Food Control 120 (2021) 107503

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Validation of analytical methods for the detection of beeswax adulteration

with a focus on paraffin
Lidija Svečnjak a, *, Fernando M. Nunes b, Raquel Garcia Matas c, Jean-Pierre Cravedi d,
Anna Christodoulidou c, Agnes Rortais c, Claude Saegerman e
a
University of Zagreb Faculty of Agriculture, Department of Fisheries, Apiculture, Wildlife Management and Special Zoology, Svetošimunska cesta 25, 10 000, Zagreb,
Croatia
b
CQ-Chemistry Research Centre, Chemistry Department, University of Trás-os-Montes e Alto Douro, 5000-801, Vila Real, Portugal
c
European Food Safety Authority (EFSA), Via Carlo Magno 1A, 43126, Parma, Italy
d
UMR1331 Toxalim (Research Centre in Food Toxicology) INRAE, ENVT, INP-Purpan, UPS, Toulouse, France
e
Research Unit for Epidemiology and Risk Analysis Applied to Veterinary Sciences (UREAR-ULiège), Fundamental and Applied Research for Animals and Health
(FARAH) Center, University of Liège, Quartier Vallée 2, Avenue de Cureghem 7A, B42, 4000, Liege, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Beeswax adulteration in the apiculture sector represents a growing problem worldwide due to the lack of clearly
Beeswax defined purity criteria, the absence of official quality (authenticity) controls, and the inconsistency of the
Adulteration analytical methods used for adulteration detection. Although beeswax authentication is implemented in other
Paraffin
regulatory sectors (pharmaceutical and food industry), the classical physico-chemical analytical methods used
Stearin
Stearic acid
for determination of beeswax purity exhibit inconsistencies for the detection of adulterants. In this study, an
Analytical methods inventory was made on a comprehensive set of analytical methods and the corresponding purity criteria used for
Validation the detection of the most common beeswax adulterants (paraffin, stearin and/or stearic acid) from existing
legislations and scientific literature. The selected analytical methods (classical physico-chemical, and advanced
instrumental, i.e. chromatographic and spectroscopic analytical techniques) were weighted by three independent
experts against two criteria: feasibility and analytical performance in detecting targeted adulterants. Classical
methods for which measurement data were available (melting point and acid/saponification/ester values for
paraffin-adulterated vs. non-adulterated beeswax samples) were retained and further validated by a receiver
operating characteristic (ROC) analysis. These methods were also validated by generating the corresponding
calibration curves for paraffin detection using paraffin-beeswax mixtures containing different proportions of
paraffin (ranging from 5 to 95%, w/w). The results of the ROC analysis revealed that a tentative detection of
paraffin in beeswax can be achieved by a combination of at least two physico-chemical methods. However, for a
reliable detection of the most common adulterants in beeswax, physico-chemical methods should be com­
plemented with advanced analytical tools. i.e. GC-MS, HTGC-FID (MS) and/or FTIR-ATR spectroscopy,
depending on the expected adulterant.

1. Introduction
Beeswax has a complex role in the honey bee (Apis mellifera L.) col­
ony where it serves as a construction material for the honeycomb (comb
wax) that provides an infrastructure for brood rearing and food storage.
It also has an important role in chemical communication (Breed et al.,
1995a, Hepburn, 1998; Fröhlich et al., 2000; D’Ettorre et al., 2006),
mechanical communication (Kirchner, 1993; Sandeman et al., 1996;
Tautz, 1996; Tautz & Lindauer, 1997), and thermoregulation (Ellis et al.,
2010; Humphrey & Dykes, 2008). It can thus be assumed that the use of
good quality (genuine, authentic) beeswax in apiculture is important to
preserve its natural multi-functionality for honey bees.
Beeswax production is a complex process comprising various bio­
logical and technological transformation steps. Beeswax is secreted by
the honey bee workers in the form of wax scales and it is further being
chewed by their mandibles which results in the transformation of
texturally anisotropic scale wax into isotropic comb wax (Hepburn et al.,
2014) supporting its multi-functionality in the honey bee colony. In

* Corresponding author.
E-mail address: lsvecnjak@agr.hr (L. Svečnjak).

https://doi.org/10.1016/j.foodcont.2020.107503
Received 25 April 2020; Received in revised form 26 June 2020; Accepted 21 July 2020
Available online 25 July 2020
0956-7135/© 2020 Elsevier Ltd. All rights reserved.
L. Svečnjak et al.
apiculture, comb wax is commonly being recycled by melting the hon­
eycomb and wax capping (after extraction of honey) and by removing
foreign matter by melting it with boiling water which enables the pro­
duction of crude beeswax - the final product being further commer­
cialized. Beeswax recycling may also include industrial-scale
decontamination methods for removing acaricides and heavy metals
from beeswax (Navarro-Hortal et al., 2019), as well as discoloration, i.e.
bleaching methods (Serra-Bonvehí & Orantes Bermejo, 2016). Nowa­
days, crude beeswax is used by various industry types, such as phar­
maceuticals, cosmetics and food industry (e.g. food additive, packaging
and coating) (Bogdanov, 2017a), along with its major (re-) use in
apiculture in the form of comb foundations (Bogdanov, 2017b). Comb
foundation represents a thin layer of beeswax intended for further use in
apiculture (insertion in the beehives within wired frames) as a founda­
tion for further comb wax construction by the bees (the cycle continues:
wax scales - comb wax - crude beeswax - comb foundation). Although it
is difficult to present exact figures on beeswax use, production and trade,
it can be stated that the comb foundation production is probably the
major use of beeswax (Bogdanov, 2017b; Crane, 1990; Krell, 1996).
There are only a few official statutory documents from the European
Union and FAO/WHO providing a definition for beeswax, its description
and quality (purity criteria). However, these documents only refer to its
uses in the pharmaceutical sector as pharmaceutical grade beeswax
defined by European Pharmacopoeia - Ph.Eur. 10th Edition, Supplement
10.2 (Council of Europe, 2020) and in the food industry as food additive
E901 (Commission Regulation, 2012; Commission Directive, 2009; Eu­
ropean Food Safety Authority (EFSA), 2007). Under those regulatory
sectors, beeswax authentication relies on several classical
physico-chemical analytical methods and corresponding
physico-chemical criteria (range values) defining pure beeswax.
Most of the physico-chemical analytical range values characterizing
pure beeswax in the existing legislations are generally accepted and
widely used for beeswax research. The most commonly used methods
are the determination of the melting point, acid value, saponification
and ester value (Tulloch & Hoffman, 1972; Tulloch, 1973; Puleo & Rit,
1992; Bernal et al., 2005; Serra Bonvehı, 1990; Serra Bonvehí & Orantes
Bermejo, 2012; Maia & Nunes, 2013, Svečnjak et al., 2015, 2019a),
while some other methods have been used sporadically for the purpose
of beeswax authentication, namely, determination of beeswax density
(specific gravity), peroxide value (Bernal et al., 2005; Bogdanov, 2004),
ash content and iodine value (Bernal et al., 2005; Puleo & Rit, 1992;
Serra Bonvehı, 1990).
While the quality control of beeswax in pharmaceutical and food
industry is currently covered by legislation, beeswax material used in
apiculture (as comb foundations and crude beeswax used for their pro­
duction) is not supported by any legislative framework. A proposal of
purity criteria for beeswax used in apiculture based on physico-chemical
parameters from existing legislation (Council of Europe, 2020) com­
plemented with additional analytical testing (determination of refrac­
tive index, water content and ester/acid ratio) has been initiated by the
world-wide network on honey and bee product science, the Interna­
tional Honey Commission (IHC), a decade ago (Bogdanov, 2009,
2017b). However, this initiative remained at a proposal level.
Currently, beeswax and its products used in the apiculture sector are
classified as animal by-products (ABP) not intended for human con­
sumption and sub-categorized as category 3 material (Commission
Regulation, 2011; Regulation (EC), 2009). This category includes ABPs
that do not present a potential risk for the food chain and, therefore,
beeswax is not subject of any quality control before being marketed. This
represents a vulnerable entry point to adulteration. Consequently,
adulterated beeswax is frequently re-entering the apiculture sector via
uncontrolled comb foundation trade. Comb foundations are the major
target of adulteration which further leads to adulteration of crude
beeswax. As reported in numerous studies, the most commonly used
adulterant is paraffin wax (Tulloch, 1973; Bogdanov, 2004; Bernal et al.,
2005; Serra Bonvehí and Orantes Bermejo, 2012; Maia et al., 2013;
2
Food Control 120 (2021) 107503
Svečnjak et al., 2015; Waś et al., 2016; Svečnjak et al., 2018; Tanner &
Lichtenberg-Kraag, 2019; Špaldoňová et al., 2020), while stearin and/or
stearic acid appears sporadically, especially in recent years (Reybroeck
& Van Nevel, 2018, p. 115; Svečnjak et al., 2018; Tanner &
Lichtenberg-Kraag, 2019). In chemistry, the term stearin (tristearin)
corresponds to a tristearic acid triglyceride but commercially available
stearin can also refers to stearic acid or a mixture of stearic acid and
palmitic acid. These fatty acids are commonly used for beeswax
adulteration.
In addition to the lack of legislation and defined purity criteria,
another challenge in determining the beeswax purity is the reliability of
the most commonly used physico-chemical methods (i.e. determination
of the melting point, acid, saponification and ester value) for detecting
adulterants in beeswax. As reported by Bernal et al. (2005), the mini­
mum amount of the most common beeswax adulterants (paraffin, stearic
acid, tallow and carnauba wax) that can be detected by
physico-chemical methods is relatively high (between 2% and 50%,
depending on the type of adulterant, as well as the method applied) and
the determination of physico-chemical parameters does not guarantee
beeswax purity (i.e. the absence of adulterants).
Furthermore, as indicated by Svečnjak et al. (2019a), one of the
factors that may affect the analytical range values (primarily saponifi­
cation value, and consequently, ester value and ester/acid ratio) is an
exposure of beeswax to high heat treatment (Tulloch, 1973) applied as
an integral part of the comb foundation production process (exposure to
125–130 ◦ C) necessary to kill the spores of the heat-resistant Paeniba­
cillus larvae, a bacteria responsible for the American foulbrood. A recent
report by Špaldoňová et al. (2020) revealed small changes in the
chemical composition of beeswax (i.e. simultaneous decrease in car­
boxylic acid groups and an increase in hydroxyl esters and palmitic acid
methyl ester) caused by the heating treatment (i.e. exposure to 100 ◦ C
for 60 min). The results indicate that the heating process does not affect
the quality of beeswax significantly but the effects of the most commonly
used heating treatments during the comb foundation production process
(125–130 ◦ C) have yet to be investigated and correlated with the values
of the physico-chemical parameters. Some deviations in analytical range
values may also arise from a different geographical origin of the beeswax
(Beverly et al., 1995), different A. mellifera subspecies producing the
wax (Beverly et al., 1995; Fröhlich et al., 2000; Tulloch, 1980), comb
age (Waś et al., 2014), and/or minor natural chemical variability of
beeswax among colonies (Breed et al., 1995b; Svečnjak et al., 2015;
2019a). Still, the overall analytical deviations of the physico-chemical
parameters are not yet fully explained. Sampling details are often not
available in the scientific literature, so it can be assumed that anomalous
analytical values may also be related to questionable sampling and/or to
the origin of the beeswax samples.
Aiming to find robust and more reliable analytical methods for
determining beeswax authenticity, advanced instrumental analytical
methods have been developed and utilized in the last decade for the
detection and quantification of adulterants in beeswax: gas chroma­
tography (GC) coupled with different detectors (Jiménez et al., 2009;
Serra Bonvehi & Orantes Bermejo 2012; Waś et al., 2015, 2016), and
Fourier transform infrared spectroscopy (FTIR) coupled with Attenuated
Total Reflectance (ATR) accessory (FTIR-ATR technique) (Maia et al.,
2013; Svečnjak et al., 2015; 2019a; Tanner & Lichtenberg-Kraag, 2019).
Both analytical methods proved to be more reliable for beeswax adul­
teration detection compared to physico-chemical methods as they pro­
vide good detection limits (LOD<5%) for the most commonly used
adulterants (i.e. paraffin, stearin/stearic acid). However, they have not
yet been validated by inter-laboratory comparisons and, consequently,
they are not yet incorporated in the current legislation on beeswax.
The aim of this study was to assess the effectiveness and reliability of
the available analytical methods for the determination of beeswax pu­
rity with a focus on the detection of adulteration with paraffin as the
most commonly used adulterant in apiculture. Based on the statistical
validation of the analytical methods and the data available in the
L. Svečnjak et al.
literature, a practical recommendation for the use of the analytical
methods (and corresponding purity criteria) showing the best perfor­
mance in detecting beeswax adulteration with paraffin was provided.
2. Materials and methods
The assessment of the analytical methods used for the detection of
beeswax adulteration was performed on four levels (Fig. 1).
2.1. Inventory of classical and advanced analytical methods for detecting
beeswax adulteration with paraffin and stearin/stearic acid
An inventory of classical physico-chemical and advanced chro­
matographic/spectroscopic analytical methods for detecting beeswax
adulteration with paraffin and stearin/stearic was selected based on 2
eligibility criteria: 1) implementation of the methods in existing legis­
lation, 2) employment of the methods on research level (scientific
literature data). The methods listed in this inventory were considered for
further evaluation.
2.1.1. Classical physico-chemical analytical methods
Classical physico-chemical analytical methods for the determination
of beeswax purity (absence of paraffin and of stearin/stearic acid) were
implemented and described in pharmacy (Council of Europe, 2020) and
food industry (Commission Regulation, 2012; Commission Directive,
2009; EFSA, 2007; Joint FAO/WHO Expert Committee on Food Addi­
tives, 2005). The methods utilized on research level and supported by
scientific literature data comprising reports on physico-chemical mea­
surements for authentic and adulterated beeswax samples (Serra Bon­
vehı, 1990; Bernal et al., 2005; Serra Bonvehí and Orantes Bermejo,
2012; Maia & Nunes, 2013, Svečnjak et al., 2015) were used to
consolidate the list of the following methods of interest: determination
of melting point (drop point), specific gravity (density), acid value,
saponification value, ester value, ester/acid ratio, peroxide value, and
iodine value.
2.1.2. Advanced instrumental analytical methods
Two advanced chromatographic and spectroscopic analytical
methods enabling reliable detection and quantification of paraffin and
stearin/stearic acid in beeswax have been considered as methods of
interest: gas chromatography coupled to mass spectrometry (GC-MS)
(Svečnjak et al., 2019a; Waś et al., 2016, 2015)., as well as
high-temperature GC with flame ionization detection (HTGC-FID,
alternatively complemented with MS) (Jiménez et al., 2009; Serra
Bonvehi & Orantes Bermejo 2012), and FTIR-ATR spectroscopy (Maia
et al., 2013; Svečnjak et al., 2015; 2019a; Tanner & Lichtenberg-Kraag,
2019).
The inventory of selected classical and advanced analytical methods
Fig. 1. Flow diagram of the methodology to assess analytical methods for
beeswax adulteration detection.
3
Food Control 120 (2021) 107503
of interest was further complemented by extracting additional method
information relevant for the detection of paraffin and stearin/stearic
acid in beeswax. For this, the measurement uncertainty indicators
(anomalous values reported for pure beeswax), and crucial methods’
advantages and disadvantages in terms of their feasibility and analytical
performance in detecting targeted adulterants were considered. In­
ventory list was used as a foundation for the construction of evaluation
grid for assessing the analytical methods by weight analysis.
2.2. Assessment of analytical methods by weight analysis
Weight analysis was performed to select a set of analytical methods
and corresponding purity criteria showing the best/most reliable results
in terms of detecting adulterants (based on both classical physico-
chemical and advanced analytical methods, and covering both
paraffin and stearin detection). An inventory of selected basic physico-
chemical methods and chromatographic/spectroscopic analytical sys­
tems (Tables 1 and 2) was used as input data for an evaluation grid
employed to weight the importance of the method (and corresponding
purity criteria) in detecting targeted adulterants in beeswax.
Weight analysis was performed by assessing the importance of each
analytical method via weighting factors by taking into consideration the
overall list of parameters in the constructed evaluation grid. Weighting
factors have been obtained by three international independent analysts/
assessors with experience in beeswax analysis for authentication pur­
poses, which enabled methods to be retained and weighted in impor­
tance in terms of detection of targeted adulterant types (paraffin,
stearin/stearic acid). Assessors have weighted the methods they
consider to be retained for beeswax adulteration detection based on
their knowledge/experience and parameters listed in the evaluation
grid. This relative weight was determined using the Las Vegas technique
(Gore, 1987). Briefly, these experts were given a number of points to be
distributed between the analytical methods according to the importance
of each analytical method. If all the selected analytical methods would
have been considered equivalent by experts, each of them would have
received the same number of points. Indeed, each expert distributed 200
points to the retained analytical methods (particular weight was given to
each retained method to assign its lighter or heavier importance in a
group of retained methods). This was performed separately for paraffin
and stearin/stearic acid.
2.3. Assessment of analytical methods for paraffin adulteration detection
using a receiver operating characteristic analysis
In order to assess the reliability of physico-chemical analytical
methods in terms of their ability for correct classification (paraffin
adulterated versus non-adulterated beeswax samples), two analytical
data sets on four physico-chemical measurements most commonly used
as purity criteria (acid value, ester value, saponification value, and
melting point) obtained for pure and adulterated (with paraffin)
beeswax samples have been validated, pooled and used:
I) data set A: data extracted from Maia & Nunes (2013) were sum­
marized in the Supplementary material S1 (virgin beeswax samples:
n = 12; comb wax: n = 26; comb foundations from the market: n =
33);
II) data set B: raw data set provided by Svečnjak et al., 2015 (virgin
beeswax samples: n = 18; comb foundations from the market: n =
56) were summarized in the Supplementary material S1.
Firstly, for each purity criterion, the distribution of values obtained
for all beeswax samples was presented as a boxplot regarding the status
of adulteration (i.e. adulterated vs. non-adulterated samples). Secondly,
a two-sample Wilcoxon rank-sum (Mann-Whitney) test (i.e. a non-
parametric test) was used to verify the putative discrimination of the
two sub-populations (adulterated vs. non-adulterated samples) using
L. Svečnjak et al. Food Control 120 (2021) 107503

Table 1
An inventory of analytical methods for the detection of paraffin in beeswax (used as input data for evaluation grid).
Analytical method for Purity criteria Expression of Validated Validated Anomalous Minimum Advantage Disadvantage -
beeswax adulteration the result method - method - values reported adulteration (s) - reagent- requires
detection (reference: (unit) defining supported by supported by for pure percentages free and specialized
legislation/scientific pure beeswax legislation scientific (authentic) detected rapid knowledge
literature) (absence of (Y/N) literature (Y/ beeswax in the (%)/LOD method (Y/
paraffin) N) literature (Y/N/ N)
NDA)

Determination of specific Specific gravity 0.960 g/cm3 Y Y N 10–15 Y N

gravity (density) (EFSA, (density)
2007; EC, 2009; 2012; Ph.
Eur., 2020/Bernal et al.,
2005)
Determination of melting Melting point 61-65 (◦ C) Y Y N 30–50 Y N
range (JECFA, 2005; EFSA, (drop point)
2007; EC, 2009; 2012; Ph.
Eur., 2020/Serra Bonvehı,
1990; Bernal et al., 2005;
Serra Bonvehí and Orantes
Bermejo, 2012; Maia &
Nunes, 2013; Svečnjak
et al., 2015)
Determination of acid value Acid value 17-24(mg Y Y Y 10 N N
(JECFA, 2005; EFSA, 2007; KOH/g)
EC, 2009, 2012; Ph.Eur.,
2020/Serra Bonvehı, 1990;
Bernal et al., 2005; Maia &
Nunes, 2013; Svečnjak
et al., 2015)
Determination of Saponification 87-104(mg Y Y Y 10 N N
saponification value value KOH/g)
(JECFA, 2005; EFSA, 2007;
EC, 2009; 2012; Ph.Eur.,
2020/Serra Bonvehı, 1990;
Bernal et al., 2005; Serra
Bonvehí and Orantes
Bermejo, 2012; Maia &
Nunes, 2013; Svečnjak
et al., 2015)
Determination of ester value Ester value 70-90(mg Y Y Y 5 N N
(Ph.Eur., 2020/Serra KOH/g)
Bonvehı, 1990; Bernal
et al., 2005; Svečnjak et al.,
2015, 2019a)
Determination of ester/acid Ester/acid ratio 3.3–4.3 N Y Y 10 N N
ratio (Serra Bonvehı, 1990;
Bernal et al., 2005;
Svečnjak et al., 2015,
2019a)
Determination of peroxide Peroxide value <5(mM H2O2/ N Y Y NDA N N
value (JECFA, 2005; EFSA, kg)
2007; EC, 2009, 2012/
Bernal et al., 2005,
Svečnjak et al., 2019a)
Determination of Iodine Iodine value 7.6–13.1(g I/ N Y N 15 N N
value (Serra Bonvehı, 100g)
1990; Bernal et al., 2005;
Svečnjak et al., 2019a)
GC-MS (Svečnjak et al., Paraffin absent N Y N <3 N Y
2019a; Waś et al., 2016,
2015)
HTGC-FID/MS (Jiménez Paraffin (and absent N Y N <4 N Y
et al., 2009; Serra Bonvehi other foreign
& Orantes Bermejo, 2012) substances)
FTIR-ATR (Maia et al., 2013; Paraffin (and absent N Y N <3.5 Y Y
Svečnjak et al., 2019a, other foreign
2015; Tanner & substances)
Lichtenberg-Kraag, 2019)

Legend: Y, Yes; N, No; NDA, no data available; LOD, limit of detection; GC-MS, gas chromatography - mass spectrometry; HTGC-FID(MS), high-temperature gas
chromatography with flame ionization detection/mass spectrometry, FTIR-ATR, Fourier transform infrared spectroscopy- attenuated total reflectance; JECFA, 2005 -
Joint FAO/WHO Expert Committee on Food Additives, 2005; EFSA, 2007 - European Food Safety Authority, 2007; EC, 2009 - Regulation (EC), 2009; EC, 2012 -
Commission Regulation, 2012; Ph.Eur., 2020 - European Pharmacopoeia (Council of Europe, 2020).

4
L. Svečnjak et al. Food Control 120 (2021) 107503

Table 2
An inventory of analytical methods for the detection of stearin/stearic acid in beeswax (used as input data for evaluation grid).
Analytical method for Purity criteria Expression of Validated Validated Anomalous Minimum Advantage(s) Disadvantage -
beeswax adulteration the result (unit) method - method - values reported adulteration - reagent-free requires
detection (reference: defining pure supported by supported by for pure percentages and rapid specialized
legislation/scientific beeswax legislation scientific (authentic) detected method (Y/ knowledge
literature) (absence of (Y/N) literature (Y/ beeswax in the (%)/LOD N)
paraffin) N) literature (Y/N/
NDA)

Determination of specific Specific gravity 0.960 g/cm3 N Y N unuseful Y N

gravity (density) (EC, (density)
2009; 2012; Ph.Eur.,
2020/Bernal et al.,
2005)
Determination of melting Melting point 61-65 (◦ C) N Y N 30 Y N
range (JECFA, 2005; (drop point)
EC, 2009; 2012; Ph.
Eur., 2020/Bernal
et al., 2005)
Determination of acid Acid value 17-24(mg N Y Y 2 N N
value (JECFA, 2005; KOH/g)
EC, 2009; 2012; Ph.
Eur., 2020/Bernal
et al., 2005)
Determination of Saponification 87-104(mg N Y Y 3 N N
saponification value value KOH/g)
(JECFA, 2005; EC,
2009; Ph.Eur., 2020/
Bernal et al., 2005)
Determination of ester Ester value 70-90(mg N Y Y 5 N N
value (Ph.Eur., 2020/ KOH/g)
Bernal et al., 2005)
Determination of ester/ Ester/acid ratio 3.3–4.3 N Y Y 15 N N
acid ratio (Bernal
et al., 2005)
Determination of Peroxide value <5(mM N Y Y NDA N N
peroxide value H2O2/kg)
(JECFA, 2005; EC,
2009; 2012/Bernal
et al., 2005)
Determination of Iodine Iodine value 7.6–13.1(g I/ N Y N 15 N N
value (Bernal et al., 100g)
2005)
GC-MS (Svečnjak et al., Stearin/stearic absent N N NDA unuseful N Y
2019a; Waś et al., acid
2016, 2015)
HTGC-FID/MS (Jiménez Stearin/stearic absent N Y N <1 N Y
et al., 2009; Serra acid (and other
Bonvehi & Orantes foreign
Bermejo, 2012) substances)
FTIR-ATR (Maia et al., Stearin/stearic absent N Y N <1.2 Y Y
2013; Svečnjak et al., acid (and other
2019a, 2015; Tanner foreign
& Lichtenberg-Kraag, substances)
2019)

Legend: Y, Yes; N, No; NDA, no data available; LOD, limit of detection; GC-MS, gas chromatography - mass spectrometry; HTGC-FID(MS), high-temperature gas
chromatography with flame ionization detection/mass spectrometry, FTIR-ATR, Fourier transform infrared spectroscopy- attenuated total reflectance; JECFA, 2005 -
Joint FAO/WHO Expert Committee on Food Additives, 2005; EFSA, 2007 - European Food Safety Authority, 2007; EC, 2009 - Regulation (EC), 2009; EC, 2012 -
Commission Regulation, 2012; Ph.Eur., 2020 - European Pharmacopoeia (Council of Europe, 2020).

each purity criteria. Thirdly, a receiver operating characteristic (ROC)
curve was used to determine the best cut-off for each purity criterion
related to the known status of paraffin adulteration of the sample, as
reference. The ROC curve (probability curve) was plotted with true
positive results (Y-Axis) against the false positive results (X-Axis). The
area under the ROC curve (AUC) is the performance measurement for
the classification test at various thresholds settings. The higher the AUC,
the better the test is able to distinguish between paraffin adulterated and
non-adulterated beeswax samples (i.e. measurement of the separation of
the two sub-populations). In addition, the Youden’s index “J” is
frequently used in conjunction with the ROC curve analysis to estimate
the best cut-off, with:
Youden’s index = sensitivity + specificity − 1
The value of AUC ranges from 0 to 1 (inclusive). A zero value is
observed when a diagnostic test gives the same proportion of positive
results for groups with and without the adulteration. A value of 1 in­
dicates that there are no false positives or false negatives, i.e. the test is
perfect. In a ROC curve the calculation of the Youden’s index in all
points allows to determine the best cut-off of the test (i.e. optimal
Youden’s index).
All analyses were performed with STATA/SE 14.2 (StataCorp., Col­
lege Station, Texas, USA).
2.4. Validation of calibration curves constructed for detecting paraffin by
retained physico-chemical analytical methods
(1)
Samples of virgin beeswax, paraffin, and beeswax-paraffin mixtures

5
L. Svečnjak et al. Food Control 120 (2021) 107503

(BP) containing different proportions of paraffin were obtained. Virgin
beeswax samples were collected directly from the Apis mellifera beehives
(wild-built combs) to ensure authenticity, and melted by boiling water
to remove impurities. Paraffin was purchased from commercial chemi­
cal supplier that guarantee product’s authenticity (Paraffinum solidum,
Ph.Eur. 7,8, Kemig, Croatia).
Beeswax-paraffin mixtures containing different proportions of
paraffin (5–95%, following 5% increasing sequence of adulterant addi­
tion, w/w) were prepared by precise weighting (±0.001 g) of each
mixture constituent. Mixtures were further melted at 90 ◦ C for homog­
enization, re-solidified at room temperature, and stored in the dark at
room temperature prior to further physico-chemical analyses.
Prepared samples (3 technical replicates) were analysed by means of
four retained physico-chemical methods: determination of melting
point, acid value, saponification value, and ester value. Determination
of melting point was conducted by capillary tube-based instrument MP
55 Melting Point System (Mettler Toledo) for automatic determination
of melting point with a built-in digital camera that allows the observa­
tion of beeswax melting (method proposed by Svečnjak et al., 2019a as
easy-to-use alternative to classical capillary method).
Determination of acid, saponification and ester value was performed
according to European Pharmacopoeia 10.2 (Council of Europe, 2020)
reference methods following detailed step-by-step protocol descriptions
provided by Svečnjak et al. (2019a). Mixtures were used for the con­
struction of calibration curves exhibiting physico-chemical properties
specific for particular adulteration level (5–95%). In order to determine
the prediction strength of physico-chemical analytical methods for
paraffin share detection, performance parameters of constructed cali­
bration curves were validated by simple linear regression model.
Saponification value was determined on a smaller sampling set (for
mixtures with adulteration level 10–90%, i.e. for 10% increasing
sequence of paraffin addition, w/w) due to insufficient amount of pure
beeswax necessary for the preparation of 5%-sequence mixtures for all
measurements).
3. Results and discussion
3.1. Inventory of analytical methods used for detection of paraffin and
stearin/stearic acid in beeswax
The inventory of both classical routine and advanced analytical
methods used for the detection of paraffin (Table 1) and stearin/stearic
acid (Table 2) were summarized based on available literature data.
These inventory tables were constructed based on the general method
information (validation supported by legislation and scientific
literature), indication of measurement uncertainty (limit of detection,
reports on anomalous values for authentic beeswax), and crucial
methods advantages and disadvantages in terms of their feasibility and
analytical performance (rapidness, requirements for specialized
knowledge).
These two tables were used as input data to weight the importance of
the method and corresponding purity criteria for beeswax adulteration
detection with paraffin and stearin/stearic acid (see next section).
3.2. Weight analysis by expert elicitation
Evaluation of retained analytical methods by weighting of their
importance in detecting paraffin and stearin/stearic acid in beeswax is
presented in Table 3 and Table 4, respectively. The three analysts/as­
sessors who performed the weight analysis appeared to give moderate
importance to the classical physico-chemical analytical methods, but
they emphasized the use of advanced analytical methods, especially the
HTGC-FID (MS) and/or the FTIR-ATR spectroscopy for reliable detec­
tion of both paraffin and stearin/stearic acid.
It appears that experts identify four classical methods (i.e. melting
point, specific gravity, acid value and saponification value), and three
advanced methods (i.e. GC-MS, HTGC-FID (MS), FTIR-ATR) as useful for
the detection of beeswax adulteration with paraffin. The same experts
identified three classical physico-chemical methods (i.e. melting point,
acid value, and saponification value), and two advanced methods (i.e.
HTGC-FID (MS), FTIR-ATR) as useful for the detection of stearin/stearic
acid adulteration of beeswax.
It can be observed that although ester value is one of the most
commonly used physico-chemical parameter (as presented previously
and further in section 3.3.), it was not chosen by the assessors. This can
be explained by the fact that ester value is obtained by calculation (by
subtracting acid from the saponification value), and it can be assumed
that the assessors gave importance to the methods (determination of
acid and saponification value) that indirectly provide the result for ester
value as well.
The GC-MS was selected only for reliable detection of paraffin in
beeswax. It can be assumed that GC-MS would provide satisfactory re­
sults in detecting and quantifying stearin/stearic acid as well, but there
is still no report on this. Future research should be focused on devel­
oping the GC-MS analytical procedure for the detection and quantifi­
cation of stearin/stearic acid in beeswax.

Table 3
Weight analysis - analytical methods for paraffin adulteration detection.
Method retained and weighted (distribution of 200 points between the retained Aggregation of results
method)

Analytical method for paraffin adulteration detection Assessor 1 Assessor 2 Assessor 3 Mean S.D. Sum/600 Sum/100

Classical methods
Determination of specific gravity (density) 20 10 15.00 7.07 30 5
Determination of melting range 10 10 10 10.00 0.00 30 5
Determination of acid value 30 20 35 28.33 7.64 85 14
Determination of saponification value 20 20 15 18.33 2.89 55 9
Determination of ester value
Determination of ester/acid ratio
Determination of peroxide value
Determination of Iodine value
Advanced methods
GC-MS 40 50 40 43.33 5.77 130 22
HTGC-FID(MS) 40 50 40 43.33 5.77 130 22
FTIR-ATR 40 50 50 46.67 5.77 140 23
Total 200 200 200 600 100

Legend: S.D., standard deviation; GC-MS, gas chromatography - mass spectrometry; HTGC-FID(MS), high-temperature gas chromatography with flame ionization
detection (mass spectrometry), FTIR-ATR, Fourier transform infrared spectroscopy- attenuated total reflectance.

6
L. Svečnjak et al. Food Control 120 (2021) 107503

Table 4
Weight analysis - analytical methods for stearin/stearic acid adulteration detection.
Method retained and weighted (distribution of 200 points Aggregation of results
between the retained method)

Analytical method for stearin/stearic acid adulteration detection Assessor 1 Assessor 2 Assessor 3 Mean S.D. Sum/600 Sum/100

Classical methods
Determination of specific gravity (density)
Determination of melting range 10 20 10 13.33 5.77 40 7
Determination of acid value 40 50 35 41.67 7.64 125 21
Determination of saponification value 30 30 25 28.33 2.89 85 14
Determination of ester value
Determination of ester/acid ratio
Determination of peroxide value
Determination of Iodine value
Advanced methods
GC-MS
HTGC-FID (MS) 60 50 60 56.67 5.77 170 28
FTIR-ATR 60 50 70 60.00 10.00 180 30
Total 200 200 200 600 100

Legend: S.D., standard deviation; GC-MS, gas chromatography - mass spectrometry; HTGC-FID(MS), high-temperature gas chromatography with flame ionization
detection (mass spectrometry), FTIR-ATR, Fourier transform infrared spectroscopy- attenuated total reflectance.

3.3. Determination of the best cut-off for some classical methods for and ester value.
which data are available in the literature (for paraffin only) The distribution of values of all these parameters are statistically
different between paraffin-adulterated (N = 73) and non-adulterated (N
As presented in Supplementary Material Table 1 chosen data sets A = 72) beeswax samples (two-sample Wilcoxon rank-sum (Mann-Whit­
and B from two literature sources (Maia & Nunes, 2013 and Svečnjak ney) test; p-value < 0.0001).
et al., 2015, respectively) contained data on the four most commonly For each parameter, a ROC curve was built and the area under the
used physico-chemical measurements (acid value, ester value, saponi­ curve (AUC) was calculated in order to estimate the discriminatory
fication value, and melting point). A confirmation of authenticity or power of each parameter (Fig. 3). For the melting point value, the best
adulteration with paraffin was provided for all the samples in these data cut-off was 63 ◦ C and the AUC was 0.9261 (S.E. = 0.0211) (Fig. 3 [A]).
sets using advanced chromatographic (HTGC-FID for data set A) and For the acid value, the best cut-off was 17.3 and the AUC was 0.7419 (S.
spectroscopic (FTIR-ATR spectroscopy for data set B) analytical tools. E. = 0.0422) (Fig. 3 [B]). For the saponification value, the best cut-off
was 75.8 and the AUC was 0.8066 (S.E. = 0.0372) (Fig. 3 [C]). For
3.3.1. Assessment of the discrimination of adulterated and non-adulterated the ester value, the best cut-off was 56.59 and the AUC was 0.8091 (S.E.
beeswax samples based on each selected physico-chemical parameters = 0.0366) (Fig. 3 [D]).
Basic statistics (Table 5) and boxplots for each selected classical Although the best cut-offs for individual physico-chemical parame­
physico-chemical parameters were presented in Fig. 2 [A], [B], [C] and ters can be obtained, it should be noted that the practical use of these
[D] for respectively melting point value, acid value, saponification value values have limitations in the discrimination of adulterated and non-
adulterated beeswax when physico-chemical parameters are com­
Table 5 bined. This was validated by further ROC analysis presented in section
Basic statistics of each physico-chemical parameters for adulterated and non- 3.3.2 according to different scenarios.
adulterated beeswax samples. Analytical values causing misclassification of beeswax samples (false
[A] Melting PA NA [C] Saponification PA NA positives/false negatives) when using generally proposed physico-
point(◦ C) value (mg KOH/g) chemical purity criteria are likely to be related to several factors. It
Min 48.00 60.00 Min 4.20 57.50
can be assumed that a high rate of misclassified samples may be related
Max 66.50 67.30 Max 117.70 134.00 to the measurement uncertainty arising from the samples, i.e. minor
Mean 58.32 64.03 Mean 63.23 91.54 chemical variations naturally occurring in different beeswax samples, e.
St. Error 4.14 1.46 St. Error 29.63 14.76 g. different comb age (Waś et al., 2014), different geographical origin
Q1 56.00 63.23 Q1 44.10 82.23
(Beverly et al., 1995), or beeswax originating from different colonies
Median 59.00 64.30 Median 70.80 89.40
Q3 61.00 63.23 Q3 86.90 98.38 (Breed et al., 1995b; Svečnjak et al., 2015; 2019a). Variations in
Middle value 62 Middle value between 85 beeswax composition amongst colonies have been quantified for analyte
between Q3 Q3 for PA and Q1 for signals (corresponding to lipid-based constituents) used for the detec­
for PA and Q1 NAP tion of adulterants by FTIR-ATR spectroscopy (Svecnjak et al., 2019a).
for NA
[B] Acid value PA NA [D] Ester value(mg PA NA
Those analyte signals are generally recommended to be considered
(mg KOH/g) KOH/g) when detecting adulterants (up to 3.48% for lipid constituents). Indeed,
Min 0.86 14.40 Min 1.40 31.10 analytical deviations can be caused by physico-chemical properties of
Max 25.80 30.20 Max 92.50 112.20 the adulterants themselves (such as e.g. melting point of paraffin which
Mean 15.17 20.45 Mean 47.92 71.45
can be similar to beeswax depending on the type of paraffin used for
St. Error 7.21 3.52 St. Error 23.13 14.31
Q1 9.55 18.20 Q1 34.36 61.70 adulteration), different origins of beeswax samples (different chemical
Median 16.90 19.35 Median 53.40 70.10 composition) and recycling processes (temperature treatments); not
Q3 20.26 20.90 Q3 64.50 78.53 only because of the shortcomings of the classical physico-chemical
Middle value 19 Middle value between 63 analytical methods themselves. Thus, it can be assumed that natural
between Q3 Q3 for PA and Q1 for
variations in beeswax composition arising from all the listed factors go
for PA and Q1 NAP
for NA beyond analytical uncertainties, including limits of detection and
quantification determined for given adulterants by advanced instru­
Legend: PA, paraffin adulterated beeswax samples; NA, non-adulterated
mental analytical methods (Jiménez et al., 2009; Serra Bonvehi &
beeswax samples; Q, quartile.

7
L. Svečnjak et al.
Fig. 2. Boxplots for adulterated and non-adulterated beeswax samples based on each selected physico-chemical parameters. Legend: The dashed line represents the
median value; the solid lines below and above each rectangle represent the first and the third quartiles, respectively; adjacent lines to the whiskers represent the
limits of the 95% confidence interval; small circles represent outside values.
Orantes Bermejo, 2012; Maia et al., 2013; Waś et al., 2016, 2015;
Svečnjak et al., 2015; 2019a; Tanner & Lichtenberg-Kraag, 2019). Also,
regardless of which analytical method is used for the detection of
adulterants, it is important that samples being analysed are purified and
homogenized, i.e. free of other impurities originating from the hive or
elsewhere which might cause noise and affect the analyte signals.
3.3.2. Assessment of the discrimination of adulterated and non-adulterated
beeswax samples based on combination of physico-chemical parameters
The four previous purity criteria were evaluated in combination
using a score and a ROC curve. The score was defined as the number of
positive results in the four different parameters (range: 0 to 4). Three
scenarios were investigated. The first one consisted to use the range of
values from the existing legislation and scientific literature (Table 1) for
each parameter considered. The second one was based on the middle
value between the quartile 3 of the paraffin adulterated beeswax
8
Food Control 120 (2021) 107503
Fig. 3. Receiver operating characteristic (ROC) curve
for the melting point [A], acid value [B], saponifica­
tion value [C] and ester value [D]. Legend: Points are
observed values. The curve (in black) with its 95%
confidence interval (in grey) is based on fitted values
using a binomial model. Dashed line represents the
situation when the number of true positive equals the
number of false positive. In this case the area under
the curve equal 0.50 (i.e. uninformative testing pro­
cedure). The ROC curve is plotted with true positive
results denoted as Sensitivity (Y-Axis) against the
false positive results denoted as 1-Specificity (X-Axis).
samples and the quartile 1 of non-adulterated beeswax samples
(Table 5). The third one was based on the estimation of the cut-off using
a classification tree analysis (Salford Predicitve modeler software, Sal­
ford Systems. San Diego. CA, USA). The classification tree analysis is a
complex data mining process (for more details, see e.g. Saegerman et al.,
2011) that permits to split the data into two nodes with maximization of
pure data (i.e. adulterated vs. non-adulterated samples) in each terminal
nodes (Table 6).
The ROC curve for the three scenarios (described in Table 6) are
presented in Fig. 4. It appears that area under the ROC curve was
significantly higher (Chi-square (2 df) = 10.89; p-value = 0.004) for the
scenario 3 (AUC = 0.9380; S.E. = 0.0210) in comparison with the sce­
nario 1 (AUC = 0.8307, S.E. = 0.0362) and 2 (AUC = 0.8501; S.E. =
0.0342). The cut-off for the two first scenarios was equal to two (i.e. at
least two analytical methods amongst the four). However, this cut-off
was equal to four for the third scenario.
L. Svečnjak et al. Food Control 120 (2021) 107503

Table 6 descriptions for all analytical methods used for beeswax adulteration
Three scenarios tested for the purity criteria retained. detection have been covered in this paper. As recommended by Svečnjak
Scenario tested Purity criteria Expression of the Value of the et al. (2019a), a set of several classical physico-chemical measurements
retained result parameter should always be combined (at least three). For reliable confirmation
Scenario 1 (range of Melting point 1 for the value 60–65 and quantification of adulterants in beeswax, physico-chemical mea­
value for NA (◦ C) inside the range surements should be complemented with chromatographic and/or
beeswax samples Acid value (mg and 0 for the value 17–24 spectroscopic analysis. This is in close agreement with the results of this
according to Table 1) KOH/g) outside the range study, promoting at least two physico-chemical methods (scenario 1)
Saponification 87–104
value (mg KOH/
that should be complemented by advanced analytical methods, GC-MS,
g) HTGC-FID(MS) and FTIR-ATR, respectively. In case of unavailability of
Ester value (mg 70–90 advanced analytical instrumentation (e.g. in developing countries), it is
KOH/g) recommended to use a substitutional set of at least four
Scenario 2 (cut-off Melting point 1 for the value ≥ 62
physico-chemical parameters for tentative beeswax adulteration
value to discriminate (◦ C) cut-off and 0 for the
PA and NA beeswax Acid value (mg value < cut-off 19 detection.
sampled according to KOH/g)
Table 5) Saponification 85 3.4. Validation of calibration curves for classical analytical methods
value (mg KOH/
retained for the paraffin adulteration detection
g)
Ester value (mg 63
KOH/g) The calibration curves for paraffin detection using four selected
Scenario 3 (cut-off Melting point 1 for the value ≥ 62.75 physico-chemical methods (melting point, acid value, saponification
value to discriminate (◦ C) cut-off and 0 for the value and ester value) was validated by linear regression modelling was
PA and NA beeswax Acid value (mg value < cut-off 15.55
presented in Fig. 5. These curves reflect a relationship between physico-
sampled according to KOH/g)
regression tree Saponification 75.75 chemical measurements and the known concentrations of the targeted
analysis) value (mg KOH/ adulterant (paraffin) in beeswax.
g) The correlation between each parameter and the percentage of
Ester value (mg 56.25
paraffin-beeswax adulteration were highly significant (p-value <
KOH/g)
0.0001) and between 0.9043 (for melting point) and 0.9962 (for acid
Legend: PA. paraffin adulterated beeswax samples; NA. non-adulterated value). All these parameters can be considered as satisfactory (i.e. >
beeswax samples. 0.90) for tentative detection of paraffin in beeswax. However, it should
be noted that for the purpose of detection accuracy and precision, the
It appears that the third tested scenario (i.e. cut-off of each method prediction strength <0.98 can be considered as partially satisfactory.
determined by a regression tree analysis) has a significantly higher This is supported by Bernal et al. (2005) who reported high detection
discriminatory power (higher AUC) than the two other scenarios. Also, limits for most physico-chemical analytical methods (5–50% for
the best cut-off (score) combining methods was higher with the third paraffin, 3–30% for stearic acid). This indicates that an exclusive use of
scenario and corresponds to the use of all combined methods. However, the physico-chemical parameters is not sufficient to guarantee beeswax
currently, the main weakness was the access to limited datasets, as well purity (i.e. the absence of adulterants).
as limitations mentioned in section 3.3.1. For this reason, the first sce­ Additional studies should be carried out to test the robustness of the
nario was proposed. In this scenario intended to identify a unique cut-off proposed model (ROC analysis and calibration curves) for the detection
for each method, the proposal is to use a range of values for the physico- of beeswax adulteration with stearin/stearic acid.
chemical methods that characterize non-adulterated beeswax. Because Finally, it should be highlighted that beeswax is a natural animal-
of the limited datasets currently available, this option (ranges of values) derived product that cannot be produced artificially. Thus, the apicul­
is more suited. For this scenario, it is recommended to use at least two ture sector represents the only source of genuine (pure) beeswax.
classical physico-chemical methods. In the same time, in order to Allowing the entrance of adulterants in the apicultural sector by not
consolidate the original results presented in this work, it is also rec­ performing analytical authenticity control, contaminated beeswax will
ommended to improve open access to raw data for the different physico- eventually end up in the pharmaceutical and food industry, which raises
chemical measurements. This would allow transparency and more the question of public health and food safety issue as highlighted by
robustness of the proposed purity criteria. Finally, because the beeswax monitoring the issue in the media (Rortais et al., 2020, in press). Food
trade includes Southern and Northern countries, the use, of at least, two safety is jeopardized by direct contact of beeswax of questionable origin
different physico-chemical methods in combination with more advanced with honey, as beeswax (honeycomb) represents the first natural pack­
methods like HTGC-FID (MS) or FTIR-ATR (especially in developed aging material for honey (Svečnjak et al., 2019b). Moreover, recent
countries) is recommended for reliable detection of adulterants in reports by Svečnjak et al. (2019b) on comprehensive analysis of honey
beeswax. ripened in beeswax adulterated with paraffin indicate compositional
According to Bernal et al. (2005), the most reliable combination of changes and chemical alterations in contaminated (paraffin-based)
classical physico-chemical measurements to discriminate between pure ripening media. Recently, EFSA (2020) provided a detailed report on
and adulterated beeswaxes is the determination of acid, iodine and risk assessment of beeswax adulterated with paraffin and/or stearin/­
peroxide values. Given that iodine value is not substantiated by legis­ stearic acid when used in apiculture and as food (honeycomb).
lation, nor sufficiently covered by the scientific literature, and that Considering the widespread use of beeswax (originating from apicul­
peroxide value is supported only partially (Joint FAO/WHO Expert ture) in various sectors (such as pharmaceuticals, cosmetics and food
Committee on Food Additives, 2005; Commission Regulation, 2012), industry), there is an urgent need for routine analytical testing of
these physico-chemical parameters should be validated before their use beeswax used in apiculture. This should be supported by an effective
in detecting beeswax adulteration. legislative framework. Along with the beeswax adulteration issue,
Recently, a comprehensive overview of the beeswax research and a another challenging aspect of beeswax quality in terms of pesticide
proposal for the standardization of the analytical methods used for the residues, including varroacides (Wallner, 1999; Chauzat & Faucon,
beeswax authentication was provided by the COLOSS (Prevention of 2007; Orantes-Bermejo et al., 2010; Chauzat et al., 2011; Ravoet et al.,
Honey Bee Colony Losses) network within the BEEBOOK chapter on 2015; Wilmart et al., 2016; Boi et al., 2016; Porrini et al., 2016; Kast
beeswax research (Svečnjak et al., 2019a). Detailed protocol et al., 2020), should also be considered.

9
L. Svečnjak et al. Food Control 120 (2021) 107503

Fig. 4. Receiver operating characteristic (ROC)

curve for the combination of purity criteria accord­
ing to three scenarios for the cut-off of physico-
chemical analytical methods. Legend: The three
scenarios are described in Table 6. Points are
observed values. The curve (in black) with its 95%
confidence interval (in grey) is based on fitted values
using a binomial model. Dashed line represents the
situation when the number of true positive equals
the number of false positive. In this case the area
under the curve equal 0.50 (i.e. uninformative
testing procedure). The ROC curve is plotted with
true positive results denoted as Sensitivity (Y-Axis)
against the false positive results denoted as 1-Speci­
ficity (X-Axis).

10
L. Svečnjak et al.
Fig. 5. Effect of different levels of beeswax adulteration with paraffin on the melting point [A], acid value [B], saponification value [C] and ester value [D].
A validation of analytical methods was demonstrated in this study,
with an emphasis on physico-chemcal methods as revealed numerous
challenges. This was performed on the most commonly used physico-
chemical analytical methods (supported by both the legislation and
the scientific literature) and the corresponding purity criteria based on
which a recommendation on the most reliable analysis approach was
recommended. However, these preliminary findings should be further
investigated (for other adulterants), and supported by inter-laboratory
comparisons organized and managed by an independent third party
on the EU and/or international level.
4. Conclusions
In this study, an inventory of a comprehensive set of analytical
methods (classical physico-chemical, and advanced instrumental tech­
niques) and the corresponding purity criteria used for the detection of
beeswax adulteration was established. This was achieved by retrieving
the methods using an evidence-based data (covered by both legislation
and scientific literature). The importance of selected methods was
weighted in terms of their reliability and effectiveness in detecting tar­
geted adulterants (paraffin and stearin/stearic acid). Further validation
of the analytical methods was performed by a statistical analysis based
on the available datasets on paraffin adulteration using a combination of
the four most commonly used physico-chemical analytical methods and
corresponding purity criteria (i.e. melting point, acid value, saponifi­
cation value, and ester value).
The results of the multi-analysis approach (weight analysis, ROC
analysis, construction of calibration curves) presented in this study
revealed that an exclusive use of the physico-chemical parameters is not
sufficient to guarantee the purity of beeswax in the apiculture sector (i.e.
the absence of paraffin). Therefore, for reliable detection (and quanti­
fication) of paraffin and stearin/stearic acid (or other adulterants), it is
recommended to complement at least two physico-chemical methods
with advanced instrumental analytical methods, HTGC-FID(MS) and/or
FTIR-ATR. Further validation should be supported by inter-laboratory
comparisons organized and managed by an independent third party
on the EU and/or international level.
The current categorization of beeswax (APB category 3) and the lack
11
Food Control 120 (2021) 107503
of regulations controlling its authenticity in the apiculture sector allow
the entry of various adulterants of questionable origin in apiculture. This
poses a risk to both honey bee health and human health given that
apiculture represents the only source of genuine beeswax that supplies
other sectors (pharmaceuticals, cosmetics and food industry). Thus,
there is an urgent need for routine analytical testing of beeswax used in
apiculture. This should be supported by a legislative framework gov­
erning beeswax quality (authenticity) control in the apiculture sector.
Disclaimer
The views or positions expressed in this article do not necessarily
represent in legal terms the official position of the European Food Safety
Authority (EFSA). EFSA assumes no responsibility or liability for any
errors or inaccuracies that may appear. This article does not disclose any
confidential information or data. Mention of proprietary products is
solely for the purpose of providing specific information and does not
constitute an endorsement or a recommendation by EFSA for their use.
CRediT authorship contribution statement
Lidija Svečnjak: Conceptualization, Methodology, Investigation,
Validation, Formal analysis, Resources, Writing - original draft, Visual­
ization, Writing - review & editing. Fernando M. Nunes: Investigation,
Methodology, Formal analysis, Resources, Writing - review & editing.
Raquel Garcia Matas: Investigation, Data curation, Writing - review &
editing. Jean-Pierre Cravedi: Investigation, Writing - review & editing.
Anna Christodoulidou: Investigation, Writing - review & editing.
Agnes Rortais: Investigation, Writing - review & editing, Validation,
Supervision. Claude Saegerman: Conceptualization, Methodology,
Formal analysis, Visualization, Writing - original draft, Supervision,
Validation, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
L. Svečnjak et al.
Acknowledgments
The authors wish to thank to Ewa Waś, PhD (Research Institute of
Horticulture, Apiculture Department, Puławy, Poland) for participation
in weight analysis by providing her expert opinion in weighting the
importance of analytical methods for beeswax adulteration detection.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodcont.2020.107503.
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