Acaricide Residues in Beeswax. Implications in Honey, Brood and Honeybee and Influence of Landscape Around Colonies

doi:10.21203/rs.3.rs-2247080/v1

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Acaricide Residues in Beeswax. Implications in Honey, Brood and Honeybee and Influence of Landscape Around Colonies

https://doi.org/10.21203/rs.3.rs-2247080/v1

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published 09 Mar, 2023

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To make beekeeping sustainable, the management of bee colonies to produce bee products financially viable without compromising the life of bees must implement acceptable practices such as the treatment of hives with appropriate products. Occasionally, the use of acaricides to treat the hives against varroosis is uncontrolled and can accumulate in the hives endangering the bee colonies. In this work, a screening of seven acaricides was carried out in different apiaries in Andalusia (Spain). Their distribution in beeswax, brood, honey, and bee, was evaluated in different times considering the influence of the environment (agricultural, urban and forest) surrounding the colonies. It was found that beeswax was highly polluted but honey, brood and bees had acceptable levels, below their respective MRL or LD50, past a certain period after varrocide treatments. The landscape management around the hives treated with products against Varroa does not influence the hive contamination. Acaricides banned for their use against Varroa, such as chlorfenvinphos, cypermethrin and especially acrinathrin, were found in the hives analysed.

Acaricides

beeswax

honey

honeybee

brood

apiary location

Beeswax is a natural product composed of a mixture of lipophilic compounds constructed and moulded in hexagonal shape by worker honeybee mandibles (Bogdanov, 2004) to build the combs for honey and pollen storage and brood cradle. However, the production of beeswax, in general, is not the goal of beekeepers, therefore, manufactured wax sheet foundations, from recycling old combs, are provided to honeybees to extend and form the cells. Nonetheless, beeswax can remain in the hive several years without being changed and, due to its lipophilic character, accumulate non-polar pesticides from diverse sources such as agriculture and veterinary treatment (Mullin et al., 2010; Chauzat and Faucon, 2007).

The Varroa destructor is an ectoparasite present in beehive all year that feeds on haemolymph of larvae and adult honeybees. This parasite has spread worldwide and is considered a major threat for apiculture. It is believed that it is one of the main promoters of bee colony loss. Therefore, it is necessary to treat the hives against varroosis, because without treatment the colony will die in 3 to 5 years (Gregorc et al., 2018). In most cases, the mite is controlled using synthetic acaricides such as amitraz, fluvalinate and coumaphos that are the most frequently applied. However, the continuous use of these chemicals over the same hive could cause the collapse of honeybee colonies (Rial-Otero et al., 2007). The chemical structure of the acaricide determines its physico-chemical properties such as solubility, molecular mass and lipophilicity that affect its accumulation in beeswax or in other parts of the hive. Water soluble acaricides tend to accumulate in honey while lipophilic ones do so in beeswax. The application of acaricides in the hives can be by direct application of a drip solution, by fumigation or by strips impregnated with the acaricide that are placed in the comb frames of a hive. These apicultural practices could result in the accumulation of acaricides in beeswax and in other beehive products by diffusion or by migration from beeswax in contact with them. In addition, Varroa resistance to acaricides could occur (Rinkevich, 2020; Benito-Murcia et al., 2021). The recycling of beeswax can be insufficient to eliminate all the chemicals accumulated in it because they resist the melting temperature (Ravoet et al., 2015). Acaricides will remain in the beeswax foundation sheets and go back into the hive and the new acaricide application could result toxic for the proper development of the colony.

While maximum residue limits (MRLs) in honey are established for certain acaricides used against varroosis, there are no MRL for beeswax despite the fact it is used in food and pharmaceutical products. In Europe there is no quality control on beeswax associated to its use.

There are many studies on acaricide levels on beeswax from different parts of the world (Calatayud-Vernich et al., 2017; El Agrebi et al., 2020; Kast et al., 2021; Lozano et al., 2019; Mullin et al., 2010; Perugini et al., 2018) but fewer studies evaluate its distribution or possible transfer from wax to the rest of the hive (honey, brood and bee) (Kast and Kilchenmann, 2022; Murcia Morales et al., 2020) and their relation with the hives environment (Fulton et al., 2019; Calatayud-Vernish et al., 2018). Thus, the aims of this work were to obtain an overview of the acaricides found in hives in apiaries from Andalusia (Spain), determining their concentration levels in beeswax and other hive components (brood, honey, and bee) at different times and also evaluating the possible influence of the environment (agricultural, urban and forest) surrounding the colonies on the acaricide levels. The acaricides analysed were, coumaphos and its metabolite chlorferon, chlorfenvinphos, acrinathrin, cypermethrin and amitraz. Coumaphos and chlorfenvinphos are organophosphate compounds, acrinathrin and cypermethrin are pyrethroids and amitraz is an amidine. As amitraz is usually undetected in beeswax and honey samples due to its short half-life in these matrices (Shimshoni et al., 2019), this compound was analysed indirectly through its metabolites N-(2,4-Dimethylphenyl)-formamide (DMF) and N-(2,4-dimethylphenyl)-N-methylformamidine) (DMPF).

2.1. Origin of the samples

The sampling was performed in sixteen honeybee hives (H1-H16) initially located on the same apiary in Andalusia, Spain (L1) (December 2017). In March 2018, these hives were moved to seven different locations (2 hives in each location) leaving two hives in the initial site L1 (Fig. 1). Land use in locations L1-L8 is described in Table 1. In June 2018, a second sampling was conducted in the hives sited in their new locations. Samples of wax, honey, brood, and honeybees were collected by beekeepers from each colony in December 2017 and June 2018. Approximately, 20 broods, 100 honeybees and 5 g of honey, were collected in each hive. Brood samples taken for this study were in the larval stage, with a development of six days. All the samples were stored at -20°C until analysis.

Table 1

Land use of the locations were apiaries were placed
Location	Apiary environment	Land use
L1	Mountainous landscape. Mediterranean-continental vegetation, pines, holm oaks	Forest
L2	Urban landscape: ornamental garden	Urban
L3	Dryland crops: olive and Mediterranean vegetation, near a highroad	Farming
L4	Grassland, scrub with a stream and a highroad in the proximity.	Forest-pastureland
L5	Irrigations crops: citrus	Farming
L6	Riparian vegetation and dryland crops: olive	Farming
L7	Dryland crops: olive. Mountainous landscape: pines and holm oaks.	Agroforestry
L8	Rural grassland within the city and near a large river	Urban-rural

2.2. Reagents and materials

All pesticide standards were obtained from Sigma-Aldrich (St. Louis, USA): coumaphos, acrinathrin, chlorfenvinphos, cypermethrin and DMPF with purity ≥ 98%; and chlorferon and DMF with 97% purity. Individual stock solutions (1000 mg/L) were prepared in acetonitrile and stored in amber vials at − 20°C. Individual standard solutions and standard-mix solutions, used for the optimization and calibration respectively, were prepared by an appropriate dilution of the stock standard solutions with acetonitrile.

Magnesium sulphate (MgSO₄), sodium acetate (NaAc), formic acid and ammonium formate were obtained from Sigma-Aldrich (Steinheim, Germany). PSA and C18 sorbents were purchased from Scharlab S.L (Barcelona, Spain), and Z-Sep from Agilent (Santa Clara, CA, United States). LiChrosolv acetonitrile was provided by Merck (Darmstadt, Germany). High purity water was obtained with a Milli-Q water purification system (Millipore, Milford, MA, USA).

2.3. Extraction procedure

2.3.1. Beeswax

Beeswax samples were free of beebread, honey, cocoons, or brood before their analysis. The beeswax extraction method was based on Niell et al. (2014). Briefly, beeswax pieces of 15 cm² were cut as small as possible and mixed to make a homogenised sample. Then, 1 g of beeswax and 5 mL of acetonitrile were added to a centrifuge tube of PP (polypropylene) and the tube was heated in a water bath at 80°C. When beeswax was melted, the tube content was homogenised for 30 s by vortex agitation, followed of sonication for 5 min using an ultrasound water bath at 60°C. These procedures were repeated five times to ensure an efficient extraction of pesticides. Afterwards, the sample was centrifuged at -4°C and 5000 rpm for 15 min. The supernatant was collected in a PP tube and stored overnight in a freezer at -20°C followed by centrifugation for a good separation of the beeswax and the solvent. Supernatant was diluted with acetonitrile (1:1, v/v). Then, an extract aliquot was cleaned up with PSA and C18 (50 mg of each sorbent by ml extract). The tube with the sorbents was shaken for 3 min in a vortex and centrifuged at 5000 rpm for 10 min. Finally, the supernatant was filtered using 0.22 µm nylon filter. And 1 mL of filtrate and 0.01 mL of a solution of acetonitrile with 5% of formic acid were mixed in a vial before chromatographic analysis.

2.3.2. Honeybee and brood

Sample preparation of honeybee and brood was carried out using a modified ultrasound-assisted QuEChERS method (García-Valcárcel et al, 2019). One gram of sample (honeybee or brood) and 5mL of MilliQ water were added to a centrifuge tube and let stand for 5 min. Afterward, honeybees or broods in the tube were fragmented by agitation in a horizontal shaker with two agate balls and 5 mL of acetonitrile for 10 min. Fragmentation was followed by sonication for 5 min, and a mixture of 4 g of MgSO₄:NaAc (4:1, w/w) was added to the tube. The tube was shaken vigorously in vortex for 1 min and centrifuged for 5 min at 5000 rpm and 4˚C. An aliquot of supernatant was transferred to a tube containing C18 and PSA in the case of brood (50 mg of each sorbent by ml of supernatant) and MgSO₄ (150 mg/ml) and PSA (100 mg/ml) in the case of honeybees. The tube was shaken by vortex for 1 min and centrifuged for 5 min at 5000 rpm at -4˚C. The supernatant was filtered using 0.22 µm nylon filter. In a vial, 1 mL of filtrate and 0.01 mL of a solution of acetonitrile with 5% of formic acid were mixed before quantitation by HPLC-MS/MS.

2.3.3. Honey

Honey (1 g) and 7mL of MilliQ water in a centrifuge tube were vortexed for 1 min. Then, 10 mL of acetonitrile containing 5% of formic acid were added to the tube and shaken by hand followed by sonication in an ultrasonic water bath for 10 min. A mixture of 4 g of MgSO₄:NaAc (4:1, w/w) was added and shaken for 30 s followed by centrifugation at 5000 rpm and 4°C for 15 min. Then, an aliquot of the extract was cleaned up by adding 0.15 g MgSO₄, 0.05 g of PSA and 0.05 g of Z-Sep for each ml of extract. Afterward, filtration using 0.22 µm nylon filter was performed.

2.4. Quantitation analysis

Quantitation of acrinathrin and cypermethrin in the different samples was performed by gas chromatography-mass spectrometry (GC-MS) and by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the rest of the acaricides.

Analysis by GC-MS was performed in a gas chromatograph (Agilent 7890A) coupled to a mass spectrometer (HP 5977A) equipped with an automatic injector. Separations were performed by using a HP-5MS column, (30 m×0.25 mm i.d. 0.25µm film thickness) from Agilent (Torrance, CA, USA). Helium (purity 99.995%) was used as carrier gas at a flow rate of 1.4 mL/min. Operating conditions were as follows: 2 µL extracts were injected in a simple-taper glass liner with deactivated glass wool, with the autosampler in pulsed splitless mode (40 psi for 0.75 min). The injection port temperature was 250°C. The column temperature was maintained at 70°C for 1 min, then programmed at 30°C/min to 280°C (held for 7 min) and then at 40°C/min to 300°C. The total analysis time was 15.5 min. The mass spectrometric detector was operated in electron impact ionization mode with an ionizing energy of 70 eV. Ion source and transfer line temperatures were 230 and 280°C, respectively. Retention time and mass spectra of all analytes were acquired in the full scan mode (mass range from 50 to 600 m/z). Selected Ion Monitoring (SIM) mode was employed for quantitative analysis, using one target and two qualifier ions to identify each analyte. Table S1 lists the mass spectrometric parameters and retention time of acrinathrin and cypermethrin by GC-MS.

An Agilent liquid chromatograph (HPLC 1200 series) equipped with an automatic injector, a degasser, a quaternary pump and a column oven combined with an Agilent 6410 triple quadrupole (QQQ) mass spectrometer with an electrospray ionization interface (Agilent Technologies, Waldbronn, Germany) was used for the analysis of chlorferon, coumaphos, chlorfenvinphos, DMPF and DMF. The electrospray ionisation source was operated in positive mode with a gas temperature of 300°C, a gas flow of 9 L/min, a nebulizer pressure of 35 psi and a capillary voltage 4000 V. Nitrogen was used in the nebulizer and in the collision cell. The chromatographic column was EVO-C18 of 100×3mm i.d. and 2.6 µm, 100 Å particle size (Phenomenex, Torrance, CA, USA). The mobile phase consisted of A (acetonitrile with 0.1% formic acid) and B (ammonium formate 5mM), the flow rate was of 0.4 mL/min and the injection volume was 10 µL. The gradient elution program used was as follows, starting at 50% of solvent A, increased to 80% in 10 min, to 95% in 3 min and kept constant for 2 min. Return to initial conditions in 7 min and keep constant for 8 min. The QQQ mass spectrometer was operated in selected reaction monitoring (SRM) and two optimised SRM transitions were monitored for each acaricide (Table S2). Data were processed using the Mass Hunter Workstation Software for qualitative and quantitative analysis (Agilent Technologies, Palo Alto, CA, USA).

Matrix-matched external calibration method was used to quantify the acaricides in honeybee, brood, and honey. But, as it was difficult to find a wax sample without the selected pesticides, a standard addition method was employed for quantitation of pesticides in beeswax by adding different concentrations (0, 20, 100 and 500 ng/mL) of the target analytes to different aliquots of the same beeswax extract. In addition, the standard addition method could compensate matrix effects due to the wax.

The method LOQ (limit of quantification) was set as the minimum concentration that can be quantified with acceptable accuracy (between 70–120%) and good precision (RSD < 20%). Due to the difficulty of finding blank beeswax samples, these samples were analysed, and the target pesticides found in them were considered to calculate LOQ, that should be the lowest spiked level validated accomplishing SANTE criteria ((SANTE/11813/2017, 2017).

Recovery and precision of the methods were conducted at two levels, 2 and 20 µg/kg for brood, honey, and bee and at 10 and 1000 µg/kg for wax. Four replicates of each concentration level were carried out for recovery check. Repeatability and reproducibility were evaluated for both spiking levels; over 1 and 4 days, respectively. Linearity was evaluated by spiking blank extracts with a standard solution at six concentration levels (from 2 to 1000 µg/kg). If the area of a positive finding was above the linear range, the sample was diluted to an appropriate concentration and reinjected in a new curve.

2.5. Acaricide risk assessment

Hazard quotients (HQ) in wax were determined as the sum of all acaricide residues detected in wax (µg/kg) divided by their respective contact LD₅₀ (µg/bee) in each beeswax sample. LD₅₀ were taken from Sanchez-Bayo and Goka (2014) and PPDB/VSDB (Pesticide Properties DataBase /Veterinary Substances DataBase). The risk to honeybee and broods was evaluated by comparison of the LD₅₀ with the residue levels for each acaricide found in bees and brood. The contamination of honey was established by comparison of the acaricide levels in honey and its MRL.

3.1. Method validation

The LOQ was 2 µg/kg for all compounds in honey, brood, and honeybee. Recoveries for brood, honey and honeybee were between 71 and 108%, with good precision, RSD between 1 and 19% (repeatability) and between 2% and 21% for reproducibility. Recoveries in wax were in the range of 80–110% with RSD between 3% and 17% (repeatability) and 5–20% (reproducibility). LOQ in beeswax was 10 µg/kg for all compounds. Matrix matched calibration curves showed good correlation coefficients R² > 0.98 in the range of 2–1000 µg/kg.

3.2. Acaricide residues in beeswax

3.2.1. Levels in Beeswax in December and June

From the total acaricides analysed in all waxes collected in December 2017, coumaphos was the most abundant followed by DMF, acrinathrin, chlorfenvinphos and DMPF with percentages of 52%, 28%, 11%, 4% and 3%, respectively. The rest of compounds analysed in wax were present in a percentage < 3%.

Considering the total compounds analysed in all waxes in the different locations in June 2018, the most abundant acaricide was coumaphos with 33% of the total acaricides, followed of acrinathrin with 32%, DMF with 25% and chlorfenvinphos with 7%. Chlorferon, DMPF and cypermethrin represented less than 5% of the total.

Coumaphos, amitraz (as DMF and DMPF), chlorfenvinphos and acrinathrin were present in all the waxes collected in December, chlorferon was found in > 87% and cypermethrin in 31% of the beeswax samples in December (Table 2). For the positive compounds found in waxes in December, the ranges of concentration found were very wide, indicating a high variation between hives although all waxes were collected from hives located at the same site and sampled at the same time. The mean concentration of coumaphos was about twice that of amitraz and approximately six times that acrinathrin for the set of waxes where these compounds were quantified (Table 2).

Table 2

Summary of compounds quantified in beeswax (n = 16) at the initial location in December 2017 and at the final locations in June 2018 ^a
Compounds	Positive (%)		Minimum (µg/kg)		Maximum (µg/kg)		Mean (µg/kg)		Median (µg/kg)
	Dec.	June	Dec.	June	Dec.	June	Dec.	June	Dec.	June
Chlorferon	87.5	81	24	22	1532	174	225	76	112	66
Coumaphos	100	100	323	255	35678	6687	5958	1617	3506	807
Chlorfenvinphos	100	100	55	46	1409	1845	445	315	401	176
Amitraz (DMF)	100	75	374	28	24880	17799	3210	1597	714	134
Amitraz (DMPF)	100	69	29	10	3266	1298	322	159	95	48
Acrinathrin	100	100	120	107	4278	4447	1214	1541	687	553
Cypermethrin	31	0	62		93		78		81
^a Minimun, maximun, mean and median values of positive samples (> LOQ)

Coumaphos, chlorfenvinphos and acrinathrin were present in all the waxes collected in June. DMF and DMPF (amitraz metabolites) were present in 75% and 69% of the samples, respectively. Chlorferon was present in 81% of the samples and cypermethrin was not quantified in any wax sample at any location in June 2018. The mean concentration values of coumaphos, DMF and acrinathrin were similar, between 1.5 and 1.6 mg/kg, when positive samples for these acaricides were considered (Table 2).

The amount of chlorferon was always inferior to that of coumaphos for the same wax sample and the levels of DMF were higher than that of DMPF within the same wax. Nevertheless, no correlation between chlorferon and coumaphos or DMF and DMPF were found.

Considering the average of the 16 wax samples in December and in June, a decline in the concentration of each acaricide over time was detected (Table 2). The most pronounced decrease was observed for coumaphos followed by amitraz with a 75% and 63% drop, respectively, in relation to their initial mean levels. On the other hand, chlorfenvinphos mean concentrations were similar in both sampling times and acrinathrin mean concentrations were slightly higher in June. Nevertheless, considering the median values, all acaricides values decreased with time. The mean of total acaricide load in the total waxes sampled in December was 11.4 mg/kg while in those sampled in June it was 4.8 mg/kg, in other words, 42% of the initial load of pesticides remains six months later.

3.2.2. Levels in the different locations

The differences in acaricide concentrations for the same hives in the initial location in December and in the final locations in June are shown in Fig. 2. Cypermethrin was not present in any beeswax sample collected in June and was only detected in five hives in December with a low mean concentration level of 24 µg/kg, so this compound was not presented in the Fig. 2.

The acaricide with the highest concentration in most of the waxes analysed, both in December and in June, was coumaphos (coumaphos + chlorferon). The highest losses of coumaphos occurred at sites L1 (hives H1-H2), L4 (hives H7-H8), and L7 (H13-H14) in relation to the initial levels in December, as only 2–14% of the initial coumaphos concentration in beeswax remained in these locations. Chlorfenvinphos levels in June increased between 1.5 and 2.2 times in sites L1 (H1-H2), L3 (H5-H6) and L5 (H9-H10) whereas in the rest of hives the levels decreased. Acrinathrin residues increased sharply in 50% of the trial sites in June (sites L1 (H1-H2), L3 (H5-H6), L4 (H7-H8) and L7 (H13-H14)). Amitraz (DMF + DMPF) levels decreased between 99% and 67% in relation to the initial amount in all locations except in site L3 (H5-H6) where amitraz increased seven-fold from the initial level in December.

Except for beeswax sited in location L3 (H5-H6), a decrease in the total load of acaricides was found over time in each location (Fig. 3). At this site (L3), an important increase in the amount of amitraz and acrinathrin and to a lesser extent of chlorfenvinphos was observed in relation to their content in December (Fig. 2).

3.2.3. Pesticide risk assessment in waxes

Given the high toxicity of acrinathrin in adult honeybees, contact LD₅₀ = 0.084 µg/bee, this compound contributes the most to the HQ score (Fig. 4) whereas coumaphos with a LD₅₀ = 20 µg/bee and amitraz with a LD₅₀ = 50 µg/bee were the acaricides that less contribute to the HQ value. Chlorfenvinphos, despite its low wax levels, also contributes to raising the HQ value due to its LD₅₀ = 0.55 µg/bee. HQ values in wax were often > 5000 (Fig. 4). Beeswax in locations L4 and L7 presented the highest concentrations of acrinathrin.

3.3. Acaricide residues in honey, brood and bees

3.3.1. Levels of acaricides in December and June

Of the seven acaricides analysed, none of the honey, brood or bee samples contained cypermethrin or chlorfenvinphos. Acrinathrin was only quantified in one sample of honey (24.5 µg/kg in H15) and one of brood (14.4 µg/kg in H3) in the original location in December. These hives contained the highest acrinathrin load concentration in wax (Fig. 2). Chlorferon was quantified in 3 of the 16 honey samples (H2, H8 and H10) and in one bee sample (H15) at the original site, but was not quantified in brood. This compound was also quantified in one of the 16 honey samples at the exposure locations (H11). In December, coumaphos was present in 69%, 25% and 25% of honey, brood and bee samples, respectively, whereas in June its presence in honey and bees was reduced to 19% and 6%, respectively, and increased in brood up to 50%. Amitraz (DMF + DMPF) was present in all honey and bee samples analysed and in 87% of broods at the initial site L1 and in June, its presence decreased to 94%, 7% and 12% for honey, brood and bee, respectively.

Table 3 summarises the mean concentrations of coumaphos and amitraz in the hives analysed at the initial and final sites. Honey was the matrix with the highest amount of both acaricides in December (initial site) and June (exposure sites). Concentrations of amitraz were higher than those of coumaphos for both sampling times and for all matrices. Levels of coumaphos and amitraz were higher in the initial site than in the exposure locations for all three matrices, except for brood where the mean amount of coumaphos was similar in the initial and final apiaries (Table 3). In the initial site, amitraz content in bees was higher than in brood but at the exposure sites similar amounts of amitraz were obtained in bee and brood with values very low.

Table 3

Coumaphos and amitraz average concentration levels (µg/kg) in samples, corresponding to two different hives, for honey, brood, bee and wax placed in the different locations in December and June.
	Coumaphos								Amitraz
	Honey		Brood		Bee		Wax		Honey		Brood		Bee		Wax
Location ^a (Hives)	Dec.	June	Dec.	June	Dec.	June	Dec.	June	Dec.	June	Dec.	June	Dec.	June	Dec.	June
L1-L1(H1-H2)	11.5	n.d^b	n.d	5.6	n.d	n.d	8665	800	32	73	15	n.d	63	n.d	728	196
L1-L2(H3-H4)	14.2	n.d	2.4	2.5	3.2	n.d	2054	1017	2195	24	36.4	n.d	93	n.d	22179	87
L1-L3(H5-H6)	4.4	n.d	9.7	2.0	1.8	n.d	1011	761	571	9	32.5	2.9	387	2.7	1305	9619
L1-L4(H7-H8)	3.4	n.d	2.3	1.5	n.d	n.d	4595	661	171	11	9.1	n.d	50	n.d	649	n.d
L1-L5(H9-H10)	12.6	5.6	n.d	n.d.	n.d	2.25	3857	2918	72	23	6.2	n.d	138	3.2	990	210
L1-L6(H11-H12)	8.9	15.7	n.d	1.2	1.0	n.d	4577	3472	32	25	8.4	n.d	94	n.d	993	113
L1-L7(H13-H14)	0.0	n.d	n.d	5.9	n.d	n.d	19548	416	32	122	5.3	n.d	100	n.d	571	189
L1-L8(H15-H16)	9.7	n.d	n.d	1.0	3.0	n.d	3352	2892	64	24	35.4	n.d	78	n.d	847	41
Mean^c	8.1	2.7	1.8	2.5	1.1	0.3	5957	1617	396	39	18.5	0.4	125	0.7	3533	1307
^a Location December 2017- Location June 2018
^b n.d.: not detected (< LOD)
^c If the acaricide, coumaphos or amitraz (DMF + DMPF), was not detected in a sample it was considered 0 for the determination of mean.

3.3.2. Influence of the exposure location.

The highest concentration of coumaphos at the initial site was 14.2 µg/kg for honey, followed by brood (9.7 µg/kg) and bee (3.2 µg/kg). At the exposure sites in June, the concentration of coumaphos was, in general, lower than in the initial site with some exceptions: in location L6 (H11-H12) an increase in honey and brood in relation to the initial values was noted (Table 3) and in location L7 (H13-H14) where a considerable increase in brood (from not detected to 5.9 µg/kg) was observed. In addition, in hives H9-H10, coumaphos was quantified in bee in June but not in December. No correlation were found between coumaphos levels in wax and the other matrices.

In December, when all hives were in location L1, all matrices contained amitraz, with the highest concentration values for honey and the lowest for brood. While in June, amitraz was found in all honey samples, at lower levels than in December, but found only in brood samples from one site and in bee samples from two sites. It is necessary to point out the high levels in honey from hives (H3-H4) and (H5-H6) collected in December, with values exceeding the MRL value of 200 µg/kg for amitraz in honey. In addition, bees contained relatively high concentrations of amitraz in the initial apiary (Table 3). The highest amitraz concentration values in beeswax in December (> 1.2 mg/kg ) correspond to the highest initial values of this compounds in honey, brood and bee.

In December, the total load of acaricides in honey, bee and brood in most hives was between 120 and 200 µg/kg with the exception of the hives H3-H4 and H5-H6 with about 2300 µg/kg and 1000 µg/kg, respectively (Fig. 5). These values were due basically to the high amount of amitraz found in honey at this initial time. In June, the total load was between 12 and 120 µg/kg, which represents a reduction up to 99% of the initial load in some hives (H3-H4).

3.3.3. Potential impact of acaricides in the bee colonies

The highest concentration of coumaphos found in brood was 9.7 µg/kg that corresponds to 1.2 µg/larva (one brood weighs 125 mg) lower than its contact LD50 of 2.7µg/larva (Dai et al 2017). For honeybee, 0.25 µg/bee was the highest concentration found (one bee weighs 100 mg, approximately), lower than its LD50 = 24 µg/bee according to Stoner and Eitzer (2013) or LD50 = 20.29 µg/bee reported by Sánchez–Bayo and Goka (2014). The highest concentrations of amitraz were 4.55 µg/larva and 38.7 µg/bee that did not exceed the values of their LD50 (14.83 µg/larva (Dai et al 2017) or 50 µg/bee (PPDV)). For acrinathrin no LD50 values were found for larva but the concentration of acrinathrin found in a brood sample was 14.4 µg/kg (1.8 µg/larva), much higher than the LD50 available in the literature for this compound in adult bees.

4.1. Levels in Beeswax in December and June

In Spain, the Royal Decree 608/2006 establishes that is mandatory the application of at least one treatment per year (autumn) against varroosis. In view of the results obtained from the wax sampling in December, it seems the hives were treated in autumn with coumaphos and, in some cases with amitraz, judging by the high concentrations of these compounds found and by the percentage of individual acaricide found in relation to the total amount of acaricides detected. These treatments (coumaphos and amitraz) are in accordance with the principal veterinary treatments in Spanish apiaries (Calatayud-Vernich et al 2017). The mean concentration of coumaphos (coumaphos + chlorferon) in the 16 hives sited in location L1 was 6.0 mg/kg, which is similar to those reported for coumaphos in other works where waxes from Spanish hives were analysed (Calatayud-Vernich et al., 2017 and 2018; Murcia-Morales 2020). A mean concentration of 0.8 mg/kg for amitraz (DMF + DMPF) was observed which was similar to the concentration level (0.74 mg/kg) reported by El Agrebi et al. (2020). Concentration levels in hives H3 and H4 were excluded from this mean because they were excessively high (Fig. 2), probably due to the application of amitraz in these hives close to the samplings. Chlorfenvinphos and acrinathrin were detected in all the beeswax samples collected in December. These acaricides are not allowed in beekeeping as treatments against Varroa but chlorfenvinphos is used as acaricide in other livestock species and acrinathrin is used in agriculture. Mean concentration levels in waxes from this apiary were relatively low, 1.21 mg/kg and 0.44 mg/kg for acrinathrin and chlorfenvinphos, respectively. Therefore, these compounds were most likely already present in the foundation sheets employed in these hives, which were obtained from recycled waxes where they can accumulate and persist long time after their application (Mullin et al., 2010; Lozano et al., 2019). Acaricides can resist the melting temperature when wax is recycled and accumulate in beeswax. The lipophilic character of the compound together with its half-life in beeswax determines its potential accumulation in it. The higher lipophilicity of acrinathrin (log Kow = 6.3) in relation to the rest of compounds, could explain its persistence in wax.

In June, a decrease of the concentration of all acaricides in wax was observed except for acrinathrin (Fig. 2), which increases its residual level in some hives. Although this acaricide is not allowed in apiculture, it is likely that it was applied illegally in these hives between both samplings. This probable illegal treatment has also been reported in some Spanish apiaries (Jimenez et al., 2005; Orantes-Bermejo et al., 2010; Serra-Bonvehi and Orantes-Bermejo, 2010; Calatayud-Venish et al., 2018). In December, the detection frequency of cypermethrin was low (20%) with a mean concentration of 0.08 mg/kg. This compound, with a log Kow = 6.6, has a high affinity for wax, so its presence could be due to the use of recycled waxes that did not eliminate cypermethrin successfully during its treatment. Nevertheless, cypermetrin was not found in wax samples in June probably due to its low presence in December and to its heterogeneous distribution in the wax (Kast et al., 2020), so that in the random June sampling, no pieces of wax containing cypermethrin were sampled.

From December to June, the content of coumaphos (coumaphos + chlorferon) decreased 73%, chlorfenvinphos 30% and amitraz (DMF + DMPF) 64%. Coumaphos and chlorfenvinphos with similar lipophilicity (log Kow of 3.9 and 3.8, respectively) dissipated at different rates. This is most likely due to a recent treatment with coumaphos whereas chlorfenvinphos was already in the wax and strongly retained in it before coumaphos was applied. In addition, amitraz with log Kow of 2.1 and 2.9 for DMF and DMPF, respectively should have dissipated over time in greater proportion than coumaphos. However, the dissipation of amitraz was slower than that of coumaphos. This apparently contradictory result may be due to a new application of amitraz close to the June sampling, which increased the value of the mean in this month (Fig. 2).

4.2. Distribution of acaricides in beeswax depending of the location

High spatial and temporal differences among acaricide concentration values in beeswax from different colonies were observed (Fig. 2). In the initial location L1, except hives H3-H4 and H13-H14 that had a total load of acaricides 3–4 times higher than in the other hives, the average total load was around 7000 µg/kg (Fig. 3). In the case of hives H3-H4, an extraordinarily high amount of amitraz was found and in the case of hives H13-H14 a high amount of coumaphos was detected (Fig. 2), which could point out an extra treatment with these acaricides at the initial location, L1. In June, hives placed in locations L2 and L7 decreased their acaricide load to 9% and 17% of their initial load, respectively. In the rest of the hives the decrease was close to 35%.The faster dissipation rate of amitraz and coumaphos in these hives could be due to a sampling of wax in December close to the placement of the treatment strips (Kast et al., 2020) or to the geographical location of the hives in June whose characteristics (weather) eases this dissipation.

Acrinathrin, a highly lipophilic compound, was found at higher levels in beeswax from hives located in L1, L3, L4 and L7 (from 2.3 to 4.7 times higher) in June than when there were sited in L1 in December (Fig. 2). Acrinathrin is employed as miticide-insecticide in agriculture, which could justify the amounts of this compound in L3 and L7, sites with olive groves, where acrinathrin could have been applied to combat the olive fly and transported by honeybees to the hives. Nevertheless, the high amount (about 3000 mg/kg) found in L1 and L4, which are surrounded with pine forests and Mediterranean vegetation, suggests the use of this pyrethroid by beekeepers, as it has already been pointed out in other works on Spanish waxes (Calatayud-Vernich et al., 2018).

4.3. HQ wax values for the beeswax samples analysed

HQ wax was calculated for samples only collected in June because in December the hives had just been treated. Figure 4 shows that in all locations, except L6, hives contained beeswax with HQ > 5000, which is considered a high pesticide load and is related with a queen replacement and a higher colony mortality, therefore represents an elevated toxicity for bees (Trainor et al 2016). Acrinathrin, due to its high toxicity (LD₅₀ 0.17 µg/bee), was the acaricide that most contributed to the high HQ in wax. Agrebi et al. (2020) provided recommendations on the re-use of beeswaxes, suggesting not to recycle and reuse waxes when the HQ value is > 5000.

4.4. Acaricides inside the hives.

Acaricides found in beeswax can migrate by diffusion/partition to other beehive components such as brood, beebread, and honey. Bees can be contaminated by contact as they walk on hive frames and orally by eating beebread or honey in contact with wax or by chewing beeswax to build the combs (Tremolada et al., 2004; Lozano et al., 2019; Murcia-Morales et al., 2020). The rate of migration depends on the solubility of the compound in each beehive matrix. The high prevalence of coumaphos found in honey in December could be due to the closeness of treatment with this compound to the sampling date or to bees carrying coumaphos in their bodies contaminating the honey. Nevertheless, the residue levels detected were in the range of those found in the literature (< 40 µg/kg) (Maver and Poklukar, 2003; Valdovinos-Flores et al., 2016). These levels decreased until they disappeared in almost all honey samples in June. In any case, these levels are lower than the MRL established for coumaphos in honey making it fit for consumption.

In brood samples collected in December, a scarce number contained coumaphos whereas in June in practically all localities, brood had residues of coumaphos (Table 3). Brood is rich in lipids, so coumaphos as a fat-soluble compound could accumulate in this matrix more than in honey. Coumaphos can migrate from wax to brood directly by contact or indirectly by migration into the larval diet. Although the concentration levels in brood were similar in December and June, there was a higher prevalence of coumaphos in June than in December. This could be explained by a migration of this lipophilic compound from beeswax to brood after a certain time from its treatment. Levels of coumaphos in brood were much lower than those reported by Bajuk et al. (2017) (100–250 µg/kg) after coumaphos treatment and of the same order as those found by Murcia-Morales et al (2020) in hives untreated with coumaphos (from 1.5 to 12 µg/kg). The migration to brood could negatively affect the development of bee colonies althoughthe levels found in brood were lower than its LD₅₀.

Honeybees are in contact with acaricides applied in the hives and, therefore, exposed to them. However, the determination of these compounds in bees could be underestimated due to biotransformation and rapid excretion in honeybees, as well as, the possible disorientation of contaminated bees to return to their hives (Calatayud-Vernich et al., 2018). In this study, a low coumaphos concentration in bees was obtained with the maximum values in December (mean 1.1 µg/kg).

Amitraz (DMF + DMPF) was found in all honey, brood, and bees samples analysed in December and was the acaricide with the highest concentration of all compounds assayed. The highest concentration was found in honey in December, 396 µg/kg, which decreased to 39 µg/kg in June. The high value of this compound in wax indicated that this treatment was applied near December in the hives H3-H4 and probably in H5-H6 and then transferred to honey in these hives reaching values above the allowed MRL (> 200 µg/kg). Average concentration in bees was 125 µg/kg in December that practically disappeared in June. In accordance with Mullin et al. (2010), amitraz metabolites bioaccumulate in bees in a greater extent than coumaphos. In brood, amitraz was also present in all the samples analysed in December at concentrations that ranged from 8.4 to 36.4 µg/kg, which are similar (from 1.9 to 35 µg/kg) to those found by Murcia-Morales et al. (2020) two months after the amitraz application in hives. Amitraz residues in brood and bee decreased to its practical disappearance in June but remained in honey. This could be due to the more hydrophilic character of honey that would have more affinity for DMF and DMPF that present low Kow values.

Coumaphos and amitraz were the main acaricides used to control Varroa in the analysed hives, although it appears acrinathrin was also employed in some hives. The low levels of chlorfenvinphos and cypermethrin found in beeswax suggest that these acaricides come from recycled or reused waxes in the hives but not from a recent treatment. Levels of coumaphos and amitraz in beeswax decreased with time after treatment and were independent of the landscape surrounding the colonies.

There is evidence that contaminated beeswax by application of coumaphos and amitraz in hives can contaminate honey and brood in contact with it although the levels found did not reach the MRL values for honey or the LD₅₀ for brood.

High HQ values obtained for beeswax indicate a possible negative effect in the bee colony particularly due to the acrinathrin content, but only one sample of sixteen brood samples contained it. Kast and Kilchenmann (2022) pointed out that coumaphos concentrations up to 20 mg/kg in wax are not lethal to larvae. In this work, none beeswax analysed exceeded this value for coumaphos.

In order to achieve a sustainable beekeeping, monitoring of the state of the hives in terms of pesticide content should be carried out on a regular basis. Results of this work showed that it is necessary to reduce the load of varrocides in waxes by their correct application and to use only authorised acaricides for the Varroa treatment. In addition, alternative treatments against Varroa or products with lower lipophilic character should be developed. Putting in practice these advices together with other beekeeping practices will provide hive products with benefits to the society while maintaining biological resources on ecosystems, such as plant biodiversity and a healthy bee population.

Author contribution: Conceptualization: A.I. García-Valcárcel; Methodology: E. Miguel; Formal analysis and investigation: A.I. García-Valcárcel, E. Miguel and B. Albero; Writing - original draft preparation: A.I. García-Valcárcel; Writing - review and editing: A.I. García-Valcárcel and B. Albero, Supervision: A.I. García-Valcárcel.

Funding: This work was funded by the National Institute for Agricultural and Food Research and Technology (INIA-CSIC) in Spain, Projects AT2017-002 and RTA2017-00058-C04-01.

Data availability: Data will be made available on reasonable request

Ethical approval: Not applicable

Consent to participate: All authors consent to participate

Consent for publication: All authors consent to publish.

Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Acknowledgments

The authors acknowledge the collaboration of beekeepers.

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Acaricide Residues in Beeswax. Implications in Honey, Brood and Honeybee and Influence of Landscape Around Colonies

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Origin of the samples

2.2. Reagents and materials

2.3. Extraction procedure

2.3.1. Beeswax

2.3.2. Honeybee and brood

2.3.3. Honey

2.4. Quantitation analysis

2.5. Acaricide risk assessment

3. Results

3.1. Method validation

3.2. Acaricide residues in beeswax

3.2.1. Levels in Beeswax in December and June

3.2.2. Levels in the different locations

3.2.3. Pesticide risk assessment in waxes

3.3. Acaricide residues in honey, brood and bees

3.3.1. Levels of acaricides in December and June

3.3.2. Influence of the exposure location.

3.3.3. Potential impact of acaricides in the bee colonies

4. Discussion

4.1. Levels in Beeswax in December and June

4.2. Distribution of acaricides in beeswax depending of the location

4.3. HQ wax values for the beeswax samples analysed

4.4. Acaricides inside the hives.

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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