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Adulteration has been one of the main quality issues of beeswax production and represents a long-term and increasing problem worldwide. However, there are still no internationally standardized analytical methods or legislation regulating beeswax authenticity control. This problem is not referring only to the aspects of uncontrolled contamination via comb foundation trade but also to the fact that adulterants are being accumulated in the beekeeping technology through comb foundation production process. After developing an analytical procedure for routine beeswax adulteration detection using Fourier transform infrared spectroscopy coupled with attenuated total reflectance recording technique (FTIR-ATR), we have conducted a systematic monitoring of the situation on the comb foundation international market in the period from 2014 to 2016. The results revealed that more than 70% of analyzed samples were adulterated with paraffin (other adulterants were observed sporadically). An amount of paraffin that remains in the honeycomb built on adulterated foundations, after its full construction and melting was also assayed in order to determine residual quantities. It was observed that in average >50% of paraffin remains in the newly built and melted raw wax material. Such material is commercially being used for further comb foundation production and thus, re-enters the beekeeping technology by insertion in the hives. The results obtained in this study indicate “chronic” circulation of adulterants in the comb foundation production process and consequently a large-scale beeswax contamination problem.

Paraffin is the most commonly used beeswax adulterant due to its specific physico-chemical properties, low price, availability and lack of internationally standardized analytical methods and regulations controlling beeswax authenticity. Consequently, the accumulation and circulation of paraffin in comb foundation production process is inevitably. A causal effects of paraffin adulteration on the quality and chemical composition of the comb constructed on foundations contaminated with paraffin, as a material that further enters comb foundation production process, have not yet been investigated. Also, there are no data on the amount of paraffin that remains in the honeycomb after it was built on adulterated foundations. Therefore, the aim of this study was to determine the residual paraffin share in the honeycomb constructed on paraffin-based foundations. Comb foundations containing 90% of paraffin were placed in 5 Apis mellifera colonies (3 frames / LR hive) situated at experimental apiaries in Croatia at the beginning of May 2014, and left until the honey bees finished comb construction. In order to determine the exact proportion of residual paraffin in constructed combs, samples of adulterated foundations and combs built on them were recorded separately (n=30) by FTIR-ATR spectroscopy. Combs constructed on experimental paraffin foundations were melted separately prior to spectroscopic analysis. PLSR model was used for determination of paraffin share in analyzed samples. The results have revealed that 47-67% of paraffin remains in the newly built comb indicating that honey bees can annul at best only 50 % of paraffin by their work (addition of virgin beeswax).

Although beeswax adulteration represents one of the main beeswax quality issues, there are still no internationally standardised analytical methods for routine quality control. The objective of this study was to establish an analytical procedure suitable for routine detection of beeswax adulteration using FTIR-ATR spectroscopy. For the purpose of this study, reference IR spectra of virgin beeswax, paraffin, and their mixtures containing different proportions of paraffin (5 - 95%), were obtained. Mixtures were used for the establishment of calibration curves. To determine the prediction strength of IR spectral data for the share of paraffin in mixtures, the Partial Least Squares Regression method was used. The same procedure was conducted on beeswax-beef tallow mixtures. The model was validated using comb foundation samples of an unknown chemical background which had been collected from the international market (n = 56). Selected physico-chemical parameters were determined for comparison purposes. Results revealed a strong predictive power (R2 = 0.999) of IR spectra for the paraffin and beef tallow share in beeswax. The results also revealed that the majority of the analysed samples (89%) were adulterated with paraffin; only 6 out of 56 (11%) samples were identified as virgin beeswax, 28% of the samples exhibited a higher level of paraffin adulteration (>46% of paraffin), while the majority of the analysed samples (50%) were found to be adulterated with 5 - 20% of paraffin. These results indicate an urgent need for routine beeswax authenticity control. In this study, we demonstrated that the analytical approach defining the standard curves for particular adulteration levels in beeswax, based on chemometric modelling of specific IR spectral region indicative for adulteration, enables reliable determination of the adulterant proportions in beeswax.

Although Apis mellifera beeswax adulteration has been one of the main quality issues of its production and processing for more than a decade (Bogdanov, 2004), there are still no internationally standardized analytical methods for routine beeswax authenticity control. However, the analytical methods defining beeswax composition criteria for routine authenticity testing based on several classical physico-chemical parameters (Bogdanov, 2009) determined in accordance with European Pharmacopoeia (2008) and the Methods of German Society for Fat Science (DGF), have been employed and nationally established in some countries. Recent report on the feasibility study of a single-reflection Attenuated Total Reflectance Fourier transform infrared spectroscopy (ATR-FTIR) for the detection of beeswax adulterants by Maia et al. (2013), revealed good detection limits of four of the most commonly employed beeswax adulterants ; paraffin, microcrystalline wax, tallow and stearic acid. The reference IR spectra of pure beeswax, main adulterants and different adulteration mixtures, i.e. samples of virgin beeswax (brace combs collected from experimental hives), main adulterants (paraffin, beef tallow) and prepared mixtures of virgin beeswax containing different proportions (5-95%) of paraffin/tallow (n=36), were recorded by FTIR spectroscopy using single-reflection high temperature heated diamond ATR system. Furthermore, in total 27 of comb foundation samples collected from beeswax manufacturers and/or specialized beekeeping shops present in Croatian and neighbouring countries market, were recorded aiming to determine their authenticity based on previously obtained reference IR spectra. Selected classical physico-chemical analyses (determination of acid number, ester number, ester/acid ratio, saponification number and melting point) were performed (EP, 2008) on all samples in order to assess the relationship between IR spectral information and physico-chemical measurands of analysed samples. A factor-based calibration method, Partial Least Squares Regression (PLSR) was used to determine the prediction strength of FTIR-ATR spectroscopy for physico-chemical parameters and proportion of adulterants in studied samples. In this study we demonstrated that an IR fingerprinting enables reliable identification of different beeswax adulteration levels based on indicative IR spectral variations observed in studied specimens. The results of analyses conducted on comb foundations collected in the local beeswax market revealed disturbingly large number of adulterated samples (>50% of analysed samples were adulterated with paraffin) and high level of adulteration (presence of 45-92% of paraffin). The PLSR model performance parameters showed strong predictive power of FTIR-ATR spectroscopy for classical physico-chemical data (calibration R2>0.90) and for adulteration proportion (R2=0.99), which indicates analytical reliability of this method for routine quality control of A. mellifera beeswax. The advantages of this method over commonly used classical ones are that it is fast, reliable, cheap and easy-to-use. Also, FTIR-ATR measurements require no sample preparation (reagent-free method) and only small amount of the sample (0, 1g), which further supports the applicability of FTIR-ATR spectroscopy for routine beeswax analysis.