Comparison of transfer of different sulphonamides from contaminated beeswax to honey

The honey bee (Apis mellifera L.) population, and thus the manufacture of honey and other apiculture products, may significantly decline because of bacterial or protozoal diseases in honey bees. American and European foulbrood are the diseases that affect adult honey bees most severely and widely. They cause significant losses in apiaries and are a grave economic problem. These infections in bee colonies have usually been controlled by using sulphonamides (1). However, no maximum residue limits (MRLs) in honey were set for sulfonamides or other antimicrobial substances in the Commission Regulation (EU) No. 37/2010 (3), and thus, they are not permitted for treating honey bees. They can only be applied in apiculture within a cascade system (6), according to which the veterinarian prescribes only a veterinary medicinal product with an allowed pharmacologically active substance and sets a withdrawal period long enough to guarantee that the honey does not contain residues in amounts harmful to human health. Although this rule has been adopted, 14 honey samples monitored under Directive 96/23/EC were non-compliant for antibacterial residues in 2021 (4). Honey was the most frequently non-compliant animal product with antibacterials (0.96%). The antibacterial classes with the highest instances in honey were sulphonamides and tetracyclines. Most of these non-compliant results were due to the presence of sulphamethazine, sulphathiazole, sulphacetamide, sulphachloropyrazine, sulphadimethoxine, and sulphamonomethoxine.

Depletion of residues of pharmacologically active substances in honey is not time-dependent, in contrast to their depletion in mammalian or avian organisms due to pharmacokinetic behaviour. When the residues are found in honey, they mostly stay there (5). Because of this, using sulphonamides illegally may cause their residue accumulation in honey and other apiculture products like beeswax (7, 10). Apart from honey, beeswax is a valuable beehive product. It is used by the food, cosmetic and pharmaceutical industries in a wide range of applications that require high-quality beeswax. The product is classified as an authorised food additive (E 901) in the EU and is listed in the European Pharmacopoeia (2). Since it is a natural product, there should not be any additives in it.

However, beeswax can dissolve or integrate toxic compounds, which could be released long afterwards when the beeswax is used to produce pharmaceuticals or cosmetics, is eaten, or is given to honey bees as a wax foundation. Previous work has focused only on sulphamethazine, which can contaminate honey during the next season if it stays in the comb’s wax after being used in the hive (11). To the best of our knowledge, no similar studies have been carried out for other sulphonamides. Therefore, the aim of the research was to compare the migration of 10 sulphonamides from contaminated beeswax to honey.

In order to have comparable results, the sulphonamide-tainted wax foundations were prepared in the same way as drug-containing wax foundations used in similar experiments (10, 12) by adding analytes at 10,000 μg/kg to blank beeswax melted at 80°C. Although the melting point for beeswax is 62–64°C, increasing the temperature to 80°C was necessary to contaminate the beeswax homogeneously without subjecting the added substances to high temperatures over an extended period. As it transpired, some portions of the added sulphonamides were lost nevertheless, because of thermal degradation. Sulphamonomethoxine was the most stable sulphonamide and degraded 47%, while SMT was revealed to be the least stable, with a loss of 80%. Our results are in line with those obtained by Reybroeck et al. (11), in which 62% of SMZ was lost in the preparation of wax foundation because of thermal degradation: in our experiment, 59% SMZ was degraded (Table 2). Sulphonamides were less stable than nitroimidazoles such as metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ) and ipronidazole (IPZ), which only degraded in the range from 3 to 28% in an analogous study (9).

The analysis of sulphonamides in honey indicated that all the tested compounds could be transferred from contaminated beeswax to honey. During the experiment, the sulphonamide levels in honey appeared to be relatively stable despite their quantities declining subsequent to the four-month point after reaching earlier maximum concentrations. The observed decrease in sulphonamide concentrations after four months could be attributed to the possible degradation of compounds in both honey and beeswax. The stability data obtained by Posyniak et al. (10) showed that the concentration of STZ, SCA and SMZ in honey was not significantly affected by an incubation period of at least 28 days at 34°C, but the stability of sulphonamides in beeswax is unknown. In a similar experiment that lasted four months, the SMZ concentration increased during the first month that the frames were kept in the incubator, while from the second month, the SMZ residues in honey were rather constant (11). When comparing the initial quantity of sulphonamides that was transferred from contaminated beeswax foundations to honey, SDM showed the largest transfer rate (42.6%), whilst SCA showed the lowest (8.6%) (Table 3). Although Reybroeck et al. (11) found that SMZ transfer to honey from beeswax contaminated at the same initial spiking level was 56.9%, we found a lower transfer rate for this antimicrobial of 25.5%. It was also found that in the same conditions, the maximum transfer of the tested sulphonamides was lower than that of MNZ (89.4%), RNZ (54.6%), 2-hydroxymethyl-1-methyl-5-nitroimidazole (79.4%) and hydroxymetronidazole (99.5%) and higher than that of DMZ (2.7%) and IPZ (2.0%) (9). It should be taken into account that the calculations assumed no analyte losses in honey and beeswax during all the transfer studies. However, the different stability levels among the sulphonamides at 35°C might have impacted calculated transfer values, just as the low level of sulphonamides in honey might be due to low thermal resistance.

According to Martinello et al. (7), when sulphonamides were applied to honey bee colonies, beeswax was more contaminated than honey and the honey bees. The physicochemical properties of sulphonamides determine their accumulation in the different parts of the hive. For the prediction of sulphonamides’ physicochemical properties such as pKa and lipophilicity, Calculators & Predictors software was used (Chemaxon, Budapest, Hungary). Sulphonamides have an amphoteric nature with at least two pKa values, meaning that they can behave either as an acid or a base, depending on the pH of the medium. The predicted sulphonamide values of pKa1, the strongest basic value, were in a range from 1.95 (SDM) to 2.17 (SMM), while those of pKa2, the strongest acidic value, were in a range from 5.60 (SCA) to 7.15 (SMM) (Table 4). At the pH of honey, which in the study was 4.2, 95.3–99.4% of the particles of the sulphonamides were in unionised form (Table 4). Honey is a hydrophilic sugar solution, and beeswax is a hydrophobic substance; therefore, sulphonamide lipophilicity is influential upon their redistribution between the two. The predicted values of the logarithm of the n-octanol/water partition coefficient (logP) of the sulphonamides were in a range from −0.26 (SCA) to 1.26 (SDM) (Table 4). Since the sulphonamides in honey are dominantly present in their neutral forms, their lipophilicity in this matrix is not affected by the pH. The values of the logarithm of the n-octanol/water distribution coefficient (logD), which quantifies the degree of ionisation at a given pH, are consequently nearly the same as logP. A compound with a negative logP value is more hydrophilic, while a positive value means a more lipophilic compound. By their predicted logP values, all the tested sulphonamides except SDM were considered hydrophilic and could be expected to accumulate in honey, whereas lipophilic SDM should tend to accumulate in beeswax. However, in our experiment, lipophilic SDM with logP of 1.26 transferred in the highest proportion (42.6%) from contaminated beeswax foundations to honey, while hydrophilic SCA with logP of −0.26 did so in the lowest (8.6%) (Table 3). This might be explained by sulphonamides having different stabilities and by the possible loss of analytes in honey and beeswax that could have occurred during the experiment. Additionally, since there is not an experimentally determined logP value available for every sulphonamide and the calculated logP values were a wide span, it was impossible to predict these compounds’ beeswax/honey partitioning correctly.

Because residues of sulphonamides, as well as other veterinary substances and plant protection products, could be transferred from contaminated wax combs to stored honey and pose a health risk to consumers, Wilmart et al. (12) suggested that action limits should be applied to the presence of residues in beeswax in order to regulate possible dangers in the food chain. This suggestion is supported by the present researchers. It is also recommended that beekeepers either recycle their beeswax for use in the production of wax foundations or demand a certificate when purchasing wax foundations from commercial operations that convert wax, as most of these facilities utilise wax with unknown origins.

This investigation’s findings lead us to conclude that every tested sulphonamide can migrate from beeswax in combs to honey stored in those combs, with the highest migration potential for SDM and the lowest for SCA. Consequently, honey contamination may occur through the use of sulphonamide-contaminated beeswax in wax foundations. Therefore, sulphonamides need to be monitored in this matrix to guarantee the high safety and quality of beeswax as a product, beeswax as comb foundation, and honey.