0. Abstract

Beeswax has traditionally been considered a simple construction material for comb building. Recent scientific data, however, show that it constitutes an active biological compartment capable of influencing colony health through structural, toxicological and epidemiological mechanisms. This synthesis analyses the effects of comb ageing, contaminant accumulation, pathogen persistence and the implications of the wax circuit.

Morphometric studies demonstrate that comb age leads to a progressive reduction in cell volume and in the weight of emerging workers, with observed correlations to brood surface area, longevity and colony performance. In parallel, wax acts as a cumulative reservoir of lipophilic substances (acaricides, pesticides) and heavy metals. Sublethal effects on worker longevity and drone sperm viability have been documented following developmental exposure. Combs may also constitute a persistence compartment for highly resistant bacterial spores, such as Paenibacillus larvae, if sterilisation procedures do not meet validated parameters. Data on viral presence in wax indicate measurable contamination, although the relative epidemiological importance of this transmission pathway remains to be quantified.

Wax recycling, a central practice in beekeeping, thus appears as a strategic node in risk management. Open systems favour the redistribution of residues and potentially pathogens between operations, whereas closed systems limit external introduction but do not eliminate internal accumulation. Available data support the relevance of periodic comb renewal in order to limit the cumulative build-up of structural, chemical and biological constraints.

In conclusion, management of the wax circuit constitutes a major strategic lever for colony health and the quality of bee products. The realistic objective is not the complete elimination of contaminants, but the progressive minimisation of risks through controlled comb rotation, traceability of the circuit and a clear distinction between biological sterilisation and chemical decontamination.

1. Foundations and current state of the scientific debate

Wax management in beekeeping has long been approached primarily from a technical and economic perspective. Combs were considered mainly as structural supports for brood and food storage, and their renewal was based more on empirical practice than on structured biological reasoning.

Scientific work over the past two decades has profoundly modified this perception. Combs are now seen as a dynamic compartment in which structural, chemical, microbiological and epidemiological factors interact. Comb ageing, the progressive accumulation of contaminants, possible pathogen persistence and induced physiological responses in larvae constitute interdependent dimensions.

The aim of this chapter is to examine these different axes in light of available experimental and observational data, in order to clarify the biological role of wax in colony health. The goal is not to identify a single factor, but to understand a cumulative system in which wax acts as a matrix of exposure and progressive transformation of the brood microenvironment.

1.1 Wax as a biological compartment

The current scientific consensus converges towards a clear conclusion: beeswax is not merely an inert structural material, but an active biological compartment endowed with physicochemical and ecological properties that directly influence colony health.

From a physicochemical perspective, wax is a highly lipophilic matrix composed mainly of esters of fatty acids and long-chain alcohols. This characteristic confers a strong capacity to adsorb and accumulate liposoluble substances present in the environment or introduced into the hive, notably synthetic acaricides (Calatayud-Vernich et al., 2018 ; Marti et al., 2022). In contrast to honey, which is regularly renewed, wax persists for several years in the brood nest and acts as a cumulative reservoir.

However, reducing wax to simple “chemical storage” would be incomplete. Combs evolve structurally over successive brood cycles. Meng et al. (2025), in a recent systematic review, describe this process as a progressive transformation of the initial material into a composite matrix combining wax, successive layers of larval cocoons, organic debris and incorporated residues. This dynamic leads to measurable morphological modifications:

• progressive darkening linked to the adsorption of pigments and larval faecal matter,
• significant increase in cell wall thickness,
• increase in comb surface weight,
• reduction in internal cell volume,
• modification of cell geometry and comb macro-architecture.

Quantitative data illustrate the magnitude of the phenomenon: an increase in surface weight from 0.26 g/cm² to 1.32 g/cm² after seven years of use, an increase in wall thickness from 88 µm to nearly 300 µm in two years, and a reduction in cell volume from 0.31 ml to 0.18 ml in six years (Meng et al., 2025).

These changes are not purely aesthetic. They modify the brood microclimate, gas diffusion, local thermal capacity and potentially the distribution of chemical signals within the colony. They also influence the contact surface between the larva and its immediate environment, which can modulate exposure to accumulated residues.

Earlier work confirms that comb age is accompanied by a progressive decrease in internal cell diameter (Berry & Delaplane, 2001 ; Al-Kahtani, 2021). The consistency of these results across different geographical and experimental contexts strengthens the robustness of this observation.

Beyond the structural aspect, wax also constitutes a biological microhabitat. Analyses of compartment-specific microbiomes within the hive show that combs display a distinct profile compared with honey, pollen or propolis (Grubbs et al., 2015). Samples cluster primarily by compartment type rather than by colony or site, suggesting the existence of an ecological signature specific to combs. Although these lipid-biomarker-based analyses do not allow precise identification of the microbial species involved, they demonstrate that wax participates in the colony’s internal microbial ecology.

The epidemiological dimension further reinforces this interpretation. International sanitary manuals explicitly recognise that spores of Paenibacillus larvae (the agent of American foulbrood) can survive for years in hive products, including wax, and that the manufacture of foundation sheets from contaminated wax can contribute to disease spread if sterilisation procedures are inadequate (WOAH, 2023a). Even in the absence of sporulation, European foulbrood can persist via mechanical contamination of combs (WOAH, 2023b). Thus, the comb is not only a passive support: it can become a compartment of pathogen persistence.

It has also been demonstrated that fungal spores, notably Ascosphaera apis (the agent of chalkbrood ), present in contaminated foundation sheets can infect brood built on these foundations (Flores et al., 2005). This experimental observation confirms that wax can constitute a transmission vector when infectious agents are incorporated into it.

Regarding viruses, several studies have detected viral RNA in wax (De Guzman et al., 2019 ; Colwell et al., 2024). Although RNA detection does not necessarily demonstrate the presence of active infectious particles, these studies suggest that combs can contain viral material and potentially contribute to infection ecology, even if the relative importance of this route compared with transmission via Varroa destructor or nurse bees remains to be quantified.

Finally, wax plays a role in parasitic dynamics. Experimental comparisons have shown that old combs can exhibit significantly higher infestation rates by Varroa destructor than new combs, independently of simple cell width (Piccirillo & De Jong, 2004). This suggests that chemical or structural factors associated with comb age could influence the attractiveness or reproduction of the parasite.

In addition, syntheses devoted to the biology of Galleria mellonella indicate that the greater wax moth develops preferentially in old, dark combs rich in larval cocoons and organic residues (Kwadha et al., 2017). Old wax offers not only a structurally more favourable matrix for tunnelling, but also increased nutritional value linked to the accumulation of proteins and debris from brood cycles (Kwadha et al., 2017). While strong colonies generally manage to limit larval development through cleaning and defensive activity, stored material — notably old brood frames — remains particularly vulnerable to massive infestations (Charrière & Imdorf, 1999 ; Ellis et al., 2013). Regular renewal of old frames thus helps not only to reduce chemical and microbiological load, but also to decrease pressure from the wax moth.

Taken together, these elements lead to considering wax as an integrated biological matrix in which structure, chemistry and microbiology interact. It is not merely a material support for brood, but an evolving environment that durably influences the colony’s physiological and sanitary equilibria.

This conceptual evolution transforms the beekeeping perspective: comb management becomes a strategic biological parameter, and no longer only a technical or economic decision.

1.2 Structural comb ageing and impacts on workers

Comb ageing is not limited to a visual change or simple residue accumulation. It corresponds to a progressive transformation of the microenvironment in which brood develops, with measurable consequences for the morphology and performance of adult bees.

Morphometric data from controlled experimental series show that the reduction in cell volume associated with comb age translates into measurable differences in emerging workers. Meng et al. (2025) report that bees reared in old combs have a significantly lower birth weight than those from recent combs (for example 114.9 mg versus 88.0 mg in the compared series). This decrease in body weight is accompanied by morphological changes such as a shorter proboscis, reduced thoracic and abdominal dimensions, as well as reduced tarsal size and certain wing parameters.

These results are consistent with earlier work demonstrating a negative correlation between comb age and the weight of newly emerged workers (Berry & Delaplane, 2001 ; Al-Kahtani, 2021). In the study by Berry and Delaplane (2001), colonies placed on new combs produced heavier bees and larger brood areas than those placed on old combs. The study nevertheless highlights an important complexity: at some sampling times, short-term brood survival could be slightly higher in old combs, suggesting that the effects are neither uniform nor strictly linear.

The decrease in weight and body dimensions is not merely an isolated morphometric parameter. It can influence individual physiology and the functional capacities of workers. Meng et al. (2025) report associations between comb ageing and:

• a reduction in the frequency of returning with pollen,
• a decrease in total brood surface area,
• a reduction in worker longevity,
• a decline in honey and pollen accumulation at the colony level.

These correlations suggest that modification of the larval microenvironment can affect collective performance.

It should, however, be emphasised that precise causal attribution remains complex. Several mechanisms may coexist:

1. Geometrical constraints: reduction in cell diameter and depth potentially limits larval developmental space (Al-Kahtani, 2021).
2. Accumulation of chemical residues: increasing presence of lipophilic substances in the wax–cocoon matrix may modify chronic larval exposure (Wu et al., 2011).
3. Microclimate modification: thickening of walls and organic densification may influence thermal properties and gas circulation.
4. Behavioural factors: queen laying preferences and worker maintenance behaviours may be affected by comb condition.

The experimental study by Wu et al. (2011) provides an important element by showing that workers reared in combs heavily contaminated by pesticide residues display an adult longevity reduced by about four days on average, as well as altered larval development. Although this study focuses specifically on chemical contamination, it underscores that the comb can act as a vector of chronic exposure throughout the entire larval period.

It is also notable that the ageing comb structure can interact with parasitic dynamics. Piccirillo and De Jong (2004) observed that old combs had significantly higher infestation rates by Varroa destructor than new combs, independently of simple cell width. This observation suggests that comb age may influence chemical or structural signals affecting the attractiveness or reproduction of the parasite, which could indirectly modulate viral pressure within the colony.

Thus, comb ageing must be understood as a multifactorial process involving:

• measurable geometrical changes,
• progressive accumulation of organic and inorganic compounds,
• transformation of the larval microenvironment,
• potential effects on individual physiology and collective dynamics.

It would be scientifically inaccurate to claim that “old combs are always harmful.” Available data rather show a set of interconnected effects whose magnitude depends on context (subspecies, environmental conditions, parasitic pressure, treatment history). Nevertheless, the convergence of morphometric and functional results reinforces the idea that periodic comb renewal constitutes a coherent biological lever to limit the accumulation of structural and chemical constraints.

In summary, an old comb is not merely a worn structure: it progressively becomes a transformed microenvironment whose physical, chemical and biological properties can influence the quality of bees produced and, by extension, colony performance.

1.3 Environmental contaminants, residues and accumulation in wax

Beeswax constitutes one of the main compartments of contaminant accumulation within the colony. Its lipophilic nature favours the adsorption and retention of many hydrophobic substances, notably acaricides used against Varroa destructor, as well as various pesticides of environmental origin (Calatayud-Vernich et al., 2018 ; Marti et al., 2022). Unlike honey, which is harvested and renewed regularly, brood nest wax can remain in place for several years, making it a long-term cumulative reservoir.

Monitoring studies confirm the high frequency of residues in commercial wax. In an analysis of Swiss foundation wax collected from nine manufacturers (98 samples), Marti et al. (2022) detected between 7 and 14 active substances per sample, including commonly used beekeeping acaricides such as coumaphos and tau-fluvalinate. Similarly, analyses in Germany have identified up to 16–20 substances simultaneously present in commercial foundation sheets, with a predominance of persistent lipophilic acaricides (Alkassab et al., 2020). These results illustrate that wax in the commercial circuit is frequently exposed to complex mixtures of residues.

It is important to distinguish two analytical dimensions:
(1) the presence of residues, which is empirically well documented;
(2) the biological impact of these residues, which depends on concentration, combinations of substances, exposure route and developmental stage.

Regarding biological effects, Wu et al. (2011) experimentally demonstrated that workers reared in brood combs carrying high pesticide-residue loads showed a significant reduction in adult longevity (about four days on average) as well as altered larval development. Particularly importantly, the authors observed a transfer of residues to initially clean combs introduced later, confirming that wax can act not only as a source of direct exposure but also as a vector of internal redistribution.

However, the presence of residues does not necessarily imply immediate lethal effects. A dose–response study conducted under real-hive conditions showed that coumaphos concentrations up to 62 mg/kg in foundation wax did not significantly increase brood mortality compared with controls, whereas a higher concentration (132 mg/kg) strongly reduced emergence rates (Kast et al., 2023). This study provides an essential quantitative anchor: not all concentrations detected in wax automatically produce lethal effects. Sublethal effects and interactions between substances remain, however, a major area of uncertainty.

The issue of mixtures (“cocktails” of residues) is central. Multiple exposures are the rule rather than the exception in commercial circuits (Marti et al., 2022). Work on male reproduction shows that developmental exposure to mixtures of acaricides can significantly reduce drone sperm viability (Fisher & Rangel, 2018). While these results do not formally demonstrate synergy in the strict toxicological sense, they indicate that combinations of substances can affect sensitive reproductive parameters.

Effects on queens are more nuanced. McAfee et al. (2021) showed that topical adult exposure to pesticides commonly found in wax, even at concentrations higher than typical levels, did not alter queen body mass or sperm viability in their experimental protocol. In contrast, Rangel and Tarpy (2015) demonstrated that queens reared in wax cups contaminated with acaricides showed a significant reduction in the number of stored spermatozoa and their viability. These results underscore a fundamental distinction between adult contact exposure and developmental exposure during the larval stage, the latter potentially involving different physiological mechanisms.

Beyond organic pesticides, heavy metal accumulation constitutes a complementary dimension. Meng et al. (2025) observed a significant increase in concentrations of Cd, Cr, Ni, Pb and Mn in aged combs compared with recent combs. Metals accumulate notably in successive cocoon layers. Larvae reared in these environments show increased activation of genes involved in detoxification, notably those of the CYP450 family. This molecular response indicates physiologically relevant exposure, even if it does not automatically imply a measurable clinical effect.

Thus, wax should not be regarded as a simple passive substrate, but as a compartment of chronic exposure. It progressively concentrates substances originating from:

• veterinary treatments applied within the colony,
• environmental pesticides carried in by foragers,
• inorganic contaminants from atmospheric or agricultural sources,
• mixtures resulting from industrial recycling.

A crucial point, often misunderstood, concerns the distinction between sterilisation and decontamination. Thermal treatments effective for inactivating bacterial spores, notably in the context of American foulbrood (≥121 °C, for 30 minutes, at a pressure of 2 bars, according to official Swiss recommendations), do not remove persistent lipophilic residues (Kast et al., 2023 ; Swiss Federal Food Safety and Veterinary Office, 2016). Heat destroys biological pathogens but does not “reset” the chemical composition of the native wax produced by wax-secreting workers.

In synthesis, available data allow one to state with a high level of confidence that:

• wax is frequently contaminated by mixtures of residues;
• certain developmental exposures can affect longevity, reproduction or morphometric parameters;
• effects depend on dose, mixtures and biological context;
• wax acts as a long-term cumulative reservoir;
• usual thermal procedures do not eliminate chemical contaminants.

The issue is therefore not only the presence of residues, but their management over time. Accumulation dynamics progressively transform wax into a compartment of chronic exposure, giving comb renewal and wax-circuit management strategic importance within an integrated colony health approach.

1.4 Molecular adaptation and physiological response: adaptation does not mean absence of risk

Chronic exposure to contaminants present in old combs is not limited to morphometric or reproductive effects observable at the level of the individual or the colony. It also induces measurable molecular responses reflecting activation of detoxification systems and stress-regulation pathways.

Meng et al. (2025) report that larvae reared in old combs show a significant increase in the expression of genes involved in detoxification, notably those of the cytochrome P450 (CYP450) family, glutathione S-transferases (GST) and carboxylesterases. These enzymes play a central role in the biotransformation of xenobiotic compounds, including lipophilic acaricides and certain environmental pesticides.

The activation of these systems is regulated by several molecular signalling pathways, notably the AhR/ARNT, MAPK and CncC/Keap1 axes, known to be involved in responses to oxidative stress and persistent organic contaminants. Induction of these pathways constitutes a biological indicator of exposure, even in the absence of clinically visible short-term effects.

The metabolic mobilisation involved may generate indirect physiological costs, potentially affecting individual or collective performance in the medium term. Thus, molecular adaptation reflects a real biological interaction with the wax environment, but does not guarantee the absence of delayed effects. For this reason, activation of detoxification pathways should be interpreted as a biological signal of exposure, not as proof of harmlessness. It indicates that the wax compartment acts as a source of measurable environmental constraint at the cellular level.

In summary, molecular data reinforce the idea that old and/or contaminated combs constitute a biologically active environment. The adaptive response of larvae demonstrates a real physiological interaction with the contaminants present. However, adaptation does not mean complete neutralisation of risks. Continuous mobilisation of detoxification mechanisms may entail subtle biological costs capable of affecting individual performance and colony dynamics over the long term.

1.5 Honey and wax quality: implications of comb ageing

The effects of comb ageing do not concern only bee biology and the colony’s sanitary dynamics. They also extend to the quality of hive products, in particular honey and wax itself. This dimension is often underestimated in beekeeping practice, even though it has economic, sensory and toxicological relevance.

Meng et al. (2025) report that honey stored in old combs shows measurable changes in certain physicochemical parameters. Observed differences include a significant increase in hydroxymethylfurfural (HMF) in honey produced from aged combs (28.8 mg/kg versus 18.4 mg/kg in recent combs), as well as changes in carbohydrate profiles. HMF is a classic indicator of thermal degradation or honey ageing; its increase in old combs suggests that the storage microenvironment could influence the chemical stability of the product.

It is important to emphasise that these values remain dependent on environmental context, harvesting practices and storage conditions. Nevertheless, the observed correlation between comb age and certain honey-quality parameters indicates that the wax matrix is not a neutral container, but an environment capable of interacting with the stored product.

The same work also reports higher concentrations of heavy metals in honey originating from old combs (Meng et al., 2025). Although the exact transfer mechanisms are not fully elucidated, prolonged physical proximity between honey and a wax–cocoon matrix enriched with contaminants could contribute to these differences. These observations should be interpreted cautiously, but they underscore that comb management may have indirect implications for food quality.

Regarding wax itself, ageing leads to a progressive modification of its chemical composition. Meng et al. (2025) describe a relative increase in shorter-chain components and an alteration of the overall lipid profile in old combs. These changes may influence mechanical properties, thermal stability and material plasticity. They may also modify interactions between wax and the lipophilic substances adsorbed within it.

The issue of adulteration of commercial wax constitutes a distinct but closely related element of product quality. Alkassab et al. (2020) experimentally demonstrated that the addition of stearin to beeswax can cause significant brood disturbances. At around 20% adulteration, colonies showed delayed acceptance of foundation sheets, repeated removal of young larvae and a marked reduction in capped brood. These effects differ qualitatively from those observed for acaricide residues at realistic concentrations, which tend to manifest as chronic and sublethal impacts.

This distinction is essential:
– adulteration represents an acute problem related to material quality;
– residue accumulation corresponds rather to a progressive phenomenon of chronic load.

In open recycling circuits, these two dimensions can combine: mixing waxes from multiple operations, presence of persistent residues and risk of introducing adulterated wax. Monitoring analyses (Marti et al., 2022) confirm that commercial foundation sheets can contain complex residue profiles. Traceability and transparency of circuits thus become determining elements of final quality.

Another often neglected aspect concerns sensory perception. Meng et al. (2025) mention an organoleptic deterioration of honey stored in old combs. Although these observations require further validation and may depend on floral terroir, they reinforce the idea that final product quality depends not only on floral resources and extraction techniques, but also on the structural condition of the storage support.

In summary, available data suggest that:

• comb age can influence certain physicochemical parameters of honey;
• contaminants accumulated in wax can potentially interact with the stored product;
• the chemical composition of wax changes over time;
• adulteration of wax represents a risk distinct from residues but equally relevant;
• management of the wax circuit has not only sanitary but also quality implications.

These elements indicate that the structural state of combs can influence certain physicochemical parameters of honey. Wax should therefore not be considered only as a passive container, but as an environment capable of interacting with the stored product.

Management of comb renewal thus takes on broader significance: it concerns not only colony biology, but also the quality of bee products.

2. The optimal wax cycle

The previous analyses have shown that wax changes over time under the combined effect of structural modifications, chemical accumulation and possible biological persistence. The central question then becomes how to manage this dynamic. The wax circuit is not a simple logistical flow, but an organisational system that can either limit or amplify these processes. Recycling, batch mixing, traceability and sterilisation parameters directly influence the accumulation trajectory. This chapter examines the different possible configurations of the circuit — open, closed or intermediate — in order to assess their sanitary and toxicological implications in light of available scientific data.

2.1 The wax recycling issue

Wax recycling has historically been a pillar of beekeeping. However, in light of current knowledge, it must be analysed as a mechanism for redistributing accumulated loads. From an economic and ecological perspective, recovering old combs and transforming them into new foundation sheets appears rational and sustainable. In light of current knowledge, recycling must be analysed as a redistribution mechanism. Substances adsorbed in old combs do not disappear during melting; they are incorporated into the new wax matrix and may be distributed homogeneously throughout the processed batch.

The first dimension of the problem concerns cumulative accumulation of lipophilic residues. As shown above, wax acts as an adsorption compartment for many acaricides and environmental pesticides (Calatayud-Vernich et al., 2018 ; Marti et al., 2022). When old combs are melted and then reintroduced into the circuit as new foundation sheets, accumulated substances do not disappear. On the contrary, they may be redistributed homogeneously within the recycled batch.

Meng et al. (2025) explicitly note that recycling old combs leads to reintroduction of heavy metals, persistent acaricides and organic pollutants into the new wax matrix. This dynamic creates a phenomenon of “chemical memory” within the wax circuit: even if treatment practices change, historical residues may persist for several recycling cycles.

Analyses of commercial wax corroborate this reality. Marti et al. (2022) showed that commercial foundation sheets regularly contained multiple acaricide residues, sometimes at non-negligible concentrations. Alkassab et al. (2020) also highlighted the simultaneous presence of many residues in samples of German commercial wax. These observations indicate that mixing waxes originating from multiple operations mechanically amplifies the diversity of residues present in the final product.

This issue is intensified in so-called “open” systems, where waxes from different beekeepers are collected, melted together and redistributed without strictly traceable batch separation. In such a system, contamination from a single operation can theoretically be diluted, but also diffused on a large scale.

The second dimension concerns the persistence of pathogens. International sanitary standards recognise that spores of Paenibacillus larvae can survive for long periods in wax (WOAH, 2023a). In the absence of adequate sterilisation (≥121 °C for 30 minutes, under pressure, according to Swiss recommendations), standard thermal processing of wax does not guarantee complete inactivation of spores (Swiss Federal Food Safety and Veterinary Office, 2016). Thus, recycling without validated thermal control can constitute a dissemination vector.

It is essential here to distinguish two levels:

• simple melting of wax (removal of solid impurities),
• validated sterilisation (inactivation of resistant spores).

Confusion between these two processes remains frequent in practice (see ch. 3.5).

The recycling issue is not limited to residues and spores. Adulteration constitutes a third risk factor. Alkassab et al. (2020) showed that adding stearin to wax can cause acute brood disturbances, including delayed acceptance of foundation sheets and major larval losses. In open circuits under strong economic pressure, the temptation to adulterate wax to reduce costs represents an additional risk.

The analysis must nevertheless be nuanced. Recycling as such is not intrinsically problematic. In a closed system, with strict traceability, batch separation and validated sanitary control, wax recirculation can be controlled. The problem arises when:

• wax provenance is heterogeneous,
• traceability is absent,
• thermal treatments are not validated,
• no residue analyses are performed.

Meng et al. (2025) insist that intensive recycling without sufficient renewal favours the progressive increase of contaminant load. This is a cumulative phenomenon rather than a one-off event.

Finally, it should be noted that recycling can also influence the physical structure of new foundation sheets if the source wax is highly degraded. Although melting homogenises the material, certain mechanical and compositional properties can evolve over time, modifying plasticity or thermal stability.

In synthesis, wax recycling represents a strategic node in colony sanitary management. It can be:

• a tool for controlled circular valorisation,
• or a vector for cumulative recirculation of contaminants.

The question is therefore not “to recycle or not to recycle,” but “how to recycle.” The answer depends on the structure of the wax circuit, the level of sanitary control, traceability and the sterilisation procedures implemented.

2.2 Open systems and closed systems: risk architectures of the wax circuit

The question of recycling cannot be dissociated from the organisational structure of the wax circuit. The distinction between open systems and closed systems is not a mere logistical preference. It corresponds to two fundamentally different risk architectures with respect to chemical and biological contaminants.

An open system is characterised by collection and melting of wax from multiple operations, followed by redistribution in the form of standardised foundation sheets. This model is historically widespread in Europe and economically efficient. It allows pooling of volumes and access to industrial processing equipment, including autoclaves capable of reaching the temperatures required for inactivation of Paenibacillus larvae spores (Swiss Federal Food Safety and Veterinary Office, 2016).

However, from an epidemiological and toxicological viewpoint, the open system introduces a mixing phenomenon across colonies. Each final batch becomes the result of heterogeneous sanitary and chemical histories. If a single initial input contains a high load of residues or spores that have not been inactivated, this load can be redistributed widely.

Analytical data confirm that commercial foundation sheets frequently contain complex mixtures of residues (Marti et al., 2022 ; Alkassab et al., 2020). This does not necessarily mean concentrations reach toxic thresholds, but it demonstrates that the open system structurally tends to homogenise and redistribute existing loads.

From a microbiological perspective, international standards explicitly recognise that contaminated wax can constitute a vector of American foulbrood dissemination if it is not properly sterilised (WOAH, 2023a). Simple thermal processing (melting) does not guarantee inactivation of spores, which requires validated parameters (≥121 °C for 30 minutes, under pressure, in Swiss recommendations). Actual effectiveness depends on process control, equipment calibration and documentary control.

A closed system, by contrast, is based on strict separation of wax flows at the operation level. Cappings wax and removed combs are melted and reused exclusively within the same apiary or within a group of operations sharing a controlled sanitary history. This model offers several structural advantages:

• complete batch traceability,
• limitation of inter-operation mixing,
• direct control of treatment practices,
• coherence between exposure history and reuse.

In a closed system, any contamination remains confined to the production unit. There is no interregional amplification. However, a closed system does not automatically eliminate internal accumulation issues. If old combs are systematically recycled without sufficient renewal, residue loads can continue to increase progressively (Meng et al., 2025).

In other words, the closed system reduces the risk of external introduction, but does not neutralise the risk of internal accumulation.

The comparison between open and closed systems must therefore be formulated in terms of risk architecture:

• the open system entails a higher risk of horizontal diffusion (between operations);
• the closed system concentrates risk within a unit but provides better traceability and better capacity for targeted management.

A third intermediate model, sometimes described as semi-closed or controlled, consists of using an external processor while requiring:

• strict batch separation,
• documentation of sterilisation parameters,
• periodic residue analyses,
• contractual guarantee of absence of adulteration.

This model seeks to combine the technical advantages of industrial infrastructure with the traceability principles of the closed system.

The temporal dimension must also be integrated. In open circuits, dilution can sometimes reduce the concentration of a specific contaminant at a given time, but it increases the overall diversity of residues present. In closed circuits, diversity may be lower, but the concentration of a historically used compound may remain stable or increase if no renewal is implemented.

Thus, no architecture is intrinsically “perfect.” The evaluation depends on:

• regional disease prevalence,
• historical treatment practices,
• degree of industrial control,
• available analytical capacity.

Current scientific literature allows one to state with a high degree of confidence that:

• wax can carry persistent spores if it is not properly sterilised (WOAH, 2023a);
• commercial wax frequently contains residue mixtures (Marti et al., 2022 ; Alkassab et al., 2020);
• intensive recycling favours cumulative accumulation (Meng et al., 2025).

Consequently, the choice of wax circuit model should be considered a strategic risk-management decision rather than a simple logistical question.

2.3 Renewal intervals and biological thresholds

The question of comb exchange intervals is one of the most sensitive points in wax circuit management. Historically, renewal recommendations were based mainly on empirical or practical considerations (darkening, mechanical strength, aesthetic aspects). Recent work now allows this issue to be addressed from a more structured biological perspective.

Comb ageing is a cumulative process involving structural, chemical and microbiological modifications. Morphometric and toxicological data suggest that these modifications follow a progressive trajectory rather than an abrupt threshold. However, some studies attempt to identify functional breakpoints.

Meng et al. (2025), in their systematic review, propose temporal reference points drawn from comparative literature: for Apis mellifera, renewal beyond three years of use would be biologically justified; for Apis cerana, the proposed threshold is much shorter (more than six months or beyond eight brood cycles). These values are specific to the contexts studied and should not be mechanically transposed to all European conditions. They nonetheless indicate that biologically relevant thresholds exist in relation to cumulative accumulation of constraints.

The work of Berry and Delaplane (2001) shows that significant differences in brood area and bee weight appear when comparing colonies placed on new versus old combs. Although these studies do not define a precise temporal threshold, they confirm that modifications become functionally detectable.

Al-Kahtani (2021) shows that reduction in internal cell diameter and in worker weight progresses in a near-monotonic manner with comb age. This continuity suggests that this is not a binary phenomenon (new vs old), but a biological gradient.

The central question thus becomes: from what level of accumulation do the combined cumulative effects become sufficiently significant to justify renewal?

Several dimensions must be taken into account:

1. Structural accumulation
Wall thickening and reduction in cell volume progress with each brood cycle (Meng et al., 2025). Even if a colony can tolerate these changes for several cycles, successive accumulation can durably alter the larval microenvironment.

2. Chemical accumulation
Heavy metals and persistent acaricides increase over time in combs (Meng et al., 2025 ; Marti et al., 2022). Recycling without external renewal maintains this accumulation. Even in the absence of immediate lethal effects, chronic exposure constitutes a continuous physiological constraint.

3. Parasitic dynamics
Piccirillo and De Jong (2004) observed significantly higher infestation rates by Varroa destructor in old combs than in recent combs. Although the exact mechanism is not fully elucidated, this suggests that comb age may indirectly influence parasitic pressure.

4. Colony resilience
Documented sublethal effects (Wu et al., 2011 ; Fisher & Rangel, 2018) show that developmental exposure can affect longevity or reproductive quality without causing immediate collapse. The question then becomes one of cumulative resilience: a colony subjected to moderate but persistent constraints may exhibit increased vulnerability to additional stressors.

It is important to emphasise that no study currently allows definition of a universally valid threshold applicable to all regions and all practices. Environmental conditions, parasitic pressure, treatment history and bee subspecies strongly influence dynamics.

Thus, rather than a rigid threshold, the literature suggests a logic of progressive management:

• the older the comb, the more structural and chemical constraints increase;
• these constraints potentially interact with one another;
• regular renewal mechanically reduces cumulative accumulation.

In this perspective, the three-year interval often mentioned for Apis mellifera appears as a biologically coherent reference point rather than an absolute standard (Meng et al., 2025). It corresponds to a compromise between practical feasibility and limitation of accumulation.

It is also important to distinguish comb types. Brood nest combs, subjected to repeated brood cycles and successive cocoon layers, accumulate structural and chemical modifications more rapidly than honey super combs used mainly for honey storage. Differentiated management can therefore be scientifically justified.

In summary, the question of renewal intervals should be understood as a mechanism of temporal regulation of the wax compartment. Periodic renewal does not aim to achieve absolute purity, but to limit cumulative accumulation of structural, chemical and biological constraints.

No universal threshold can be fixed, as dynamics vary with environmental and sanitary context. Nevertheless, converging data show that periodic renewal mechanically reduces cumulative load and constitutes a rational management lever.

3. Practical recommendations

The previous sections have shown that wax changes under the combined effect of structural transformations, chemical accumulation and possible biological persistence. The following recommendations derive directly from these findings. They aim to coherently reduce the progressive risks identified, without claiming to achieve absolute purity.

3.1 Active management of comb age

Comb ageing is associated with:

• a progressive reduction in cell volume (Al-Kahtani, 2021 ; Meng et al., 2025),
• a decrease in the weight of emerging workers (Berry & Delaplane, 2001 ; Meng et al., 2025),
• increasing accumulation of heavy metals and lipophilic residues (Meng et al., 2025 ; Marti et al., 2022).

Beyond these structural and toxicological effects, old wax also shows increased vulnerability to certain pests of beekeeping equipment. Syntheses devoted to Galleria mellonella show that dark combs rich in cocoons and organic residues constitute a particularly favourable substrate for larval development. Regular renewal of frames thus also helps to limit wax moth pressure, notably during equipment storage. These observations justify active management of comb age.

In practice:

• Implement a systematic rotation of brood frames (e.g. 3 to 5 frames per year), corresponding approximately to an annual renewal of about one third of brood frames and a fraction of super frames.
• Critically assess combs that have exceeded about three years of use for Apis mellifera (Meng et al., 2025), taking into account sanitary and environmental context.
• Prioritise removal of combs that are strongly darkened, thickened or show marked wall densification.

The objective is not aesthetic, but biological: to limit the cumulative accumulation of structural and chemical constraints.

3.2 Management of residues and heavy metals

Wax acts as an accumulation compartment for acaricides and environmental contaminants (Calatayud-Vernich et al., 2018 ; Marti et al., 2022). Residues can exert sublethal effects on longevity or development (Wu et al., 2011), while high concentrations can affect brood emergence (Kast et al., 2023).

In practice:

• Avoid repeated and prolonged use of persistent lipophilic acaricides without concomitant comb renewal.
• Integrate frame renewal into the overall strategy for controlling Varroa destructor.
• Consider wax as a potential bioindicator of the environment: high loads may reflect external pressures.

It should be recalled that thermal treatments aimed at biological inactivation do not eliminate persistent chemical residues (Kast et al., 2023). This distinction is developed in detail in section 3.5.

3.3 Distinguish residues from adulteration

Problems related to residues and those related to wax adulteration must be distinguished.

Acaricide residues, at concentrations usually observed in commercial circuits, are mainly associated with chronic or sublethal effects (Wu et al., 2011 ; Fisher & Rangel, 2018).

By contrast, adulteration by addition of stearin can cause acute effects on brood, including larval removal behaviour and a reduction in capped brood (Alkassab et al., 2020).

In practice:

• In cases of strongly spotty brood without an obvious infectious or parasitic cause, consider wax quality as a possible factor.
• Demand increased transparency and traceability when purchasing foundation sheets.

3.4 Choice of wax circuit

The configuration of the wax circuit directly influences the management of chemical and microbiological risks.

Open systems involve mixing waxes from multiple origins, with potential redistribution of residues and, in the case of inadequate thermal control, spores (WOAH, 2023a ; Marti et al., 2022).

Closed systems offer better traceability and limit external inputs, while still requiring regular renewal to avoid internal accumulation.

In a closed system, it is biologically relevant, where possible, to prioritise the reintroduction of cappings wax from supers. This wax, having not undergone repeated brood cycles or successive accumulation of larval cocoons, generally has a lower structural and chemical load than brood nest combs. Its preferential use for producing new foundation sheets helps to limit cumulative internal accumulation.

In practice:

• Where possible, favour a closed or semi-closed circuit with batch separation.
• If using an external processor, verify documentation of sterilisation parameters and separation of flows.

3.5 Sterilisation versus decontamination

It is fundamental to distinguish two technically and biologically different objectives:
inactivation of pathogens and reduction of chemical contaminants.

Reliable inactivation of Paenibacillus larvae spores requires, according to official Swiss recommendations, treatment of at least 121 °C for 30 minutes under controlled pressure (Swiss Federal Food Safety and Veterinary Office, 2016 ; Agroscope, 2018). This parameter corresponds to autoclave sterilisation using saturated steam under pressure.

Classic beekeeping melting devices — solar wax melters, water baths, open steam boilers — do not exceed 100 °C at normal atmospheric pressure. They allow wax liquefaction and clarification, but do not guarantee inactivation of highly resistant spores. Prolonged melting therefore does not constitute validated sterilisation.

A domestic pressure cooker can reach temperatures close to 120 °C. However, it guarantees neither precise control of temperature within the wax mass nor thermal homogeneity comparable to a professional autoclave. Due to the specific thermal properties of molten wax, insufficiently heated zones may persist. Sporocidal effectiveness therefore cannot be considered validated.

It should also be recalled that properly performed sterilisation does not eliminate persistent chemical residues. Lipophilic acaricides and certain environmental contaminants remain present after thermal treatment (Kast et al., 2023). Biological inactivation and chemical decontamination rely on distinct mechanisms.

Thus, sanitary wax management relies on a clear understanding:

• Do not equate “melted wax” with “sterilised wax”.
• Do not equate “sterilised wax” with “chemically clean wax”.

3.6 Integrate parasitic dynamics

Old combs may show higher infestation rates by Varroa destructor than recent combs (Piccirillo & De Jong, 2004).

In practice:

• Integrate comb renewal into an overall strategy to reduce parasitic pressure.
• Consider that wax-circuit management may indirectly influence viral dynamics, even if the exact magnitude of this effect remains to be quantified (De Guzman et al., 2019 ; Colwell et al., 2024).

3.7 Strategic objective

The strategic objective is not the total elimination of contaminants, but the progressive reduction of structural, chemical and biological constraints.

Coherent management relies on regular comb rotation, control of the wax circuit, distinction between biological sterilisation and chemical decontamination, and vigilance regarding the quality of foundation sheets.

5. Conclusion

Scientific data converge towards a clear recognition of combs as a biologically active compartment within the colony. Comb ageing modifies nest structure, influences worker development and is accompanied by a cumulative increase in contaminants. Added to this are indirect sanitary implications, notably regarding parasitic pressure and management of stored equipment.

The issue is therefore not aesthetic, but systemic: management of the wax circuit constitutes a central lever of biosecurity and beekeeping sustainability. Planned frame renewal, combined with a controlled wax circuit, represents today the most coherent approach in light of available knowledge.

See also:

• Six scientific reasons not to use old frames
• Wax and combs
• Beeswax contamination
• Aide-mémoire: 4.4.1 Melt the frames
• Aide-mémoire: 2.1 American foulbrood

 

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