| Abstract | Pčelinji vosak proizvod je medonosne pčele (Apis mellifera L.) koji pčele sintetiziraju
u vlastitom organizmu. Saće u košnici građeno je od voska, a čini gnijezdo u kojemu se razvija
pčelinje leglo i mjesto u kojemu pčele pohranjuju prikupljenu hranu. Vosak se u intenzivnom
pčelarstvu kontinuirano reciklira preradom starog saća u satnu osnovu koja je nužan materijal
pri uspostavljanju proizvodnih pčelinjih zajednica kao i pri api-biotehnološkim postupcima
pripreme pčelinjih zajednica za glavne medonosne paše ili primjene biološko-tehnoloških
načina kontroliranja ektoparazitskih bolesti poput varooze. Pčelinji je vosak složena smjesa
estera viših masnih kiselina, slobodnih masnih kiselina, ugljikovodika, alkohola i drugih tvari,
a saće u košnicama liposolubilan je materijal koji poput spužve zaprima rezidue niza različitih
onečišćivača iz okoliša. U preporukama pčelarskih radova navodi se redoviti godišnji remont
za najmanje 30 do 40 % ukupnog saća pojedinog pčelinjaka, što podrazumijeva zamjenu starog
saća za novu satnu osnovu. Primjenom tehnologije lijevanja voska s produljenom fazom
hlađenja i taloženja utvrđena je mogućnost uklanjanja znatne količine teških metala iz sirovog
voska, kao i koncentriranja esencijalnih elemenata koji se troše tijekom razvoja pčelinjeg legla
u saću. Stoga je cilj ovog istraživanja bio utvrditi prisutnost i kretanje koncentracija 16
esencijalnih i toksičnih elemenata: Ag, As, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Se, V
i Zn u uzorcima voska tijekom prerade saća u satnu osnovu, primjenom analitičke metode
induktivno spregnute plazme s masenom spektrometrijom (ICP-MS) kao višeelementne analize.
Rezultati su pokazali da je kod većine elemenata primijenjena tehnika dugotrajnog
hlađenja i taloženja voska tijekom njegove prerade dala pozitivan učinak s obzirom na znatno
smanjenje visokih koncentracija, posebice teških metala i toksičnih elemenata, prilikom
odbacivanja četvrtog sedimentiranog sloja voska prije druge faze njegove prerade. Ukratko,
statistička obrada razlika u koncentracijama elemenata primjenom Kruskal-Wallisova testa
između četiri razine otopljenog voska, R1, R2, R3 i R4, uzorkovanih nakon prve faze prerade
(nakon 1. dana) pokazala je statistički značajne razlike (p < 0,05) za sve pretraživane elemente
(Ag, As, Ba, Cd, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Se, V i Zn), uz iznimku Fe, za koji je utvrđeno
da nema statistički značajne razlike (p > 0,05).
Međusobnom usporedbom koncentracija elemenata utvrđene su četiri razine voska (R1,
R2, R3, R4) uzorkovane nakon 7. dana, u drugoj fazi prerade. Utvrđene su statistički značajne
razlike (p < 0,05) za elemente Ag, As, Ba, Cd, Co, Cu, Fe, Hg, Mn, Mo, Ni, Pb, V i Zn. Iznimka
su Cr i Se za koje nisu nađene statistički značajne razlike između koncentracija četiri razine
uzorkovanja voska. Također, utvrđene su statistički značajne razlike između koncentracija
pretraživanih elemenata za iste slojeve voska uzorkovane 1. i 7. dana prerade za: Hg i Mo
za R1; Hg, Cu i Mo za R2; Ag, Cr, Hg, Zn i V za R3; Ag, As, Ba, Cd, Co, Cu, Hg, Mo, Mn,
Pb, Se, Zn i V za R4. Jedino u slučaju Fe i Ni nisu utvrđene značajne razlike između istih slojeva
uzorkovanih u ta dva navrata uzorkovanja.
Tijekom prve faze prerade voska koncentracija pretraživanih elemenata u slojevima
voska koji služe za izradu satne osnove u usporedbi sa slojem koji je odbačen iz daljnje prerade
smanjena je za: Cr 64,02 %; As 88,61 %; Hg 83,94 %; Cd 81,6 %; Pb 86,11 %; Mn 97,14 %;
Se 91,70 %; Ni 64,02 %; Co 97,1 %, Ag 93,72 %; Ba 92,89 %; Mo 89,77 %, te V 94,54 %.
Pretraživanje saća i/ili sirovog voska primjenom tehnike ICP-MS na prisutnost i kvantifikaciju
teških metala i metaloida može biti jedan od pokazatelja onečišćenja okoliša te pomoći
pčelarima pri izboru primjerene lokacije za smještaj ili preseljenje pčelinjaka. |
| Abstract (english) | Beeswax is a product of honeybees (Apis mellifera L.) which they synthesize in their
organism. It is of utmost importance for the honeybee colony's physiology and the health, and
safety of honey and bee pollen stored in honeycomb cells in which they also ripen. Honeycombs
in the hive are built of wax, forming a nest in which honeybee brood develops and where bees
store collected food, nectar, honey and bee pollen. Wax is continuously recycled in intensive
beekeeping by processing old (dark) honeycomb to comb foundations, which are a necessary
material in the establishment of productive honeybee colonies as well as technological
procedures for the preparation of honeybee colonies for the main honey pastures. It is crucial
that honeycombs, as well as food supplies in the hive, are not contaminated with xenobiotics
from the environment.
Beeswax represents a complex mixture of esters of higher fatty acids, free fatty acids,
hydrocarbons, alcohols and other substances. Honeycomb in hives represents a liposoluble
material that, like a "sponge", receives residues of many different environmental pollutants.
Therefore, beeswax is considered to be an important bioindicator when collecting data on the
degree of environmental pollution with heavy metals and/or metalloids. The recommendations
of beekeeping practice specify a regular annual overhaul for at least 30 to 40% of the total
honeycomb of each apiary, which implies the replacement of the old honeycomb for a new
comb foundation, obtained by processing wax, i.e. by melting the old honeycomb. Successful
replacement of old honeycombs with new comb foundations is crucial for achieving and
maintaining quality production and is in line with good beekeeping, veterinary and
environmental practices.
The effects of toxic elements on the reduced pollinating activity of insects, especially
honeybees, as well as their reduced longevity and survival can also be significant after their
exposure to concentrations of toxic metals, which are lower than the prescribed minimum risk
concentrations for humans. In the hive, as a result of the possible long-term accumulation of
toxic metals such as cadmium (Cd), zinc (Zn), chromium (Cr), nickel (Ni), mercury (Hg) and
arsenic (As), and the inability to decompose them in honeycombs, reproduction disorders can occur as well as other physiological functions of the honeybee colony. Some trace elements,
such as copper (Cu), zinc (Zn), manganese (Mn), iron (Fe), cobalt (Co) and selenium (Se)
represent micronutrients that are essential for the development of honeybee brood and the
functioning of the whole honeybee colony. However, in high concentrations they also become
toxic.
The usual wax processing procedures are also not sufficient for complete, or probably not
even satisfactory cleaning. In contrast, the application of beeswax casting technology with
prolonged cooling and deposition has established the possibility of removing a significant
amount of heavy metals from raw wax.
Biochemical analyzers use the spectrophotometric method to measure analytes in
combination with appropriate reagents, calibrators, and materials for quality control of sample
analysis. Spectrophotometric analyzers are analytically precise and require a high level of
repeatability. The analytical method of inductively coupled plasma with mass spectroscopy
(ICP-MS) represents a modern method used to measure metals, metalloids, and other elements
in biological samples. As an ionization source, inductively coupled plasma is used, and
detection takes place by mass spectrometry. They start from the fact that in the literature are
only a few and insufficiently confirmed results on the effectiveness of the implementation of
wax processing technologies and the quality of the final product, research on this issue is
necessary and scientifically justified. Therefore, this study aimed to determine the presence and
movement of concentrations of selected essential and toxic elements in wax samples during
honeycomb processing in comb foundations, using the ICP-MS technique as a multielement
analysis.
Wax samples were collected in a craft for processing beeswax in comb foundations.
Samples of dissolved crude beeswax were taken during the process of its processing in comb
foundations by the method of prolonged cooling and sedimentation (n = 48). The first set of
liquid wax samples was taken after 24 hours (I) from the beginning of the three-level deposition
phase directly from the tank. Samples were taken using a rake with a long handle or dropped
from the lower parts of the tank. Namely, on the tank there are two openings (taps) between
which there is a 7% altitude difference, and from which samples from layers 3 and 4 were
collected, while samples from layers 1 and 2 were collected with a rake before pouring on the
roller through which the finished comb foundations come out. The wax was taken from the
surface layer (R1a), from the middle layer of melted wax (R2a), and from the bottom (R3a) of
the tank (Figure 14). After the first sampling was carried out, a dark precipitate of contaminated beeswax (R4) was completely released from the tank, which was removed from the processing.
Then, using a pump, the remaining bright beeswax was poured into a clean steel tank and left
for re-deposition and slow cooling. The following sampling was carried out after seven days
(II) from the beginning of the repeated deposition phase of beeswax that was maintained at 75
°C. When taking the second set of samples, sampling was repeated from the surface level (R1b),
from the middle beeswax layer (R2b), and sediment on the bottom of the tank (R3b).
In laboratory conditions, 48 beeswax samples were prepared and analyzed on two occasions
(after 24 hours and after seven days) during the processing of honeycombs in comb foundations.
The samples were qualitatively and quantitatively analyzed on 16 essential and toxic elements
(Ag, As, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Se, V and Zn), using the ICP-MS
technique.
The obtained results of concentrations of analyzed elements were statistically processed by
Small Stata 13.1 (StataCorp LP, 4905 Lakeway Drive, USA). The concentrations of elements
in beeswax samples are shown as the minimum (Min) and maximum (Max), mean (SV) and
standard deviation (SD). In statistical processing, the Shapiro-Wilk test was used to test the
distribution of data, while the Kruskal-Wallis test examined differences in element
concentrations between layers R1 to R4 in the same sampling term, i.e. days 1 and 7 (I, II), and
Wilcoxon rank-sum (Mann-Whitney) test of differences in element concentrations between the
same layers sampled in the first and second phases of beeswax processing.
The results show that in most elements applied the method of long-term cooling and
deposition of wax during its processing gave a positive effect due to a significant decrease in
high concentrations, especially heavy metals and toxic elements, when removing the fourth
sedimented layer of wax before the second stage of processing.
In summary, the statistical processing of differences in element concentrations using the
Kruskal-Wallis test between the four levels of dissolved wax R1, R2, R3 and R4 sampled after
day 1 (I) of processing showed statistically significant differences (p < 0.05) for all elements
(Ag, As, Ba, Cd, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Se, V and Zn) except for Fe which was found
to have no statistically significant differences (p > 0.05).
By mutually comparing the concentrations of elements determined in four beeswax levels
(R1, R2, R3, R4) sampled after day 7 (II) in the second phase of processing, the Kruskal-Wallis
test determined statistically significant differences (p < 0.05) for the elements Ag, As, Ba, Cd,
Co, Cu, Fe, Hg, Mn, Mo, Ni, Pb, V and Zn. The exception is Cr and Se for which no statistically significant differences were found between the concentrations of four levels of beeswax
sampling.
Also, statistically significant differences were found between concentrations of analyzed
elements for the same beeswax layers sampled on day 1 (I) and day 7 (II) for Hg and Mo for
R1; Hg, Cu and Mo for R2; Ag, Cr, Hg, Zn, and V for R3; Ag, Ba, Cd, Co, Cu, Hg, Mo, Mn,
Pb, Se Zn and V for R4. Only in the case of Fe and Ni have significant differences been found
between the same layers sampled on these two occasions of sampling.
Beeswax is a honeybee product that is not food and is rarely searched from an
ecotoxicological point of view. It is mostly used in the framework of good beekeeping practices
and biosecurity measures because the success of beekeeping and the productivity of honeybee
colonies largely depend on the effective replacement of the old honeycomb and its processing
into new comb foundations. Since the honeycomb is kept in the hive for several consecutive
years, and is built of extremely liposoluble material, it can serve as a bioindicator for collecting
data on the degree of environmental pollution by hazardous pollutants. For example, heavy
metals and other toxic elements can accumulate in honeycombs over long periods, causing
unintended consequences for consumers of honeybee products or more often honeybee
colonies. With the accumulation of certain heavy metals in plants, through pollen and nectar,
pollinating insects become exposed to the harmfulness of environmental pollution. Namely, the
negative effects of heavy metals on the development and care of honeybee brood, learning and
memory, the longevity of adult bees, the overall productivity of the colony, and behavioral
patterns when taking food are known. It has been found that honeybees do not distinguish
essential micronutrients such as Zn from highly toxic elements such as Pb or As, or do not react
with altered behavior to the concentrations introduced into their body corresponding to the
relevant field doses.
Although it is known that beeswax is purified during processing, metals are particularly
resistant to high temperatures during processing and can accumulate for years since it is a
constant beekeeping practice to recycle honeycombs into comb foundations that are reused in
beekeeping production. The concentrations of most of the searched elements decreased in the
following order: R4 > R3 > R2 > R1. The established concentrations of Hg were about seven
times lower in wax samples taken from layers R1 to R3 compared to concentrations in
sedimented beeswax from layer R4. The concentration values of Pb in the beeswax layer R4,
which is excluded after the first phase of wax processing, were up to one hundred and fifty
times higher than in the light layers of beeswax layer R1, and its concentrations increased significant differences were found between the concentrations of four levels of beeswax
sampling.
Also, statistically significant differences were found between concentrations of analyzed
elements for the same beeswax layers sampled on day 1 (I) and day 7 (II) for Hg and Mo for
R1; Hg, Cu and Mo for R2; Ag, Cr, Hg, Zn, and V for R3; Ag, Ba, Cd, Co, Cu, Hg, Mo, Mn,
Pb, Se Zn and V for R4. Only in the case of Fe and Ni have significant differences been found
between the same layers sampled on these two occasions of sampling.
Beeswax is a honeybee product that is not food and is rarely searched from an
ecotoxicological point of view. It is mostly used in the framework of good beekeeping practices
and biosecurity measures because the success of beekeeping and the productivity of honeybee
colonies largely depend on the effective replacement of the old honeycomb and its processing
into new comb foundations. Since the honeycomb is kept in the hive for several consecutive
years, and is built of extremely liposoluble material, it can serve as a bioindicator for collecting
data on the degree of environmental pollution by hazardous pollutants. For example, heavy
metals and other toxic elements can accumulate in honeycombs over long periods, causing
unintended consequences for consumers of honeybee products or more often honeybee
colonies. With the accumulation of certain heavy metals in plants, through pollen and nectar,
pollinating insects become exposed to the harmfulness of environmental pollution. Namely, the
negative effects of heavy metals on the development and care of honeybee brood, learning and
memory, the longevity of adult bees, the overall productivity of the colony, and behavioral
patterns when taking food are known. It has been found that honeybees do not distinguish
essential micronutrients such as Zn from highly toxic elements such as Pb or As, or do not react
with altered behavior to the concentrations introduced into their body corresponding to the
relevant field doses.
Although it is known that beeswax is purified during processing, metals are particularly
resistant to high temperatures during processing and can accumulate for years since it is a
constant beekeeping practice to recycle honeycombs into comb foundations that are reused in
beekeeping production. The concentrations of most of the searched elements decreased in the
following order: R4 > R3 > R2 > R1. The established concentrations of Hg were about seven
times lower in wax samples taken from layers R1 to R3 compared to concentrations in
sedimented beeswax from layer R4. The concentration values of Pb in the beeswax layer R4,
which is excluded after the first phase of wax processing, were up to one hundred and fifty
times higher than in the light layers of beeswax layer R1, and its concentrations increased following: R1 < R2 < R3 < R4. Cd concentrations increased in the same order, and the
established values in lighter layers of beeswax were lower or similar to previously published
results. The content of As, Hg, Cd and Pb in beeswax layers transferred to the second stage for
further processing moved within the maximum permissible levels following the regulations for
food additives. The highest concentration of Ni was measured in beeswax samples from the R4
level, and its value was about fifty times higher compared to the measured value in beeswax
samples from layer R1. For the metals Co, Cu, Mn, Fe, Se and Zn, concentrations decreased in
the following order R4 > R3 > R2 > R1. In doing so, the mean concentrations of metals in the
beeswax layer R4 were higher compared to the concentrations determined in layers R1: Co
about 250 times, Cu about 230 times, Mn about 230 times, Fe 130 times, Se 14 times, and Zn
about 300 times.
Manganese, Se and Co are essential trace elements and micronutrients necessary for a
range of metabolic functions, but potentially toxic in larger quantities. In this study, the
concentrations of these tested elements also decreased in order: R4 > R3 > R2 > R1. In doing
so, the mean values of lighter layers of beeswax (R1-R3) did not differ statistically significantly
from the values in the same layers of the second phase of beeswax processing. However,
compared to lighter layers after day 1 of beeswax sedimentation in the tank, they were found to
be 97.14% (Mn); 91.70% (Se); and 97.1% (Co) of a higher concentration of an element in the
R4 layer that was excluded from further processing. After day 7 of sedimentation, an additional
12.84% (Mn) was excreted from processing compared to the first phase of processing in R4;
15.621% (Se); and 8.25% (Co).
For the group of elements Ag, Ba, Mo and V, according to our knowledge, there is no
published data available so far. For all of these elements, a statistically significant difference in
the concentrations of analyzed elements was found between the four beeswax levels during the
first and second phases of honeycomb processing into comb foundations (p < 0.05). In doing
so, in layer R4 compared to lighter layers of beeswax (R1 – R3) after 24 hours of sedimentation,
93.72% (Ag) was determined; by 92.89% (Ba); for 89.77% (Mo) and 94.54% (V) higher
concentration of individual elements. |
