Removal of Neonicotinoid Residues from Beeswax Using an Eco-Friendly Oxalic Acid Treatment: A Sustainable Solution for Apicultural Decontamination

Abstract

Beeswax is widely used in apiculture and can accumulate neonicotinoid residues due to the intensive use of systemic pesticides in agriculture. These contaminants pose potential risks to honeybee health and may indirectly affect the quality and safety of hive products such as honey, pollen, and royal jelly. This study evaluates several decontamination methods for neonicotinoid removal from contaminated beeswax, including modern techniques (microwaves, ultrasonic baths, and magnetic stirring with heating) and conventional approaches based on heat, agitation, and water—either pure or acidified. Among these, the traditional method that uses an aqueous oxalic acid solution proved highly effective, removing over 99% of neonicotinoid residues after two treatment cycles, even at wax quantities up to 200 g. The treatment also improved the colour and physical properties of the wax and was well tolerated by bees, according to a qualitative acceptance test. The simplicity, low cost, and absence of hazardous by-products make this method suitable for scale-up and adoption in real apicultural practices. These findings support the development of accessible and sustainable strategies for the decontamination of wax matrices that may otherwise act as long-term reservoirs of pesticide residues in the food chain.

1. Introduction

Bees play a crucial role in global ecosystems and agricultural productivity, as they are the primary pollinators responsible for the reproduction of many plants, essential both for the growth and development of natural wild flora and for crops of human interest [1]. Despite their proven and recognized importance, the accelerated global decline of bee populations has become a serious threat to biodiversity, food security, and agricultural sustainability [2].

The destruction of their natural habitat, deforestation, global climate change, diseases, infections by mites and pathogens, and the widespread use of pesticides are some of the main stressors that negatively influence the quality of life of bees [3,4,5,6,7]. The intensive and uncontrolled global application of pesticides has become a determining factor in the loss of bee colonies [5,8,9].

The persistence of pesticides in beeswax poses a significant problem because this product acts as a natural bioaccumulator of contaminants [10,11,12], which could be affecting the bee population. Studies have shown that pesticides can migrate from beeswax to other products, such as honey and propolis [13,14,15]. These matrices are capable of retaining pesticides for a prolonged period [8,16,17]. Within the broad spectrum of pesticides, neonicotinoids are one of the most widely used families of insecticides in the world [18,19] and have been associated with the disappearance of honeybees throughout the world [20,21,22].

Residue of neonicotinoid insecticides has been found in different bee products, such as honey [23,24,25,26,27], bee bread [28,29,30,31], and beeswax [5,26,28,29,30,31,32].

Bees are affected by these types of compounds [28,33,34,35,36,37,38,39], with sublethal effects such as loss of olfactory memory, alterations in brain metabolism [40,41], lack of activity and communication capacity [42,43], and low foraging activity of bees and within the hive [44,45]. Additionally, significant reductions in bee weight [39] and shorter durations of visits during larval development [46] are among the most commonly reported effects. These problems are also observed in bumblebees and solitary bees, which attribute their low reproductive rates and reduced activity to neonicotinoids, especially imidacloprid [47].

The process of recovering beeswax for reuse in hives, as well as its various uses as a raw material in the manufacture of products of human interest, has been carried out for over 1500 years [48]. In the beekeeping industry, one of the most important applications is stamped beeswax, which is obtained after cleaning the beeswax by decantation, followed by the formation of sheets and stamping to achieve the hexagonal forms of the cells [49,50].

Proper cleaning and purification of beeswax would ensure that none of the contaminant residues that accumulate in it negatively impact the health and quality of life of animals or humans. Very few cleaning and decontamination methods have been proposed in scientific literature. These are mostly laboratory-scale and are primarily used for small-scale preparation of beeswax samples for analytical quantification or monitoring.

The most efficient beeswax cleaning and purification methods reported to date are those developed by [51,52,53]. Solvent extraction and adsorption using porous solids are the chemical principles underlying these approaches, enabling the removal of up to 95% of the pesticides present in the beeswax. These proposals involve several stages, complex equipment, challenging operations, and require working with large volumes of hazardous chemical compounds. Therefore, they are considered unsafe and challenging to apply in the daily work of small-scale beekeepers, who are primarily interested in simple and practical methods that ensure the reuse of beeswax and remove contaminants.

For small- and medium-scale beekeepers, and even for large beekeeping enterprises, the usual or traditional procedure for cleaning and bleaching beeswax consists of melting the wax in a double boiler at a temperature above 65 °C with water and acids (oxalic, citric, tartaric, or mixtures of these) [13,54]. The molten beeswax is poured into a container and partially submerged in warm water, while slowly mixing, and the impurities are scraped off the surface. This method has been practiced for thousands of years and passed down through generations of beekeeping families, making them more familiar with these substances and their use. Unfortunately, there is very little scientific evidence to support the effectiveness of such traditional cleaning methods in removing contaminants from beeswax. Most of the available information is informal and found on beekeeper blogs or websites. Notably, none of these procedures report pesticide removal percentages, types of contaminants removed, or results regarding the effects of these traditional methods on beeswax colour or in the subsequent acceptance of the clean wax by the bees.

This article aims to contribute to the current knowledge on beeswax cleaning and bleaching.

The study evaluated the effectiveness of different cleaning procedures, ranging from traditional methods to more recent approaches that involve a conventional microwave, an ultrasonic bath, and a magnetic stirrer with an aluminum heating plate, in removing seven common neonicotinoid pesticides from beeswax (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam). The ultimate goal is to identify a low-cost, simple, and safer alternative process for reusing beeswax free of neonicotinoid residues, which can be easily replicated by beekeepers.

2. Methodology

2.1. Materials and Chemicals

Seven neonicotinoid standards (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). LC-grade solvents, such as methanol, acetonitrile, and isopropanol, and pestican-grade solvents like n-hexane and acetone were supplied by Lab Scan Ltd. (Dublin, Ireland). Formic acid (98–100%) was purchased from Sigma–Aldrich Chemie GmbH (Steinheim, Germany).

Diatomaceous earth-packed cartridges (Isolute^® HM-N, 10 mL sample) were obtained from Biotage (Uppsala, Sweden). A 12-port solid–liquid extraction vacuum manifold from Waters Corporation (Milford, MA, USA) was used for the extractions. Auxiliary equipment included a magnetic shaker-heater (Agimatic-N), a magnetic stirrer with an aluminum heating plate (ARE-Velp Scientifica), and an ultrasonic bath (Ultrasons), all supplied by J.P. Selecta S.A. (Barcelona, Spain). An R-210/215 rotary evaporator from Buchi (Flawil, Switzerland) was also employed. Furthermore, a variable-power microwave (Panasonic NN-GD39) from Panasonic (Barcelona, Spain) was used for some of the solvent extraction methods. Nylon syringe filters (17 mm, 0.45 μm) were purchased from Nalgene (Rochester, NY, USA). Ultrapure water was obtained using Millipore Milli-RO plus and Milli-Q systems (Bedford, MA, USA).

2.2. Beeswax Sample

Blank beeswax samples (neonicotinoid-free) from ecological apiaries were provided by the Centro Apícola Regional (CAR) of Marchamalo (Guadalajara, Spain). All samples were stored in the dark at 4 °C prior to analysis. Commercial beeswax samples were provided by Agroapícola S.A. (Villa Alemana, Chile) for purification and tests in beehives.

2.3. Standard Solutions

Standard stock solutions were prepared by dissolving approximately 10 mg of powder of each neonicotinoid in 10 mL of acetone (pestican-grade) at a final concentration of 1000 mg L⁻¹. These solutions were further diluted with the same solvent to prepare the working solutions. Then blank beeswax samples were spiked with standard amounts of 1000 µg kg⁻¹ of each neonicotinoid, and it was necessary to melt the beeswax when spiking with the neonicotinoid solutions to obtain homogeneous samples. This procedure has proven to be effective in previously published studies by Yáñez et al. [31].

Once the beeswax was spiked, it was mixed for 10 s to evaporate the solvent and homogenize the mixture. It was then removed from the hot bath, allowed to solidify at room temperature, and stored protected from light for subsequent cleaning tests. All samples, standard stocks, and working solutions were stored in amber glass containers at 4 °C and were stable for over one month.

2.4. LC-MS System

An Agilent Technologies (Palo Alto, CA, USA) 1100 series LC-MS system was used, consisting of a vacuum degasser, a quaternary solvent pump, an autosampler, a column oven, and a single quadrupole MS analyser with an electrospray (ESI) interface; control was by means of Agilent ChemStation software (v4.03.016).

Analyses were performed using a Kinetex^® column (2.6 μm C18, 150 × 4.6 mm i.d.), with a C18 guard column (4 × 2.0 mm i.d.); both were obtained from Phenomenex (Torrance, CA, USA). The mobile phase previously optimized and selected was 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) applied at a flow rate of 0.5 mL min⁻¹ in the gradient mode using the same methodology described by Yáñez et al. [31]. The injection volume and column temperature were set at 15 µL and 35 °C, respectively. The ESI interface was operated in positive mode. The most abundant ion of each compound was quantified in selected ion monitoring (SIM) mode, and two more ions were used to confirm the presence of each analyte as described in Yáñez et al. [31].

2.5. Sample Preparation and Analysis

The extraction conditions for beeswax were determined according to the studies described by Yáñez et al. [31]. Briefly, 2 g of the homogenized beeswax sample and 15 mL of the n-hexane/isopropanol mixture (8:2, v/v) were transferred to a glass beaker. The beeswax was dissolved after the mixture had been heated for 3 min at 50 °C, then 10 mL of water was added, and the mixture was centrifuged for 5 min (50 °C and 700 rpm). Subsequently, the aqueous phase (10 mL) was separated and loaded into the cartridges of diatomaceous earth. After 15 min, the analytes were eluted with acetone (20 mL). The extract was evaporated to dryness in a rotary evaporator and then reconstituted in 1 mL of a 50:50 (v/v) solution of water and acetonitrile. Finally, the extract was filtered using a syringe filter, and a 15 μL aliquot was injected into the LC-MS. The analytical methodology was validated in terms of selectivity, limits of quantification (LOQ), limits of detection (LOD), linearity, precision, and recovery, following different international guidelines described by Yáñez et al. [31].

2.6. Cleaning Methods (New and Traditional)

Four beeswax cleaning methods were tested to evaluate their ability to remove contaminating neonicotinoids. The first two methods are novel exploratory trials and include the use of water as an extraction solvent combined with physical treatments based on emerging sustainable technologies such as ultrasound and microwaves. These technologies use high-frequency mechanical waves, potentially improving the extraction of some substances (bath ultrasound), and electromagnetic waves to uniformly raise the temperature of the wax (microwave oven). The effects of one and two cleanings on neonicotinoid removal were analysed, as well as the exposure times in both technologies and the microwave power. These methods do not include stirring or preheating.

The other two methods evaluated are traditional methods commonly used by beekeepers, for which there is little evidence in the literature regarding their effectiveness in removing neonicotinoids, bleaching beeswax used in hives, and the acceptance of wax cleaned by bees [13,54]. These methods include cleaning using only water as a solvent and acidified water, with the stirring stage, and heating. Due to the efficiency of these traditional methods using acidified water in removing contaminants, it was not necessary to perform two cleanings on a small scale (5 g of beeswax). A detailed summary of the four methods and their variants (13 in total), as well as the main variables, equipment, and nomenclature used, is presented in

Table 1. Methods of cleaning beeswax, different solvents, equipment, and variables.

New Methods	Solvent	Equipment	Temp. or Power	Time (min)	Cleanings	Acronyms
Ultrasound + Water	Water	Ultrasonic bath	40 °C	40	I	USW/I
				80	II	USW/II
Microwave + Water	Water	Microwave oven	70 W	4	I	MW70W/I
			350 W	1	I	MW350W/I
			700 W	0.5	I	MW700W/I
			70 W	4	II	MW70W/II
			350 W	1	II	MW350W/II
			700 W	0.5	II	MW700W/II
Traditional methods	Solvent	Equipment	Temp. or power	Time (min)	Cleanings	Acronyms
Stirring + Heating + Water	Water	Magnetic Stirrer (700 rpm)	70 °C	10	I	SHW/I
					II	SHW/II
Stirring + Heating + Water + Acid	Water/Sulfuric acid (0.1% v/v)				I	SHWSA
	Water/Citric acid (0.25% v/v)					SHWCA
	Water/Oxalic acid (0.25% v/v)					SHWOA

.

The methods that showed the highest percentages of contaminant removal were selected to perform an analysis of the wax coloration (bleaching) before and after the proposed treatments, through photography. Then, the most effective method, both in the removal of contaminants and in the bleaching of the wax, was selected to perform an evaluation of its efficiency in larger quantities of wax, 50 g and 200 g. Also, with the purified wax resulting from the application of the most effective method in larger quantities, an evaluation of the acceptance by bees in experimental hives was carried out. At different stages of the experiment, 5 g, 50 g and 200 g of wax were cleaned using solvent volumes of 25 mL, 250 mL and 1000 mL, respectively. Three replicates were performed for each treatment tested. A summary of the research stages is shown in Figure 1.

2.7. Determination of Neonicotinoid Removal After Cleaning

After each cleaning, 2 g of blank beeswax spiked at 1000 µg kg⁻¹ (unpurified beeswax) and 2 g of purified beeswax were treated using the sample preparation described in Section 2.5 and were analysed using the analytical methodology validated and described in Yáñez et al. [31]. With these results, comparisons were made to determine the most efficient method for decontaminating and bleaching the wax. The effectiveness of the cleaning methods was evaluated by measuring the removal of pesticides for all seven neonicotinoids studied. The concentrations of each neonicotinoid (N_n) found in the unpurified beeswax and purified beeswax were measured using the sample preparation and the validated LC-MS analytical methodology. The percentages of removal of neonicotinoids were calculated according to Equation (1).

$${\mathrm{R}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{v}\mathrm{a}\mathrm{l}}_{{\mathrm{N}}_{\mathrm{n}}}\left(\mathbf{\%}\right)=100-\left({\displaystyle \frac{{\mathrm{C}}_{{\mathrm{p}\mathrm{b}}_{\mathrm{n}}}}{{\mathrm{C}}_{{\mathrm{u}\mathrm{b}}_{\mathrm{n}}}}}\times 100\right)$$

(1)

where N_n is each neonicotinoid studied, C_pbn is the neonicotinoid concentration (µg kg⁻¹) in purified beeswax using different cleaning methodologies (

Table 2. Qualitative indicators, their scoring scale, and the proposed acceptance levels.

Activity	Day	Point	Criteria and Scale Used
Initial activity	5	0	No bees inspecting the wax.
		1	Some bees inspecting. Less than 25% of the frame with bees.
		2	Moderate activity. Between 25 and 50% of the frame with bees.
		3	High activity. More than 50% of the frame with bees.
Beeswax stretching	20	0	No visible change in wax stretch.
		1	Minor changes. Less than 25% of the frame stretched.
		2	Moderate changes. Between 25 and 50% of the frame stretched.
		3	Significant progress. More than 50% of the frame stretched.
Final use	45	0	No cells occupied (brood, pollen, and/or nectar)
		1	Less than 25% of cells occupied (brood, pollen, and/or nectar)
		2	Between 25 and 50% of cells occupied (brood, pollen, and/or nectar)
		3	More than 50% of cells occupied (brood, pollen, and/or nectar)
Total Score in points			0	1–3	4–6	7–9
Level Acceptance			Reject	Low	Medium	High

), and C_ubn is the neonicotinoid concentration in unpurified beeswax (µg kg⁻¹). The main indicator in the comparisons made was the Mean removal efficiency, which considers the average of the individual removal of the seven neonicotinoids analysed. This evaluates the overall cleaning and purification capacity of each method, rather than the removal efficiency of each pesticide.

2.8. Analysis of the Beeswax Coloration After Cleaning (Bleaching)

After cleaning, the wax mass from each replicate (3) was combined and homogenized to obtain a representative quantity of cleaned wax. This allowed for the creation of a solid block with a diameter of 20 mm and a thickness of 5 mm for each cleaning method. This ensured uniformity and a thickness suitable for colour analysis. To evaluate the effect of cleaning methods on the colour of beeswax, photographs were taken of solid blocks of wax before and after cleaning with an LG Electronics model GW520 camera. The level of bleaching obtained and colour changes were measured using the CIELAB spectrum-based colour analysis method. The CIELAB colour space, defined by the International Commission on Illumination (CIE) in 1976, expresses colour in three coordinates [55]. The first, called luminosity (L*), ranges from 0 (black) to 100 (white). The second coordinate or parameter represents the position of the colour between red and green (a*), with negative values indicating closer to green and positive values indicating closer to red. The last parameter represents the position of the colour between yellow and blue (b*), with negative values indicating blue and positive values indicating yellow [56].

The total colour difference between images (ΔE) was calculated using Equation (2) [57], and the whiteness index (WI) was calculated using Equation (3) [58].

$$\Delta E=\sqrt{{\left({\Delta L}^{\mathrm{*}}\right)}^2+{\left({\Delta a}^{\mathrm{*}}\right)}^2+{\left({\Delta b}^{\mathrm{*}}\right)}^2}$$

(2)

$$WI=100-\sqrt{{\left({100-L}^{\mathrm{*}}\right)}^2+{\left(a^{\mathrm{*}}\right)}^2+{\left(b^{\mathrm{*}}\right)}^2}$$

(3)

where ${\Delta L}^{*}$, ${\Delta a}^{*}$, and ${\Delta b}^{*}$ are the differences between the images of the treated wax and the contaminated control wax for each colour value.

These indicators have been used to evaluate colour changes and whitening quality in other matrices such as wood [56], fruit [59], and teeth [60], but never to compare photographs of purified beeswax, which highlights the novelty of this research. Image processing and parameter calculation for image comparison were performed using Python 3.0 programming language and executed on Google Colab. The OpenCV (v4.12.0), NumPy (v2.0.2), Matplotlib (v3.10.0), scikit-image (v0.25.0), and Pandas (v2.1.4) libraries were used. OpenCV was used for image manipulation and colour space conversion. Numerical calculations and array handling were performed with NumPy. Results visualization and graph generation were performed using Matplotlib. Additionally, the Pandas library was used for storing and manipulating tabular data, including the creation of data frames and spreadsheets. The scikit-image library was also used to convert images to the CIELAB colour space. All resulting data were saved in an Excel file.

2.9. Evaluation of Purified Beeswax in Experimental Hives

Once the wax was purified (200 g for triplicate), it was left to cool at room temperature, removed from the cleaning solvent, and dried to continue to the stamping phase. For this, the clean wax was melted in a hot bath at 70 °C and was poured into the double silicone to stamp the hexagon cells (Figure 2A); after a few minutes, the wax was carefully removed from the mold and embedded in wood frames (Figure 2B). Finally, each frame with pure wax was labelled and placed in the experimental hives of the USM Bee Lab located on campus.

The beeswax, after undergoing a decontamination process, must meet certain physical and chemical properties characteristic of this type of material. However, the most important thing is that it must meet the needs of the bees. Therefore, a fundamental requirement of any research attempting to clean and/or modify beeswax for reuse in honey production is to evaluate the level of acceptance or rejection of frames stamped with the modified waxes in the hive.

There are a few methods reported in the scientific literature that evaluate the acceptance of different types of wax by bees. Currently, these methods have been carried out primarily using quantitative methods that collect data from frequent visual inspections of the hive, applying technologies that include photography with high-performance cameras and software for processing these images, among others. The purpose is to collect data and provide quantitative information that allows comparing how the bees’ behaviour has been with the different samples of stamped waxes. In these evaluations, data and information are collected on parameters such as start of honeycomb construction, start of egg laying, start of cell capping, area occupied by brood, area occupied by honey, area of cells occupied by pollen, area and percentage of tilling, and viability of brood, among others [61,62]. All these variables are very important and allow comparisons between the behaviour of bees in the hive with virgin wax as a control and with waxes treated in a chemical cleaning process. However, these methods require advanced and expensive technological equipment, as well as digital software [61,62], which could make them difficult for ordinary beekeepers to use and implement. Furthermore, these methods require frequent inspections and photographs (every 48 h) of the hive frames, which could cause stress to the hive, disrupt the results, and reduce honey production.

Therefore, there is a need for new, simple methods for assessing the level of wax acceptance in hives, preferably qualitative ones that do not depend on frequent hive inspections and that do not require advanced knowledge and technology for their application by beekeepers and researchers. This research proposes a new, simple, qualitative method for assessing bee acceptance of clean or decontaminated wax in hives.

The study was conducted in three experimental Langstroth hives, in each one, three frames with the stamped purified wax (positions 2, 3, 4) and three frames with control wax (6,7,8) were inserted, respecting the symmetry of the hive, which had a total of 9 frames. The hives located in the experimental apiary of the USM Bee Lab at Universidad Técnica Federico Santa María had similar populations of honeybees (Apis mellifera mellifera) and uniform environmental conditions. The study was conducted over 45 days between spring and summer (November and December), where the ambient temperature ranged from 23 to 30 °C.

Visual inspections of the hives were conducted on days 5, 20, and 45. This selection was made based on the behaviour and work dynamics of the bees in the hive. At 5 days, a rapid, visible, and representative initial response from the bees is expected. Day 20, coinciding with the average age at which worker bees begin to produce wax and build combs [13], allows for the assessment of stretch progress and the functional use of the comb for brood and resource storage. Finally, day 45 provides insight into the long-term acceptance, consolidation, and durability of the wax, having elapsed at least two complete brood cycles (21 days) [63], which is vital to ensure the sustained integration of the comb into the hive and its impact on overall productivity. With just three observations spaced out over time, stress, discomfort, and risks to the hive are minimized; this is a primary objective of new methods for evaluating wax acceptance in hives [61]. In the observations made, only qualitative information was collected, and values were assigned to the different proposed indicators, so they do not require advanced technologies such as digital cameras or computers for processing.

The qualitative indicators collected reflect how bees interact with the introduced waxes and were: Initial activity (general inspection behaviour such as movement and active exploration), Beeswax stretching (changes in the wax surface such as stretching, perforation, remodelling, or smoothing), and Final use (cells used for brood, honey, or pollen). A full summary of these indicators, their scoring scale, and the proposed acceptance levels is shown in Table 2.

One of the central objectives of the proposed evaluation method is to ensure its simplicity and accessibility, enabling its application by any beekeeper without requiring complex technological equipment. To facilitate implementation, minimize inter-observer bias, and maintain reproducibility, simple scale values and criteria were selected, allowing for a practical assessment of wax quality. In this context, for the semi-quantitative evaluation, a scoring system based on a three-point ordinal scale (0 to 3) adapted from Spivak and Reuter [64] was implemented. This was complemented by a simple percentage scale based on quartiles (0 = 0%, 1 = <25%, 2 = 25–50%, 3 = >50%) adapted from Arvidsson [65]. The modifications made connect the numerical scales with beekeeping terminology and the main evaluation criteria used in hive management, ensuring that the method remains simple, objective, and effective.

The total sum of the three indicators in each hive is summarized as the Total Score, with values ranging from 0 to 9. Based on the values of this variable, four levels of acceptance of the wax by bees in the hives were defined. Using this method, comparisons were made between virgin beeswax, used as a control, and cleaned beeswax.

3. Results and Discussion

The main objective of this study was to evaluate different beeswax cleaning methods. The methods analysed ranged from novel methods, employing emerging technologies, to traditional methods used mostly by medium- and small-scale beekeepers. The overall removal efficiency of seven of the most reported neonicotinoids in the different hive matrices (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) was determined. Based on this information, the methods used were compared, and the most successful were selected to evaluate their effect on wax bleaching. Then, using the method with the best results for both pesticide removal and wax bleaching, a bee acceptance test of the cleaned wax was conducted.

3.1. Determination of Contaminants in Cleaned Beeswax with the New Proposed Methods

Two new methods for purifying and cleaning beeswax were proposed and evaluated in this study. The new treatments are based on the use of only water as a solvent, without agitation, and employ mechanical (sound) and electromagnetic waves to enhance the extraction of contaminants.

For the application of mechanical (sound) waves, an ultrasonic bath at room temperature and maximum power (40 °C and 110 W) was used. This technique was chosen because several reports suggest that sound waves are one of the most promising physical methods for intensifying extraction processes. It has been used successfully in the extraction of active compounds from plant products and in the food industry [66].

The results obtained for the overall removal of neonicotinoids using water as a solvent, an ultrasonic bath, and up to two cleanings are shown in Figure 3. The removal rates for each pesticide are also highlighted for the best overall result.

The application of water and ultrasound as methods for removing pesticides from wax resulted in average total removal values below 32%. The highest extraction was obtained by applying two cleanings; however, the value obtained is much lower than the expected results and those obtained by other authors using other solvent-based methods, such as methanol and adsorbents such as activated carbon and diatomaceous earth [52,53]. Similar treatments, including water washing and ultrasonic equipment, have been successfully used to remove pesticides from cereal grains [67], the surface of fresh vegetables [68], and fruits such as strawberries [69]. In all these studies, the pesticides were only present on the surface of the samples, making their removal more efficient. In the case of beeswax, pesticides are likely to diffuse throughout the honeycomb system and accumulate within the solid matrix, not just on its surface, making their removal more complex.

Analysing the removal percentage of each neonicotinoid, it can be concluded that only nitenpyram is eliminated or decomposed during this process, as it reaches a removal percentage of more than 93%. The persistence of the other neonicotinoids or the low efficiency in their removal can be explained by the fact that the maximum temperature reached during this extraction method is 40 °C, a temperature at which the wax remains in a solid state, hindering contact and interaction with the cleaning solvent, which makes the elimination of contaminants difficult. Another negative variable of this method is the long times used (up to 80 min), and despite this, good removal of the analysed contaminants is not achieved. From these results, it can be inferred that wax purification methods are needed that include, among their variables, temperatures higher than that of wax melting and constant agitation that guarantees adequate mixing, contact, and interaction of the solvent with the wax.

The effect of electromagnetic waves on the removal of these residues was also evaluated, using water as a solvent and a standard microwave oven. Three microwave power levels were studied, with varying times determined by the speed of complete wax melting. The results obtained for overall removal efficiency using this method can be seen in Figure 4.

The use of electromagnetic waves increased contaminant removal compared to sound waves, achieving overall removal rates for the seven neonicotinoids of more than 49% in all cases. Performing two consecutive cleanings guarantees a considerable increase in the removal rates of these contaminants. The best results in wax cleaning were achieved using a power of 350 W and two consecutive cleanings. Under these conditions, removal rates exceeding 67% were achieved for each of the pesticides analysed, with nitenpyram again demonstrating the highest removal rates. This method offers several advantages over previously reported methods, including more than 67% of the seven neonicotinoids can be removed using only water as a solvent; the process is completed in a short time (1 min per cleaning); the use of chemical reagents is not necessary; and household microwaves that meet the specified power requirements can be used, among others. The disadvantages of this method are that it requires a higher power consumption depending on the amount of wax being cleaned; up to 0.5 kg of wax must be cleaned at a time, so it would be difficult to implement on a large scale unless this technology were scaled from laboratory to industrial. Despite its disadvantages, this type of technology has been used to improve pesticide removal from soil samples, achieving removal efficiencies between 50% and 90% [70], similar to the results obtained in this investigation. In beeswax, this technology has been primarily used for sample preparation and as part of quantification methods [71]. No reports have been found of its use in this matrix for the purpose of purifying, eliminating, or removing pesticides.

3.2. Determination of Contaminants in Clean Beeswax Using Improved Traditional Methods

Despite the advantages of this technology, better results could be achieved by incorporating heating with agitation and acidified solvents. Acidification of water is another traditional cleaning technique that has been widely used in the beekeeping industry for hundreds of years. Therefore, the following study consisted of using three diluted acids and evaluating their effects on the removal of these types of contaminants. Figure 5 presents the results obtained using traditional methods (heating + stirring + acidified water as the solvent), with the objective of determining whether they are more effective in removing contaminants than the new methods proposed in this research. The results obtained using only water as the solvent, along with heating and agitation, are also shown; this method would be the most cost-effective in terms of chemical reagents.

Heating and stirring the wax, even using only pure water as the solvent, guarantees high removal rates of over 95%. Therefore, it can be concluded that this type of cleaning is very effective regardless of whether pure or acidified water is used. Furthermore, these results indicate that the necessary conditions for achieving good cleaning and contaminant removal in the wax are an adequate temperature (70 °C) to ensure homogeneous melting of the wax and constant stirring to allow optimal mixing and increase contact between the solvent (any solvent) and the molten wax. The differences in neonicotinoid extraction when performing one or two consecutive cleaning cycles are minimal, on the order of 3–4% when pure water is used as the solvent. For this reason, in the subsequent methods tested, which included acidified water, only one cleaning cycle was performed, not two, as in the previous cases.

Figure 5 shows no statistically significant differences in the overall removal rates achieved for the two types of solvents used: pure water and acidified water (with oxalic acid, citric acid, and sulfuric acid). All these methods used constant agitation at 700 rpm for 10 min and a heating temperature of 70 °C. Despite no statistical differences, the use of acidified water yielded higher removal rates than pure water, with the best results obtained using water acidified with 0.25% (v/v) oxalic acid as the solvent.

Traditional wax cleaning methods, which involve heating, stirring, and acidified water as the solvent, achieved removal rates ranging from 98.4% to 99.6%, effectively eliminating almost all the pesticides under study. This demonstrates their superiority to the new methods tested in this study, although they may incur higher costs and operating risks. The pesticide removal efficiency results are comparable to those obtained by Calatayud-Vernich et al., who achieved over 95% removal for 24 different pesticides than those evaluated in this study, using an extraction method with hexane and N,N-Dimethylformamide (DMF) [51]. However, despite evidence of the high efficacy of polar solvents in wax purification, no reports have been found in the scientific literature documenting specific cleaning methods or removal efficiencies for the seven neonicotinoids tested in this study. Therefore, this research is the first to analyse and quantify a method for removing this specific group of residues from the beeswax matrix, establishing a precedent in the analysis of apicultural decontamination.

3.3. Analysis of Cleaned Beeswax Coloration

The efficacy of traditional wax cleaning methods in removing neonicotinoid residues was much greater than that obtained with the new methods proposed in this research. The best results were obtained using pure or acidified water at a temperature of 70 °C, with constant agitation at 700 rpm for 10 min, and in a single cleaning step. Given this result, it became necessary to study another important variable in wax cleaning and purification: the colour achieved after the cleaning process. The waxes obtained after the cleaning processes were compared with pesticide-contaminated wax that had not undergone any type of cleaning treatment (CONTROL). In this way, the effect of the most efficient methods for removing contaminants on wax whitening was evaluated.

Table 3. Whitening index and colour parameters of images of cleaned waxes with different methods.

	CONTROL	SHW/I	SHWSA	SHWCA	SHWOA
WI	19. 1 ± 0.64 a	21.79 ± 0.51 b	20.91 ± 0.54 b	21.29 ± 0.62 b	21.58 ± 0.93 b
L*	30.91 ± 1.14 a	37.07 ± 1.38 b	35.45 ± 1.22 b	34.27 ± 1.7 b	37.42 ± 2.39 b
a*	11.49 ± 1.52 a	8.38 ± 1.34 b	10.53 ± 1.33 ab	4.66 ± 1.41 c	10.54 ± 1.61 ab
b*	40.43 ± 1.01 a	45.62 ± 1.16 b	44.42 ± 0.99 b	42.95 ± 1.46 b	45.93 ± 1.96 b
ΔE	-	8.64	6.12	8.01	8.58

^a,b,c Different lowercase superscript letters in the same file indicate a statistically significant difference (p < 0.05) for three replicates.

shows the indicators calculated for comparing the colour in the images of wax after cleaning with respect to a contaminated control wax.

The images and data in Table 3 illustrate the visual appearance, whiteness indices, average values of the corresponding colour parameters (L*, a*, b*), and calculated colour differences (ΔE) resulting from the cleaning processes or methods to which the waxes were subjected. The comparisons and calculated parameters were made only for the central region of the wax images (enclosed in a dashed square) to avoid interference from the image background and object edges.

These images and calculated indicators show that all the cleaning processes performed caused colour changes in the wax that were perceptible even to the human eye, such as increased gloss, whitening, and a lighter yellow hue in the waxes, with the latter being a characteristic of virgin waxes.

Whiteness is defined as a colour with maximum luminosity, without hue or saturation. The higher the WI, the greater the degree of wax whiteness, and vice versa. All cleaning methods analysed achieved a statistically significant increase in WI, ranging from 9.48 to 14.08%, thereby elevating the degree of wax whiteness after cleaning. The methods that showed the highest WI values were SHW/I and SHWOA, demonstrating the potential of pure water and water acidified with oxalic acid in this property.

All traditional cleaning methods (SHW, SHWCA, SHWOA, SHWSA) resulted in significantly lighter wax with higher L* values compared to the CONTROL wax. The cleaning methods that produced wax with the highest shine and tendency to whiteness were SHW/I and SHWOA, with L* values of 37.07 and 37.42, respectively, which represents an increase of approximately 21% in this indicator compared to the CONTROL wax without cleaning treatments. The CONTROL had a higher a* value (11.49), indicating a greater tendency to redden compared to the cleaned waxes. Cleaning methods tended to reduce the red component, obtaining lower a* values. The SHWCA method achieved the greatest reduction in this parameter, obtaining a value 5.44% lower than the CONTROL. This indicator is not directly related to whitening. The CONTROL had the lowest b* value (40.43), suggesting a lower intensity of the yellow colour compared to the cleaned waxes. All cleaning methods resulted in a significant increase in the yellow component. The SHWOA method presents the highest b* value, being 13.6% higher than the CONTROL, indicating the highest yellow colour intensity among all methods.

Although there were no statistically significant differences in the colour parameters of the wax subjected to the different cleaning methods, it is worth highlighting that the SHWOA method achieved the highest L* and b* values of all the methods used, meaning that this method produces the most luminous and yellow wax. This fact could guarantee greater acceptance by bees in the future. Furthermore, this method also achieves the highest percentage of contaminant removal. No research reports have been found that quantitatively compare the effect of cleaning treatments on the colour indices of beeswax. Therefore, this research constitutes a novel and initial approach in this regard.

3.4. Evaluation of the Pesticide Removal Efficacy of the Best Method in Larger Quantities of Wax

The SHWOA method achieves the best results in both contaminant extraction and final wax coloring, all of which was experimentally proven by cleaning 5 g of wax. For this reason, it was necessary to test the effectiveness of this methodology with larger quantities of wax. Therefore, the mass of wax to be cleaned was increased 10 and 40 times. This research also evaluated the efficiency of this method (SHWOA) in removing neonicotinoids from 50 g and 200 g of contaminated wax, respectively, to corroborate whether cleaning larger quantities yielded similar overall removal percentages. Additionally, sufficient amounts of cleaned wax were obtained to conduct the stamping process for subsequent evaluation of the level of acceptance of the purified wax in experimental hives. The results obtained with larger quantities of wax and using up to two consecutive cleanings are presented in Figure 6.

Figure 6 shows the results obtained from applying the SHWOA extraction method to clean 50 g and 200 g of contaminated wax with one (I) and two cleanings (II). Overall removal efficiency above 85% was obtained in both quantities of wax, thus demonstrating that the analysed method (which uses stirring, heating, and water acidified with oxalic acid as a solvent) is efficient in eliminating the main neonicotinoids present in beeswax contaminated with pesticide residues. Using two consecutive cleaning cycles, it is possible to remove more than 99% of the contaminants analysed in 50 and 200 g of wax. Similar results have been obtained using more complex methodologies and more aggressive and dangerous solvents such as methanol, hexane, and 4-N, N-dimethylformamide [51,52]. In this research, the proposed method uses water acidified with 0.25% oxalic acid as a solvent, a simpler, cheaper, and safer alternative with great potential for larger-scale use. Furthermore, it shows good results both in decontaminating the wax and in its colour after cleaning. To fully evaluate the proposed method, all that remains is to assess the level of acceptance of the cleaned wax by bees in the hives.

3.5. Qualitative Evaluation of the Acceptance Level of Wax Cleaned by Bees in Hives

The evaluation of the acceptance level of purified or cleaned beeswax in experimental hives is a key aspect of beekeeping and scientific research, as it directly influences future colony development and honeycomb productivity. This evaluation allows for a comparison of the effects on bees and their behavior of using commercial, purified, and adulterated waxes, among others. This process depends on multiple factors, such as the chemical composition of waxes, the degree of purity, the presence of contaminants, and environmental conditions.

There are two main approaches to assessing this acceptance: quantitative and qualitative methods. Quantitative methods typically include precise measurements, such as the area of the honeycomb constructed, the number of occupied cells, or the amount of brood developed. These methods require modern technologies such as powerful cameras and computers with software capable of processing images, making them difficult to use for small beekeepers, systematic research, or field studies. Qualitative methods, on the other hand, focus on specific observations, such as the bees’ behavior toward the wax, the level of work performed, and the final use of the cells. Qualitative methods can be applied by anyone with basic beekeeping knowledge and allow for comparisons of the effects of different types of wax without incurring high equipment costs. Both approaches offer a comprehensive view for determining the suitability of wax in experimental and commercial settings. The results obtained in this research by applying a qualitative method to compare cleaned beeswax with virgin control beeswax are shown in

Table 4. Qualitative indicators and their assigned values after applying the proposed method for comparing the acceptance level of wax in the honeycomb.

Indicators	CONTROL			SWHOA
	Hives			Hives
	H1	H2	H3	H1	H2	H3
Initial activity (0–3)	2	1	1	1	1	1
Beeswax stretching (0–3)	1	1	1	3	2	3
Final use (0–3)	2	2	2	3	2	3
Total Score per hive (H1 + H2 + H3)	5	4	3	7	5	7
Mean total score	4.33			6.33
Standard deviation	0.58			1.15
Level Acceptance	Medium			Medium

.

The proposed method evaluated two types of wax: untreated CONTROL wax and SWHOA wax, obtained after a cleaning and purification process using water acidified with oxalic acid as a solvent. The study was conducted simultaneously in three hives with frames stamped with both types of wax. Acceptance indicators included initial attraction (parameters taken on day 5), work performed (parameters taken on day 20), and final use (parameters taken on day 45). The results showed that the wax obtained using the SWHOA method had a higher average score (6.33) than the CONTROL wax (4.33), suggesting that the bees generally performed better when they had this type of wax in their hives, even higher than that obtained with untreated virgin wax. The standard deviation was similar between the two groups, indicating similarity in the results achieved in the hives for both types of wax.

According to the average score, both waxes are classified at the “Medium” acceptance level (scores between 4 and 6); however, the CONTROL wax was more attractive at the beginning of the experiment, which was to be expected since this is a virgin wax without interacting with any chemicals and still has the typical sensory characteristics of these hives such as smell and colour, causing the bees to use it first, quickly and naturally. Regarding the indicators work performed in the hive and final use, the SWHOA wax significantly outperformed CONTROL, which could indicate greater functionality or ease of handling of the frames stamped with the cleaned wax.

Comparable results were obtained by Flores et al. [61], who conducted a quantitative and in-depth study on the acceptance of wax purified with methanol. They performed frequent inspections of hives of the native Spanish honeybee (Apis mellifera iberiensis), taking photographs of each frame of wax. Both studies demonstrated the effectiveness of wax cleaning with polar solvents in removing pesticides. Furthermore, bees accepted and actively worked with the cleaned wax. These arguments guarantee the successful implementation of these cleaning technologies in the beekeeping industry on a larger scale by confirming that the purification processes do not cause adverse changes in the ethological behavior of the bees or in their productive indices.

These results confirm that the proposed beeswax cleaning method, which includes heating, stirring, and acidified water with oxalic acid as a solvent, does not harm the development or productive activity of the bees in the hive. All the advantages of the proposed SWHOA method in the elimination of contaminants, in the coloration of the wax, and in the level of acceptance of the wax cleaned by the bees in the hive indicate that its use on a larger scale for the cleaning and bleaching of beeswax could be viable and efficient.

4. Conclusions

Traditional wax cleaning methods based on heating and stirring, using either pure water (SHW/I) or water acidified with oxalic acid (SHWOA), proved to be the most effective for removing neonicotinoid residues from beeswax. These methods achieved removal efficiencies greater than 95%, significantly outperforming the newly proposed techniques that used ultrasound or microwaves, which showed limited efficacy (<85%). Among all methods tested, the SHWOA method (heating, stirring, and oxalic acid–acidified water) stood out for its ability to remove more than 99% of neonicotinoids, even when applied to larger wax quantities (up to 200 g) with just two cleaning cycles. Additionally, this method enhanced wax colour, increasing whiteness and luminosity towards the lighter yellow tone typical of virgin waxes. Compared to more complex methods using hazardous solvents, SHWOA offers a safe, low-cost, and scalable alternative for wax decontamination and bleaching. The proposed qualitative evaluation method also proved effective for assessing wax acceptance in hives. Results showed that beeswax cleaned with the SHWOA method, although initially less attractive, ultimately outperformed virgin wax in hive functionality and long-term acceptance. Both waxes achieved a “Medium” acceptance level, confirming that the cleaning process does not compromise the bees’ productivity and may be safely applied at scale.