Residues of organic contaminants in beeswax

Juan José JiménezJosé Luis BernalMaría Jesús del NozalMaría Teresa Martín

Department of AnalyticalChemistry, Faculty of Sciences,University of Valladolid,Valladolid, Spain

Residues of organic contaminants in beeswax

Residues of the varroacides amitraz, chlordimeform, chlorfenvinphos, bromopropy-late, coumaphos, tetradifon, acrinathrin, and fluvalinate, the organic micro-contaminants 4,4’-DDE, 4,4’-TDE, PCB 153 and PCB 180, and the lipophilic pesti-cides lindane, chlorpyrifos and endosulfan have been determined by GC/MS in52 beeswax samples. Recoveries on spiked samples ranged from 93 to 108% anddetermination limits varied from 4 to 65 mg/kg. Lindane (0.042–0.29 mg/kg), chlorfen-vinphos (0.16–7.62 mg/kg), 4,4’-TDE (0.20 mg/kg), bromopropylate (0.041–0.12 mg/kg), tetradifon (0.032–0.58 mg/kg), acrinathrin (0.058–0.59 mg/kg), coumaphos (0.27–0.38 mg/kg), fluvalinate (0.064–5.10 mg/kg), endosulfan sulfate (0.12–0.37 mg/kg) and3-phenoxybenzaldehyde, a degradation product of fluvalinate and acrinathrin (0.080–1.47 mg/kg), were the compounds detected in beeswax. Foundation beeswax sheetscontained higher contaminant concentrations and a greater diversity of compounds inrelation to comb beeswaxes. Repeated melting in boiling water of purified beeswaxspiked with the contaminants did not substantially modify the content of most of thecontaminants in beeswax, except for amitraz and chlordimeform, showing that thecontaminants are stable and remain practically unchanged in the purified beeswax.

Keywords: Beeswax, organic contaminant, persistence, GC/MS.

1 Introduction

Nowadays, the presence of organic residues in beeswaxis a major issue for beeswax quality; moreover, a highcontent of these residues could create problems for thehealth of the bee colony and for the honey quality as well.The application of pesticides in surrounding crops as wellas the treatment of some beehives can cause the incor-poration of non-desirable compounds into beeswax,mainly of those with lipophilic character as a con-sequence of the physico-chemical properties of thematrix [1].

Acaricides used for Varroa control are the main contami-nants of beeswax, besides other, minor organic contami-nants and pesticides [1]. It has been found that beeswaxcontains relatively high concentrations of varroacides incomparison to honey [2–9]. However, propolis can con-tain more acaricides than beeswax [4, 8].

In the literature, there are different reports on the pres-ence of these acaricides in beeswax: bromopropylate [2,8, 10, 11], coumaphos [6, 8, 10, 12], fluvalinate [7, 8, 13–15], flumethrin [16], acrinathrin [17], tetradifon [10], ami-traz and chlorfenvinphos [18]. PCBs and pesticides,

mainly oganochlorine ones, have also been determined inbeeswax as they can also enter the beehive and con-taminate beeswax [19–22].

Most of the concentration data about organic contami-nants in beeswax have been obtained by gas chroma-tography (GC) with electron capture detection after car-rying out sample preparation procedures mainly based onthe use of liquid/liquid partitioning and Florisil-packedcolumns [12, 23–27]. Only one previous manuscript hasturned to the mass spectrometric (MS) detection for theunequivocal determination of residues of five acaricides ina matrix as complex as beeswax [18].

On the other hand, in the industrial manufacturing ofbeeswax, the old combs are placed in boiling water [18].After this process, the concentrations of bromopropylate,fluvalinate, coumaphos and flumethrin in the recycledbeeswax were, on average, 1.7 times higher than in theold combs [8].

In this work, an improved analytical method that involvesthe use of GC/MS and a matrix-standard calibration isapplied to the determination of residues of 15 lipophilicorganic contaminants in 52 Spanish beeswaxes, with theaim of knowing the residue level. Furthermore, whitebeeswax – from newly secreted combs – has been dis-tinguished from crude beeswax, to examine if the con-tent of contaminants could be different; this subject hasso far not been dealt with in the scientific literature.

Correspondence: Juan José Jiménez, Department of AnalyticalChemistry, Faculty of Sciences, University of Valladolid, Prado dela Magdalena s/n, E-47005 Valladolid, Spain. Phone: 134 983423262, Fax: 134 983 423013, e-mail: [email protected]

896 DOI 10.1002/ejlt.200500284 Eur. J. Lipid Sci. Technol. 107 (2005) 896–902

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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

Eur. J. Lipid Sci. Technol. 107 (2005) 896–902 Residues of organic contaminants in beeswax 897

Finally, we have considered it interesting to study theinfluence of the recycling process of the old combs withboiling water on the stability of the 15 studied contami-nants in beeswax.

2 Materials and methods

2.1 Materials and reagents

Acetonitrile, n-hexane, methanol, ethyl acetate and ace-tone were obtained from Lab-scan (Dublin, Ireland). Lin-dane, amitraz, chlordimeform, chlorfenvinphos, chlorpyr-ifos, 4,4’-DDE, 4,4’-TDE, bromopropylate, tetradifon,acrinathrin, coumaphos, fluvalinate, 3-phenox-ybenzaldehyde (PBA), 2,4-dimethylaniline (DMA), endo-sulfan, endosulfan sulfate and the polychlorinated biphe-nyls (PCBs) 153 and 180 were supplied by Dr. Ehren-storfer (Augsburg, Germany). Stock solutions ofpesticides were made in acetone or n-hexane and work-ing solutions in acetone.

Sodium hydroxide and sodium carbonate, both of ana-lytical grade, were supplied by Merck (Darmstadt, Ger-many). Heptafluorobutyric anhydride (HFBA) from SigmaChemical Co. (St. Louis, MO, USA) was used as a deri-vatizing reagent. Ultrapure water was obtained from aMilli-Q apparatus (Millipore, Bedford, MA, USA). Forsolid-phase extraction, a VacMaster sample processingstation from International Sorbent Technology (Mid Gla-morgan, UK) was used; C18E strata 500-mg cartridgeswere supplied by Phenomenex (Torrance, CA, USA).

Hot plates, mechanical shakers and vortex-mixers werepurchased from Selecta (Barcelona, Spain) and a rotaryevaporator from Büchi (Plawil, Switzerland). For samplepreparation, disposable PTFE syringe filter units, 0.50 mmpore size, were obtained from Microfiltration Systems(Dublin, CA, USA).

2.2 Beeswax sampling

Twenty-one purified comb beeswax samples were ana-lyzed. Sixteen beekeepers from different Spanish regions(Andalucía, Aragón, Canarias, Castilla La Mancha, Cas-tilla y León, Extremadura and Comunidad Valenciana)collected honey combs from the beehives and placedthem in boiling water to melt the comb beeswax. Then,most of the comb debris was isolated from the beeswaxby sedimentation, and this latter was sent to the labora-tory. Beeswax received in the laboratory was still mixedwith rests of the beehive (bees, honey, etc.) and wassubjected to an additional clean-up step. To this end, thecomb beeswaxes were also purified by melting; beeswax

was added to a beaker with boiling water, in proportion100 g beeswax/L, and heated for 20 min. Then, the mix-ture was cooled at room temperature, the beeswax (lessdense) solidified over the water, and the impurities placedat the bottom of the solidified beeswax were removedwith a scraper. The treatment was repeated twice morewith fresh water to obtain the purified beeswax.

Comb beeswaxes were classified as white or yellowones. White beeswax came directly from bee scales – forthis aim, beekeepers placed an empty frame in a beehivefrom their apiaries – whereas yellow beeswax was crudebeeswax made by beekeepers from honey combs.

Beekeepers also supplied 31 foundation beeswax sam-ples from 14 different manufacturers, which were directlyanalyzed without any purification step. All samples werekept at room temperature until their analysis.

2.3 Beeswax spiking and residue stability study

Lipophilic compounds were added to a residue-free puri-fied comb beeswax to know the stability of the contami-nants during the purification. To this aim, purified bees-wax (20 g) placed in an aluminum container was melted at70 7C on a hot plate and an acetone solution containingthe analytes was added over the melted beeswax. Then,the mixture was strongly mixed and acetone wasremoved by gentle heating. Yellow beeswax sampleswere spiked at two concentration levels, 0.4 and 4 mg/kgfor each analyte, and kept at room temperature and in thedark until their analysis.

The spiked beeswaxes (5 g) were placed in a beakercontaining 50 mL water and were melted in boiling waterfor 20 min according to the laboratory purification proce-dure described in Section 2.2. Then, after cooling andseparating the water from the beeswax, the spiked bees-wax was again melted in the beaker with two further por-tions of fresh water. After the three meltings, beeswaxeswere analyzed to measure the remaining residue content,and the three portions of water used in each experimentwere combined to study whether the residues were par-tially soluble in the hot water. Experiments were made inquintuplicate.

2.4 Residue extraction from beeswax

A beeswax sample of 0.1 g was dissolved in 10 mL n-hexane and the analytes were extracted with 10 mL ace-tonitrile, in a separatory funnel, by mechanical shaking for15 min. After that, 2 mL methanol was added to break theemulsion and the phases were allowed to separate; then,the acetonitrile fraction was collected and evaporated in a


898 J. J. Jiménez et al. Eur. J. Lipid Sci. Technol. 107 (2005) 896–902

rotary evaporator. Finally, the residue was redissolved in1 mL acetone and passed through a PTFE filter with apore size of 0.5 mm. The extract was ready for injectioninto the GC system.

2.5 Residue extraction from water used in thepurification

The three aliquots of water used in the purification ofeach spiked beeswax were collected, combined andanalyzed to determine the target compounds in solution.Then, a solid-phase extraction procedure was used inorder to achieve a clean-up; to this end, C18E cartridgeswere conditioned by successive elution of 10 mL metha-nol and 10 mL water. Next, the water sample was per-colated through the cartridge at about 5 mL/min using asuction system, and after that, 6 mL water was eluted torinse the cartridge. The solvent was removed from thecartridges by passing nitrogen through it for about30 min, and finally, the cartridge was eluted with 3 mLethyl acetate, after leaving the solid phase to soak for4 min. The ethyl acetate solution was injected into theGC system.

2.6 Sample preparation for amitraz total residueanalysis in beeswax

A published procedure was followed [25]. Briefly, bees-wax (0.1 g) was extracted with 10 mL methanol, by shak-ing for 15 min. The mixture was centrifuged to separatethe liquid phase. Then, in a separatory funnel, the extractin methanol was added to 4 mL n-hexane and 100 mL 2 MNa2CO3; the mixture was mechanically shaken, thephases were allowed to decant and the extract in hexanewas collected. A 2-mL portion of the n-hexane phase washydrolyzed with 4 mL 1 M NaOH, at 90 7C for 2 h, to con-vert all amitraz residues to DMA, and finally, an aliquot of1.5 mL was derivatized with 100 mL HFBA at room tem-perature for 5 min to obtain heptafluoro-N-(2,4-dimethyl-benzene)-butanamide, measured by GC.

2.7 GC/MS analysis

A Hewlett Packard 6890 gas chromatograph (Little FallsSite, Wilmington, DE, USA) directly coupled to a Hewlett-Packard 5973 mass spectrometer was used to determinethe contaminant residues in the extracts. The chromato-graph was fitted with a 30 m60.25 mm60.25 mm ZB-5column from Phenomenex (Torrance, CA, USA). For mul-tiresidue analysis, the oven temperature program con-sisted of: 50 7C (1 min), 5 7C/min to 325 7C (15 min); andfor amitraz total residues the temperature program was:

50 7C (1 min), 10 7C/min to 155 7C, and then 40 7C/min to325 7C (15 min). The carrier gas (helium) flow was keptconstant at 1 mL/min (equivalent to a pressure of 52.8 kPaat 50 7C). In both analytical methods, pulsed splitlessinjection (1 mL) was performed with an HP7673A auto-matic sampler at an injection port temperature of 280 7C;the pressure pulse was 172 kPa for 1 min and the purgevalve was on at 1 min; the transfer line temperature was328 7C. The MS temperatures were as follows: ion source230 7C, quadrupole 150 7C. Electron multiplier voltagewas maintained at 500 V above autotune.

Quantitation was performed by the target ion recorded inSIM mode. Two qualifier ions were used to confirm thepeak identification. Tab. 1 lists the compounds studiedand the ions monitored. The dwell time for each ion was30 ms.

A matrix-standard calibration was carried out to avoid theinfluence of the matrix on the quantitation [24], except fortotal amitraz determination in which derivatized DMAstandards were used.

3 Results

3.1 Analytical method for residues in beeswax

The analytical method applied to the multiresiduedetermination of the contaminants in beeswax is basedon a method previously developed in our laboratory [24]that used a GC-ECD determination and required aclean-up procedure according to the common guide-lines of the published methods to analyze residues inbeeswax. In this work, a selective detection by GC/MSin SIM mode allowed us to remove the clean-up step.Moreover, to reduce the quantitative errors arising fromthe matrix, which affects the extraction and GC deter-mination steps, a matrix-standard calibration was car-ried out. Thus, portions of a residue-free beeswax werespiked with increasing amounts of the analytes andsubjected to the same sample preparation procedure asthe samples; the extracts were used as calibrationstandards.

The recoveries of the analytes were measured at threespiking levels: 0.06, 0.6 and 6 mg/kg for each compound,and they ranged from 93 to 108%. The coefficients ofvariation varied from 5 to 16% (n = 5). Tab. 1 shows thedetection and determination limits for the analysis ofresidues in beeswax; they were calculated as three andten times the signal-to-noise ratio. The detection limitsranged from 1 to 20 mg/kg. Figs. 1 and 2 show chromato-grams of beeswax extracts obtained after applying thedescribed procedures.



Tab. 1. Retention times, ions monitored by GC/MS, and detection and determination limits for beeswax samples. Abun-dances [%] are shown in parentheses.

Retentiontime [min]

Target ion Qualifierion 1

Qualifierion 2

SIM groupinitiated at:

Detectionlimit[mg/kg]

Determi-nation limit[mg/kg]

Derivatizated DMA 11.38 148 (100) 120 (54) 317 (74) 6 min 7 20Chlordimeform 17.07 196 (100) 181 (84) 117 (63) 15.0 min 14 453-Phenoxybenzaldehyde 18.07 198 (100) 169 (49) 141 (39) 3 8Lindane 19.90 181 (100) 219 (88) 111 (41) 1 4Chlorpyrifos 26.69 201 (100) 314 (58) 258 (37) 24.0 min 5 16z-Chlorfenvinphos 29.55 267 (100) 323 (53) 295 (18) 27.7 min 5 12Endosulfan A 30.78 195 (100) 241 (75) 339 (35) 12 424,4’-DDE 32.75 246 (100) 320 (36) 176 (36) 31.7 min 13 40Endosulfan B 34.60 195 (100) 207 (65) 237 (80) 33.7 min 11 314,4’-TDE 35.59 235 (100) 165 (42) 320 (2) 11 36Endosulfan sulfate 37.61 272 (100) 229 (64) 387 (44) 36.5 min 13 43PCB 153 38.41 360 (100) 325 (16) 290 (68) 9 26Bromopropylate 41.91 341 (100) 339 (50) 183 (61) 40.0 min 6 20PCB 180 43.12 394 (100) 369 (13) 324 (67) 7 18Tetradifon 43.62 159 (100) 229 (60) 356 (48) 12 32Amitraz 45.75 121 (100) 293 (51) 162 (82) 44.5 min 8 29Acrinathrin 47.07 181 (100) 208 (59) 441 (2) 46.4 min 15 58Coumaphos 48.16 362 (100) 226 (78) 210 (59) 15 48Fluvalinate 1 53.25 250 (100) 181 (19) 502 (3) 51.0 min 18 65Fluvalinate 2 53.51 250 (100) 181 (20) 502 (3) 20 60

3.2 Effect of the purification on residue content

The increase in concentration of the lipophilic contami-nants in beeswax when the old combs are placed in boil-ing water to produce new beeswax has been establishedin a previous work after studying the behavior of four var-roacides [8]. This behavior can be explained because thecompounds are fat-soluble and are concentrated in asmaller material mass as a consequence of the elimina-tion of comb debris. In this respect, combs contain a dif-ferent beeswax amount depending on their age: fromabout 25% in very old combs to nearly 100% in freshlybuilt combs.

There is no previous information about the heat stabilityand hydrolysis of the contaminants during the purificationprocess by boiling water. In this context, we have spikedpurified beeswaxes with 15 varroacides or contaminantsto study their stability, avoiding the concentration effectthat takes place by removal of the debris. After the purifi-cation process, amitraz and chlordimeform were notdetected in beeswax in any experiment because of theirlability [28–30]. The determination of the total amitrazresidues in the spiked beeswax revealed that only about80% of the amitraz added was recovered; amitraz iseasily hydrolyzed and it is likely that part of its residues inbeeswax are removed as 2,4-dimethylaniline (volatile)during the water boiling.

As regards the other compounds, the recoveries of thecontaminants in the beeswaxes spiked at 4 mg/kg variedfrom 95 to 106% – indicating that they are stable andpersistent in beeswax – with coefficients of variation be-tween 3.3 and 14.8% (n = 5). The determination of orga-nophosphorous compounds and bromopropylate pro-vided the worst precision. However, at a concentration of0.4 mg/kg, the content of the pyrethroids acrinathrin andfluvalinate, and of coumaphos, decreased by 15%; therecovery of lindane fell by 10%. The precision at 0.4 mg/kg was worse compared with the higher spiking level, andvaried between 9.0 and 22.5% (n = 5).

To verify these results, the concentrations of the analytesin the waters used to purify the spiked beeswaxes werealso determined. At 4 mg/kg, chlorpyrifos, endosulfan Aand B, endosulfan sulfate and bromoproylate were notdetected in water, while the concentrations in water of theother compounds indicated that the loss of these micro-contaminants from beeswax ranged between 0.08 and2.4% – higher for coumaphos and chlorfenvinphos andlower for lindane, 4,4’-DDE, 4,4’-TDE and PCBs.

At a concentration of 0.4 mg/kg and according to theconcentrations measured in the water, coumaphos wasthe compound most removed from beeswax (7.8%), fol-lowed by lindane and chlorfenvinphos with a loss of 3–4%. It is interesting to remark that amitraz, chlordimeform



Fig. 1. Chromatograms obtained in themultiresidue analysis of a foundationbeeswax (PBA, 0.6 mg/kg; chlorfenvin-phos, 1.2 mg/kg; bromopropylate,0.08 mg/kg; tetradifon, 0.03 mg/kg; flu-valinate, 1.7 mg/kg) and a residue-freebeeswax.

Fig. 2. Chromatogram obtained in the analysis of totalamitraz residues in a yellow beeswax (0.25 mg/kg).

and PBA were never detected in the waters; the first onesare easily hydrolyzed in aqueous media and PBA is hardlyretained on ODS cartridges [31].

The results suggest that the purification process by boil-ing water does not substantially modify the initial contentof the lipophilic contaminants in beeswax when these arepresent in relatively high concentrations, while at lowconcentrations slight decreases can be obtained forsome compounds: pyrethorids, lindane and coumaphos.

3.3 Residues in beeswax

Tab. 2 lists the mean values of concentration and thenumber of detections for the monitored compounds in the52 samples (purified comb and foundation beeswaxes)according to the beeswax type: white, yellow and foun-dation sheet. The foundation beeswax sheets contained awider variety of contaminants, a higher number of con-taminants detected per sample and bigger concentra-



Tab. 2. Mean concentration referred to the positive samples and incidence of the pesticides according to the beeswax type.

White beeswaxn = 9 samples

Yellow beeswaxn = 12 samples

Foundation beeswaxn = 31 samples

Mean[mg/kg]

Range Numberof detec-tions

Mean[mg/kg]

Range Numberof detec-tions

Mean[mg/kg]

Range Numberof de-tections

Lindane – – – – – – 0.13 0.042–0.29 6z-Chlorfenvinphos 0.34 – 1 1.80 0.16–4.82 7 2.55 0.16–7.62 244,4’-TDE – – – – – – 0.20 – 1Bromopropylate 0.33 – 1 0.12 0.068–0.12 5 0.093 0.041–0.11 14Tetradifon – – – 0.065 0.054–0.076 2 0.19 0.032–0.58 4Acrinathrin 0.18 0.058–0.57 3 – – – 0.41 0.22–0.59 2Coumaphos – – – – – – 0.34 0.27–0.38 3Fluvalinate 1.01 0.44–1.86 6 1.86 0.85–5.10 9 2.01 0.064–3.44 223-Phenoxybenz-

aldehyde0.15 0.14–0.16 2 0.23 0.06–0.36 8 0.44 0.080–1.47 23

Endosulfan sulfate 0.29 0.24–0.36 4 0.13 0.12–0.14 2 0.31 0.21–0.37 4Total amitraz 0.17 0.10–0.24 7 0.27 0.15–0.56 7 0.30 0.08–0.69 12

–, below detection limit.

tions than the comb beeswaxes. So, lindane and 4,4’-TDE,a degradation product of 4,4’-DDT, were found only in thefoundation beeswaxes. The organochlorine micro-contaminants 4,4’-DDE, 4,4’-DDT and the two analyzedPCBs were also not detected in the comb beeswax; thereare nopreviousconcentration data for beeswax of the sameregions with which to compare the results. However, theresults obtained seem to suggest that there is a smallerpresence of organochlorine compounds in the beehives[19–22], likely as a consequence of the restrictions imposedon the handling of organochlorine pesticides and PCBs.

The relatively high incidence of endosulfan sulfate, adegradation product of the insecticide endosulfan, insome comb beeswaxes is also noticeable. This com-pound is supposed to have reached the beeswax as aconsequence of the foraging activity of the bees; in fact,the presence of endosulfan in some honey samples hasbeen established [32]. Endosulfan and chlorpyrifos werenot detected in the other beeswax samples.

As regards the varroacides, the foundationsheets containedhigher concentrations than the comb beeswaxes. Forinstance, this is the case for z-chlorfenvinphos, tetradifon,fluvalinate and PBA. This latter is a degradation product offluvalinate, acrinathrin and other pyrethroids, and its pres-ence in beeswax had not been ascertained before. Acrina-thrin has also been detected in beeswax, but its incidenceand concentrations were lower than those of fluvalinate.

The high accumulation of residues in beeswax foundationsheets cannot be exclusively associated with the older ageof this beeswax. In fact, the sheets arise from the recycling

of the old combs; in the manufacturing process, a batch ofhighly contaminated beeswax would cause a high residualconcentration in the yielded sheets, as has beenstated [33].

In Spain, only amitraz and fluvalinate are registered foruse against Varroa. However, the frequent presence ofchlorfenvinphos, bromopropylate and other varroacidesin comb beeswaxes is remarkable. Their detection couldbe attributed to the carrying out of treatments, nowadaysor formerly, with non-authorized products or to theimportation of beeswax batches from other countries.

Regardless of the compounds that can reach the beehiveowing to the foraging activity of the bees, the existence ofdiverse varroacide residues in white comb beeswaxobtained after introducing empty frames in the beehivesis surprising; this fact could only be explained if residuesof treatments or former contributions remained on theinside of the beehive, mainly impregnating the wood thatwas not scratched out before placing the frames [34].

The common use of beeswax in apiculture involves itscollection in the form of old combs, purification, produc-tion of foundations and use of foundations for buildingnew combs. If we take into account that there is a seriesof compounds that appear with relative frequency inbeeswax and that they are not removed after the repeatedheating at 100 7C in the presence of water, these com-pounds could be introduced again into the beehive. In thiscyclic process, new contaminant amounts could beadded to the beehive, creating an accumulative processthat leads to increasing amounts of residues in beeswax,thus affecting in particular the quality of honey as well.



Thus, analytical control of the presence of lipophilic com-pounds to guarantee the beeswax quality, regardless ofits subsequent use, is necessary. However, there are noresidue limits for contaminants in beeswax and thesehave to be introduced in the legislation.

Acknowledgments

The authors thank the Spanish Instituto Nacional deInvestigaciones Agrarias for providing funds for thisinvestigation (Projects API99–003 and API01–005).

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[Received: October 10, 2005; accepted: October 31, 2005]


Residues of organic contaminants in beeswax

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