (PW), and beeswax (BW) at different concentrations (1% to 10%). Results from this study show that the physical properties of wax/oil systems is affected not only by the concentration and type of wax used, but also by the type of oil used. In general, wax/oil systems formulated with SFOW generated crystalline networks with high enthalpies (1 to 22 J/g) and high G' values (2 to 6 × 10(6) Pa) compared with the values obtained for BW and PW. SFOW crystalline networks were characterized by needle-like crystals independently of the wax concentrations and type of oil used. BW crystalline networks, however, were characterized by different crystal morphologies (needle-like or spherulites) depending on the wax concentration and type of oil used. PW samples were characterized by a crystalline network formed by needle- and platelet-like crystals. Enthalpy values of BW and PW samples were similar (0.3 to 20 J/g), but BW samples resulted in significantly higher (P < 0.05) G' values in the 5% and 10% samples with values of 3.9 × 10(6) and 6.1 × 10(5) Pa for 10% BW and PW, respectively.

Trace-level determination of pyrethroid, neonicotinoid and carboxamide pesticides in beeswax using dispersive solid-phase extraction followed by ultra-high-performance liquid chromatography-tandem mass spectrometry

The aim of the work was to develop an analytical procedure able to quantify traces of 13 neonicotinoids and pyrethroids as well as carboxamide in beeswax at low levels (ng g−1) to evaluate the contamination. For this purpose, an efficient sample preparation procedure was developed based on solid–liquid extraction using dispersive diatomaceous earth and acetonitrile. This step was followed by a selective and sensitive analysis based on ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (ESI-MS/MS). This analytical procedure was validated based on International Conference on Harmonization guidelines. The limits of quantification ranged from 1 ng g−1 (thiamethoxam, clothianidin, imidacloprid, acetamiprid, thiacloprid and boscalid) to 40 ng g−1 (lambda-cyhalothrin). The method was then successfully applied to 60 samples of beeswax collected in several areas of France. The presence of thiacloprid, boscalid, imidacloprid and deltamethrin in beeswax was confirmed. The most frequently quantified pesticide was boscalid.

Development and validation of a liquid chromatography with mass spectrometry method to determine resveratrol and piceid isomers in beeswax.

This paper represents the first report of a liquid chromatography coupled to electrospray ionization mass spectrometry method for simultaneously analyzing resveratrol and piceid isomers (cis and trans) in beeswax. An efficient extraction procedure has been proposed (average analyte recoveries were between 89 and 95%); this involved a solid-liquid extraction using a mixture of ethanol and water (80:20, v/v) and a concentration step in a rotary evaporator. The separation of all the compounds was achieved using a C18 column and a mobile phase composed of ammonium formate 0.03 M in water and acetonitrile in gradient elution mode at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification ranged from 1.0 to 1.7 and 3.5 to 5.5 μg/kg, respectively. Finally, the proposed method was applied to analyze beeswax samples collected from experimental and organic apiaries.

A large-scale field study examining effects of exposure to clothianidin seed-treated canola on honey bee colony health, development, and overwintering success.

In summer 2012, we initiated a large-scale field experiment in southern Ontario, Canada, to determine whether exposure to clothianidin seed-treated canola (oil seed rape) has any adverse impacts on honey bees. Colonies were placed in clothianidin seed-treated or control canola fields during bloom, and thereafter were moved to an apiary with no surrounding crops grown from seeds treated with neonicotinoids. Colony weight gain, honey production, pest incidence, bee mortality, number of adults, and amount of sealed brood were assessed in each colony throughout summer and autumn. Samples of honey, beeswax, pollen, and nectar were regularly collected, and samples were analyzed for clothianidin residues. Several of these endpoints were also measured in spring 2013. Overall, colonies were vigorous during and after the exposure period, and we found no effects of exposure to clothianidin seed-treated canola on any endpoint measures. Bees foraged heavily on the test fields during peak bloom and residue analysis indicated that honey bees were exposed to low levels (0.5–2 ppb) of clothianidin in pollen. Low levels of clothianidin were detected in a few pollen samples collected toward the end of the bloom from control hives, illustrating the difficulty of conducting a perfectly controlled field study with free-ranging honey bees in agricultural landscapes. Overwintering success did not differ significantly between treatment and control hives, and was similar to overwintering colony loss rates reported for the winter of 2012–2013 for beekeepers in Ontario and Canada. Our results suggest that exposure to canola grown from seed treated with clothianidin poses low risk to honey bees.

Effect of high-intensity ultrasound and cooling rate on the crystallization behavior of beeswax in edible oils.

The objective of this study was to evaluate the effect of wax concentration (0.5 and 1%), cooling rate (0.1, 1, and 10 °C/min), and high-intensity ultrasound (HIU) on the crystallization behavior of beeswax (BW) in six different edible oils. Samples were crystallized at 25 °C with and without HIU. Crystal sizes and morphologies and melting profiles were measured by microscopy and differential scanning calorimetry, respectively, after 7 days of incubation. Higher wax concentrations resulted in faster crystallization and more turbidity. Phase separation was observed due to crystals' sedimentation when samples were crystallized at slow cooling rates. Results showed that HIU induced the crystallization of 0.5% BW samples and delayed phase separation in sunflower, olive, soybean, and corn oils. Similar effects were observed in 1% samples where HIU delayed phase separation in canola, soybean, olive, and safflower oils.

Determination of spinosad at trace levels in bee pollen and beeswax with solid-liquid extraction and LC-ESI-MS

This paper reports the use of a new LC method with a fused-core analytical column coupled to ESI-MS to determine residues of the biopesticide spinosad in bee pollen and beeswax. The method analyzes the active ingredients, spinosyns A and D, with a simple and efficient sample treatment (recovery between 90 and 105%) consisting of a solid-liquid extraction with acetone (bee pollen) or acetonitrile (beeswax). The method was validated in terms of selectivity, LOD, LOQ, linearity, and precision. The LOD and LOQ values ranged between 0.1-0.2 and 0.4-0.7 μg/kg, respectively. Moreover, the precision obtained within the linear concentration range (LOQ 500 μg/kg) was satisfactory (RSD lower than 5%). Finally, the proposed method was applied to analyze bee pollen and beeswax samples collected from apiaries located close to fruit orchards in two Spanish regions.

Quantitative Determination of Trisiloxane Surfactants in Beehive Environments Based on Liquid Chromatography Coupled to Mass Spectrometry

Organosilicone surfactants are increasingly being applied to agricultural agro-ecosystems as spray adjuvants, and were recently shown to impact the learning ability of honey bees. Here we developed a method for analyzing three trisiloxane surfactants (single polyethoxylate (EO) chain and end-capped with methyl, acetyl, or hydroxyl groups; TSS-CH3, TSS-COCH3, or TSS-H) in beehive matrices based on liquid chromatography coupled to mass spectrometry (LC-MS) and the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach from less than 2 g of honey, pollen, or beeswax. Recoveries for each oligomer (2-13 EO) were between 66 and 112% in all matrices. Average method detection limits (MDL) were 0.53, 0.60, 0.56 ng/g in honey, 0.63, 0.81, 0.78 ng/g in pollen, and 0.51, 0.69, 0.63 ng/g in beeswax. Five honey, 10 pollen, and 10 beeswax samples were analyzed. Trisiloxane surfactants were detected in every beeswax and 60% of the pollen samples. Total trisiloxane surfactant concentrations were up to 390 and 39 ng/g in wax and pollen. The described method is proved suitable for analyzing trisiloxane surfactants in beehive samples. The presence of trisiloxane surfactants in North American beehives calls for renewed effort to investigate the consequence of these adjuvants to bee health and the ongoing global bee decline.

Determination of seven neonicotinoid insecticides in beeswax by liquid chromatography coupled to electrospray-mass spectrometry using a fused-core column

A new method has been developed to measure seven neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) in beeswax using liquid chromatography (LC) coupled to electrospray ionization mass spectrometry (ESI-MS) detection. Beeswax was melted and diluted in an n-hexane/isopropanol (8:2, v/v) mixture. After this, liquid extraction with water was performed followed by a clean-up on diatomaceous material based cartridges. The compounds were eluted with acetone, and the resulting solution was evaporated until dry and reconstituted with a mixture of water and acetonitrile 50:50 (v/v). The separation of all compounds was achieved in less than 15min using a C18 reverse-phase fused-core column (Kinetex® C18, 150mm×4.6mm i.d.) and a mobile phase composed of a mixture of 0.1% formic acid in water and acetonitrile in gradient elution mode at 0.5mL/min. This method was fully validated in terms of selectivity, linearity, precision and recovery. Low limits of detection and quantification could be achieved for all analytes ranging from 0.4 to 2.3μg/kg, and from 1.5 to 7.0μg/kg, respectively. Finally, the proposed method was applied to an analysis of neonicotinoid residues in beeswax samples from apiaries located close to fruit orchards.

Studies on the Aging Characteristics in Different Parts of Beeswax-treated Duplicates during Humidified Artificial Aging

In this study, to understand the aging factor and mechanism in different partitions of the beeswax-treated volumes, the duplicated beeswax-treated volume was artificially aged at <TEX>$80^{\circ}C$</TEX> of temperature and 65% of relative humidity and then a physical and optical properties of an aged volumes was analyzed. Also, the degraded components of the beeswax samples isolated from different partitions of aged volumes was measured using a gas chromatography/mass spectroscopy (GC/MS). In results, the surface of beeswax-treated volume which is primarily affected by a main aging factors such as light, oxygen, moisture was more deteriorated than the inside of that volume. However, unlike inside of the book volume which was made from paper, the inside of beeswax-treated volume wax was also considerably deteriorated. The inside of the beeswax-treated volume is largely unaffected by the oxygen and humidity during aging due to the water repellency and the air permeation resistance of beeswax. Therefore, it is confirmed that aging factors and mechanisms in the inside of the volume are different from thats of the outside of the volume. This fact was also verified by the results of GC/MS analysis of an beeswax samples which was sampled from different partitions of aged volumes. As result as GC/MS analysis of the beeswax extracted from the outside of the aged volume, the low molecular compounds with a carbon length of <TEX>$C_9-C_{20}$</TEX> (fatty acid, etc) were increased and the compounds with a carbon length of above <TEX>$C_{34}$</TEX> (ester, etc) were also increased. But the compounds with a chain length of <TEX>$C_{21}-C_{36}$</TEX> (hydrocarbon, alcohol, etc) were decreased. In case of the aged beeswax of inside, the low molecular compounds with a carbon length of <TEX>$C_9-C_{20}$</TEX> (fatty acid, etc) and the compounds with a chain length of <TEX>$C_{21}-C_{36}$</TEX> (hydrocarbon, alcohol, etc) were increased. While, the compounds with a carbon length of above <TEX>$C_{34}$</TEX> (ester, etc) were decreased.

A novel, direct, reagent-free method for the detection of beeswax adulteration by single-reflection attenuated total reflectance mid-infrared spectroscopy

In this work, a novel, direct, reagent-free method for the detection of beeswax adulteration by paraffin, microcrystalline wax, tallow and stearic acid using single-reflection attenuated total reflectance mid-infrared spectroscopy was developed. The use of the absorbance ratios of I1739cm−1/I2852cm−1, I1714cm−1/I2852cm−1 and I1739cm−1/I1714cm−1 allows a minimum of 5% paraffin/microcrystalline wax and tallow adulteration and 0.5% stearic acid adulteration of beeswax to be detected. The upper and lower critical limits for beeswax authenticity were established from the analysis of virgin beeswax and were validated by independent analysis of real sheet and comb beeswax samples using high-temperature gas chromatography with flame-ionization detection. In addition to its simplicity with respect to sample handling, the amount of sample and the time needed are far less than those required in previously described methods, which are based on chemical analysis and chromatographic techniques. These advantages result in time and cost savings, an increase in the number of samples that can be analyzed, and, most importantly, the detection of the main beeswax adulterants using a single method.

Authentication of beeswax (Apis mellifera) by high-temperature gas chromatography and chemometric analysis

Chemical characterization and authentication of beeswax of Apis mellifera was performed by high temperature capillary gas chromatography coupled to electron impact mass spectrometry or to flame ionisation detection and chemometric analysis. Many major components (>50) of beeswax, odd and even hydrocarbons, oleofin, palmitate, oleate and hydroxypalmitate monoesters were detected, and for the first time palmitate and oleate monoesters esterified with 1-octadecanol and 1-eicosanol are reported to be present in beeswax. Unsupervised pattern recognition procedures, cluster analysis and principal component analysis, were used to find data patterns and successfully differentiate authentic and paraffin adulterated beeswax based on the chemical profile obtained. Independent assessment of beeswax quality and performance of the unsupervised classification methods were performed using classical analytical parameters. The discrimination power of the chemometric unsupervised methods for detection of paraffin adulterated beeswax was superior to the discriminating power of classical analytical parameters. Using linear discriminant analysis, classification rules for authentic and paraffin adulterated beeswax samples were developed. The model was validated by leave-one-out cross validation and showed good recognition and prediction abilities, 100% and 99%, respectively.

The antimicrobial activity of honey, bee pollen loads and beeswax from Slovakia

The aim of this study was to test the antimicrobial activity of propolis, bee pollen loads and beeswax samples collected in the year 2009 from two locations in Slovakia to pathogenic bacteria, microscopic fungi and yeasts. The antimicrobial effect of the bee product samples were tested using the agar well diffusion method. For extraction, 99.9% and 70% methanol (aqueous, v/v) and 96% and 70% ethanol (aqueous, v/v) were used. Five different strains of bacteria, i.e. Listeria monocytogenes ccM 4699, Pseudomonas aeruginosa ccM 1960; Staphylococcus aureus ccM 3953; Salmonella enterica ccM 4420, Escherichia coli ccM 3988, three different strains of microscopic fungi, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and seven different strains of yeasts Candida krusei, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Geotrichum candidum, Rhodotorula mucilaginosa, were tested. After 48 hours S. aureus was the bacterium most sensitive to the 70% ethanol extract of pollen, A. fumigatus was the most sensitive microscopic fungus (70% ethanol) and C. glabrata the most sensitive yeast (70% methanol). Microorganisms most sensitive to propolis extracts were L. monocytogenes, A. fumigatus (70% ethanol) and G. candidum (70% methanol). Most sensitive to beeswax extracts were E. coli, A. niger and C. tropicalis.

Analysis of efflorescence on surface of beeswax seals

Abstract Thirteen samples of an efflorescence collected from the surface of beeswax seals or from beeswax used for their restoration were analysed in detail. The samples were of different origin, age and storage history. The composition and the incidence of the efflorescence was correlated with a chemical composition of historical and recent beeswax samples. The composition of the crystalline layer was found to be very similar in all the samples. Linear monounsaturated alkenes containing 31 and 33 carbon atoms, more precisely ( Z )-hentriacont-10-ene and ( Z )-tritriacont-10-ene, were identified as main components. The analyses of beeswax have revealed that all compounds identified in “wax bloom” naturally occur in recent beeswax. However, the historical beeswaxes contained only traces of unsaturated hydrocarbons, if anything. The efflorescence was observed primarily on the surfaces of those samples which contained larger proportion of alkenes. The presented results could be of particular importance not only for better understanding of the described phenomenon, but also for the development of new beeswax-based materials, which would be “bloom resistant” and useful for conservation purposes.