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.