Insecticides encompass a wide variety of compounds, including organochlorines, organophosphates, carbamates, and neonicotinoids. Neonicotinoids play a significant role in agriculture due to their potent effectiveness against pests and insects, acting as neurotoxic substances in the insect’s central nervous system [47]. The primary approach commonly employed to control pests involves the application of insecticide-coated seeds, as the majority of pests reside in the soil during the planting phase [48]. However, the utilization of neonicotinoid active substances in the seeds of crops such as cotton, corn, and sunflowers has sparked a hypothesis linking this particular class of pesticides to the occurrence of colony collapse disorder syndrome [10]. Different neonicotinoids have been studied in royal jelly [3,19,34,49], due to the potential presence in crops visited by bees, but in most cases, no neonicotinoids were detected. Seven neonicotinoids (dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid and thiacloprid) were investigated in royal jelly-based products from different Spanish regions (n = 12), but no residues were detected [3]. This finding does not suggest any limitation on the method’s applicability, as the European Commission established MRLs for these compounds in honey and related matrices, including royal jelly (10–200 μg/kg) [40]. Likewise, Hong et al. (2019) [47] developed a method for the simultaneous determination of ten neonicotinoids (pymetrozine, dinotefuran, nitenpyram, thiamethoxam, flonicamid, imidacloprid, clothianidin, imidaclothiz, acetamiprid and thiacloprid) and two metabolites (4-trifluoromethylnicotinamide and N-desmethylacetamiprid) in royal jelly. They validated the method, obtaining good recoveries (73–107%), and applied it to 60 royal jelly samples, detecting no neonicotinoid insecticides.

Residues of p-dichlorobenzene (p-DCB), an insecticide used against the Galleria mellonella wax moth, have been detected in royal jelly coming from honeycombs [50]. It was found that the levels of p-DCB in honey were notably lower than those in royal jelly. In fact, in some cases, royal jelly contained hundreds of times more residues of p-DCB compared to honey extracted from the same comb. The maximum concentration of p-DCB in royal jelly was 1520 μg/kg [50]. Considering that the usual daily dosage of royal jelly is expected to be lower than 0.5 g, an individual weighing 70 kg would consume approximately 0.014% of the acceptable daily intake. While this amount is considered too insignificant to pose any health concerns for consumers, it is noteworthy that these residues in royal jelly are undesirable due to the absence of established MRLs for hive products [50].

In other studies, a method was developed and validated to determine potential residues of thiamethoxam and clothianidin in royal jelly samples (n = 11) available from an online shopping website [34]. Clothianidin was never detected, but thiamethoxam was found in three of them with a concentration comprised between 0.15 and 0.25 μg/kg. Clothianidin is an active substance in insecticidal formulations and also the most toxic metabolite of thiamethoxam, and both neonicotinoids have been demonstrated to have adverse effects on queens [51,52].

To effectively control mite infestations, beekeepers often rely on synthesized substances for crop protection and livestock. Acaricides such as amitraz, cymiazole, bromopropylate, τ-fluvalinate, flumethrin, coumaphos, and malathion have been extensively used by beekeepers worldwide. Various formulations, including Apistan^® (containing τ-fluvalinate as the active ingredient), Perizin^® (containing coumaphos), Check-Mite^TM (containing coumaphos), Bayvarol^® (containing flumethrin) and Apiguard^® (containing thymol), have gained approval in numerous European countries [53]. Another widely used acaricide in beehives is amitraz, which has attracted interest due to its degradation products, particularly 2,4-dimethylaniline [30]. However, it is important to note that while some substances like amitraz have received approval in specific countries, others like malathion have not been approved at all [10]. Since 1988, τ-fluvalinate has been employed for the control of Varroa destructor mites, which represent one of the primary pests affecting honeybees. Typically, it is administered in the form of strips or sheets placed inside beehives, allowing bees to come into contact with it as they move around. The presence of residues from these compounds poses a significant hazard to consumer health, including the potential for mutations or cellular degradation. This issue stems from both direct contamination resulting from beekeeping practices and indirect contamination through environmental sources. The latter option is associated with the presence of τ-fluvalinate in royal jelly, as the widespread use and extensive distribution of pesticides have led bees to consume contaminated flowers, subsequently transferring the contaminants to the royal jelly. In a study conducted by Karazafiris et al. (2022) [39], residues of coumaphos and τ-fluvalinate were found in royal jelly produced from colonies under chemical treatment using artificial plastic queen cells, suggesting that acaricides may be transferred from wax to royal jelly. A parallel investigation [21] performed a similar test by applying pesticides inside the beehives, either in the form of aerosol (coumaphos) or as plywood inserts (τ-fluvalinate). No residues were detected when plywood inserts impregnated with τ-fluvalinate were used, but coumaphos residues were found in the range of 10–92 μg/kg. Lastly, Notardonato et al. (2014, 2016) [54,55] screened five acaricides (τ-fluvalinate, bromopropylate, fipronil, amitraz and coumaphos) in one royal jelly sample finding 81 μg/kg of bromopropylate and in homemade honey foods. However, the results are inconclusive due to the low number of analyzed samples and the lack of information about them.

Under field conditions, bees are often subjected to prolonged exposure to multiple pesticides, which can substantially harm their colonies. Herbicides, formulated to impede the growth, development, or survival of unwanted plants, play a vital role in enabling desired crops or vegetation to flourish unhindered by competition. The herbicides are extensively utilized in certain regions, like Brazil, making them one of the most prevalent types of pesticides employed worldwide [56]. Furthermore, since herbicides are not intended for insect control, manufacturers do not provide warnings regarding the potential impact of these products on bees. Consequently, bees may be exposed to significant levels of herbicides when applied to crops, particularly genetically modified ones during the flowering period [57,58]. Faita et al. (2018) [46] investigated the impact of sublethal doses of the herbicide Roundup^® on the hypopharyngeal glands of nursing worker bees and its influence on royal jelly production. The researchers determined that the herbicide induced alterations in the cellular ultrastructure of these glands, leading to premature degeneration of the rough endoplasmic reticulum and significant morphological and structural modifications in the mitochondria. It was emphasized that these discoveries can potentially harm the growth and viability of bee colonies. In a study by Martínez-Domínguez et al. (2016) [59], a multi-class method was developed to identify and quantify more than 260 toxic substances, including pesticides. Eight nutraceutical products (two capsules and six liquid presentations) were analyzed; only one sample was contaminated with propachlor (14.9 μg/kg), but with a concentration below the corresponding MRLs (20.0 μg/kg). The authors emphasized that the presence of this herbicide in royal jelly can be attributed to the fact that the tested sample is a combination of royal jelly and pollen. Therefore, it is plausible that the pesticide could be present in the pollen portion of the sample.

Fungicides are chemical substances employed to eliminate or suppress the growth of fungi, which can cause significant harm to crops and pose risks to the health of humans and domestic animals. The majority of fungicides are toxic to humans and have the potential to induce acute or chronic issues if ingested through food consumption [10]. Although some studies have investigated the presence of fungicides in royal jelly [60], they are generally more frequently found in plants or tea [59], or in combination with other pesticides. Most conducted studies have primarily focused on the effects of insecticides, while fungicides have received limited attention [57]. A combination of miticides, fungicides, herbicides, and insecticides are tested in order to evaluate the impact of colony exposure to a multi-pesticide pollen treatment on the nutritional quality of royal jelly [45]. Milone et al. (2021) [45] detected coumaphos (4.5 µg/kg), 2,4-dimethylphenyl formamide) (4.25–5 µg/kg), thymol (79–301 µg/kg) and other residues at trace levels. Carbendazim, a benzimidazole fungicide commonly used to control the Sclerotinia sclerotiorum of rape plants during the flowering period, was sprayed on rape flowers, and its residues (77 μg/kg) were analyzed in royal jelly [61]. Similar tests were also performed studying the migration of tebuconazole between wax and royal jelly [62] and triadimefon from rape flowers to apicultural products [63]. Residues of tebuconazole were found in queen cell cups decreasing its concentration over time. The authors suggested that these concentrations do not pose a lethal risk to queens, but sub-lethal effects should be considered, as azole fungicides have synergistic negative effects on honeybees when combined with insecticides. Additionally, traces of triadimefon were detected (4 μg/kg, MRLs = 100 μg/kg), but at concentrations 10 times lower than the residues found in pollen.