INTRODUCTION
Para-dichlorobenzene (1,4-dichlorobenzene, PDCB) is a fumi- gant insecticide used for different purposes. Beekeepers world- wide, and also in Switzerland, use it to control the greater wax moth, Galleria mellonella, during the storage of beeswax combs.
Because of its toxicity (NIOSH, 2000), its use should be avoid- ed. German investigations in the early 1990s showed that a rel- atively high percentage of German honeys analysed (36–50%) contained from three to 50 µg PDCB /kg (Hamman et al.1990;
Wallner, 1992). These results led to reports in the German mass media about ‘poisonous honey’. Since then the German Bee- keeping Union has carried out regular checks on the PDCB con- tent of German honey. This quality-control activity has resulted in a decrease in the percentage of PDCB-contaminated honey from the high initial contamination rate found to a present level of zero to 6% (Wallner et al.2000, 2001). Beekeepers can con- trol wax moth efficiently using alternative, non-toxic treatments (Charrière & Imdorf, 1999). In Switzerland, positive findings by the cantonal laboratories in 1999 led to the establishment of a
‘Swiss tolerance value’ (STV) for honey of 10 µg/kg, however, worldwide there is no ‘maximum residue limit’ (MRL) for honey.
To our knowledge, no publications are available on the PDCB content of commercial beeswax, except for a summary on the PDCB levels found on the German market from 1997 (Wallner, 1997). In this study only traces of PDCB below 1mg/kg were found in comb foundation from different countries. In one study, the residues in combs were examined after use of PDCB as an acaricide against varroa (Binder et al., 1988). Honeycomb is recy- cled to produce beeswax and lipophilic non-volatile acaricides accumulate in the wax (Bogdanov et al., 1998a). In this article we present for the first time the results of the long-term mon- itoring of PDCB in Swiss beeswax. Also, we summarize the results of the honey-quality control activities of three Swiss food
control laboratories over the past five years with regard to PDCB residues in honey marketed in Switzerland.
MATERIALS AND METHODS
Honey samples
The honey samples originated from the annual food-quality con- trol activity of the cantons Schaffhausen (SH), Basel-Stadt (BS) and Basel-Land (BL) during 1997, 1998, 2000, 2001and 2002.
The food control authority of the cantons Appenzell, Glarus and Schaffhausen also analysed honey samples from other cantons in the eastern part of Switzerland, as well as from the principal- ity Liechtenstein. The imported honey samples comprised hon- eys from a total of 173 Swiss and 287 imported samples. The number of imported samples per declared country of origin was as follows: Latin America 134, European Union 79, eastern Europe 6, USA 6; Australia 3, Asia 5, and blends from different, unknown countries 53.
Wax samples
During each production year all Swiss wax manufacturers (n= 8) supply samples of wax from all production lots for testing.
The samples from each lot are then melted in proportion to the weight of each production lot to yield an annual sample for every manufacturer. The amounts produced by the manufacturers var- ied greatly: seven of the wax producers produced between 400 to 5000 kg, whilst one of them produced between 30 000 and 45 000 kg. All wax producers receive the anonymous results of the monitoring. In addition, a theoretical annual average value for the amount of beeswax produced in Switzerland is calculat- ed by weighing the annual values of each manufacturer accord- ing to the amount each individually produced. Wax produced in
Journal of Apicultural Research43(1): 14–16(2004) © IBRA 2004
*Corresponding author: stefan.bogdanov@alp.admin.ch
ORIGINAL ARTICLE
Residues of para-dichlorobenzene in honey and beeswax
S BOGDANOV,1* V KILCHENMANN,1K SEILER,2H PFEFFERLI,2THFREY,3B ROUX,3P WENK4ANDJ NOSER4
1Swiss Bee Research Centre, FAM, Schwarzenburgstr. 161, CH-3003 Bern, Switzerland
2Amt für Lebensmittelkontrolle der Kantone AR, AI, GL und SH, Postfach 786, CH-8201Schaffhausan, Switzerland
3Kantonales Laboratorium, Basel-Stadt, Kannenfeldstr. 2, CH-4012 Basel, Switzerland
4Kantonales Laboratorium, Basel Land, Hammerstr. 25, CH-4410 Liestal, Swizerland
Received 29 July 2003, accepted subject to revision 17 November 2003, accepted for publication 21November 2003
SUMMARY
Para-dichlorobenzene (PDCB) is an insecticide used in beekeeping for wax moth control. Analysis of PDCB
residues were carried out on Swiss retail market honey samples by the cantonal food control authorities in
1997, 1998, 2000, 2001and 2002. 173 Swiss honeys and 287 imported samples were analysed. On average, 30% of the Swiss honeys contained PDCB, 13% of them being above the Swiss tolerance value of 10 µg/kg. On the other hand, only 7% of the imported honeys were contaminated. The minimum values were 2 µg/kg, the maximum ones 112 µg/kg. Long-term monitoring of Swiss beeswax, carried out from 1993 to 2000, showed that most of the comb foundation beeswax produced in Switzerland is contaminated by PDCB with values ranging from one to 60 mg/kg. The results show that the reason for this contamination is the use of PDCB for the control of wax moth. These residues can be avoided as wax moth can be controlled successfully with alternative methods, carried out according to good apicultural practice.
Keywords:para-dichlorobenzene, residues, analysis, Galleria mellonella, wax moth control, honey, beeswax,
Switzerland
1994, 1995, 1996, 1997, 1998 and 2002 were analysed. The wax samples were kept in a freezer before analysis.
New wax from a model experiment
Brood-comb wax was ground and spiked with an ether solution containing PDCB. After evaporation of the solvent 50 g comb material was mixed with 100 ml water in a beaker and was boiled for 3 hours. The new wax was separated from the comb debris by sieving through a cotton cloth. The recovery of new wax var- ied between 18% and 25%. The determination of PDCB in combs and new wax was carried out as indicated below.
Honey analysis by head space sampling
The food control authority of Basel Stadt (BS) applied the fol- lowing procedure: 5 g honey sample, 2 g sodium sulfate, 2.5 ml deionized water and 50 µl of the internal standard solution (1,4- dichlorobenzene-d4from Cambridge Isotope Laboratories, 1 ng/µl in methanol) were mixed at 80 °C in a tightly sealed 10 ml headspace vial. After 2 h of incubation (80 °C) 1ml headspace gas was injected for analysis. Analyses were performed on a Voy- ager mass spectrometer (Thermo Finnigan) coupled to a gas chromatoraph GC 8000 Top (CE Instruments) and equipped with a headspace-autosampler CTC HS500 (CTC Analytics).
GC: Rtx-624 fused silica capillary column, 30 m ( 0.25 mm i.d., 1.4 µm film thickness (Restek Corporation); column tempera- ture: 60 °C (1min) at 10 °C/min to 220 °C (5 min); injector temperature: 220 °C; injection mode: split; split ratio: 1: 20;
injection volume: 1ml (headspace); carrier gas: He at 1ml/min.
MS: interface temperature: 220 °C; ionization: EI+ 70 eV; ion source temperature: 200 °C; selected ions for PDCB: m/z 146, 148; selected ions for the internal standard (1,4-dichloroben- zene-d4): 150, 152. A 3-level calibration at 0.1, 1.0 and 10 µg/kg was performed spiking 5 g residue free honey samples with 50 µl internal standard solution containing different concentrations of PDCB (0.01, 0.1and 1.0 ng/µl in methanol). The in-house val- idated method allows also simultaneous determination of thy- mol, naphthalene, nitrobenzene and benzaldehyde in the con- centration range from 5 to 1000 µg/kg.
The food control authority in Schaffhausen (SH) applied almost the same method with minor differences: instead of a 5 g honey sample, 10 g were used and mixed with 5 ml of deionized water
in a tightly closed 20 ml headspace vial. No internal standard solution was used. After an incubation time of 30 min at 70 °C, 1ml of the headspace gas was injected for analysis. A 3-level cal- ibration was performed in the range from 3 to 15 µg/kg. The detection limit of the above method was 1µg/kg.
Honey analysis by SPME sampling
A 2.0 g honey sample and 250 µl internal standard solution (1,4- dichlorobenzene-d4 from Dr Glaser AG Basel, Switzerland, D11161; 0,05 ng/ µl in methanol) were diluted with water to 50 ml volume. 1.2 ml of diluted honey sample was pipetted into a 2 ml autosampler vial. The analyses were performed by SPME- GC-MS. SPME: 1.2 ml of the diluted honey solution was pipet- ted into a 2 ml autosampler vial, placed in a SPME Autosampler (Varian 8100). Adsorption with a 5 cm 100 µm PDMS-fiber (Supelco Nr. 5-7301) for 45 minutes at 20–25 °C in the liquid phase of the vial, desorption time: 15 minutes splitless at 250 °C.
GC: J & W DB5ms fused silica capillary column, 30 m, 0.25 mm i.d., 0.25 µm thickness (Agilent); column temperature: 40 °C (0 min) to 170 °C at 3 °C/min (0 min), 25 °C/min to 290 °C (15 min). Injector temperature: 250 °C, injector mode: splitless. MS:
Saturn III (Varian); transfer line temperature: 250 °C; ionization mode: EI; ion source temperature 230°C; full scan range: 55 to 350 m/z; no multiplier delay. A 4-level calibration at 1.0, 5.0, 10.0 and 20.0 µg PDCB/kg was performed by spiking 2 g of blank honey samples with 250 µl PDCB standard solution, diluted to 50 ml, to determine the concentration of PDCB in honey. The detection limit was 1µg/ kg.
Wax analysis
The method used for wax analysis was exactly the same as described in detail for the analysis of thymol in beeswax described by Bogdanov et al.(1998b). It consists of extraction by ethanol, clean-up by freezing and centrifugation, extraction by C18 SPE columns, elution by acetone and determination by capillary GC with FID detection. All analyses were carried out in the laboratory of the Swiss Bee Research Centre (SBRC). The method was validated by adding different amounts of PDCB (Riedel-de Haën, Germany, Nr. 35775) to wax and subsequent analysis. The average recovery in the whole determination range (2 to 100 mg/kg) was 74.8 ± 5.5 % (average ± standard devia- tion, n = 18). The recoveries being the same in the whole
PDCB residues in honey and wax 15
TABLE 1. Para-dichlorobenzene residues in honey from the Swiss retail market. S: Swiss honey, I: imported honey, STV: Swiss tolerance value.
Origin 1997 1998 2000 2001 2002
I S I S I S I S I S
Number 27 28 137 13 1 23 73 16 49 93
Number positive 04 04 001 06 0 06 09 05 04 32
Percentage positive 15 14 001 46 0 26 12 31 08 34
Percentage above STV 0 11 000 15 0 09 04 12 00 18
Min–max in µg/kg of positive samples 2-11 7-65 2 4-112 0 4-56 5-58 4-37 1-4 2-112
TABLE 2. Para-dichlorobenzene residues in Swiss beeswax.
Production year 1994 1995 1996 1997 1998 2000 2002
Annual value*, mg/kg 10.9 5.2 4.6 5.4 6.9 6.7 5.2
Average, mg/kg 20.1 8.6 11.1 11.7 18.4 13.0 17.8
Min–Max, mg/kg 7.5-60.5 ≤0.7-13.8 ≤0.7-55.2 ≤0.7-26.9 ≤0.7-35.2 ≤0.7-39.3 ≤0.7-74.6
Percentage positive 100 87 62.5 87 71 75 66
Number (wax producers) 8 8 8 8 7 8 9
*annual values for Switzerland, seeMaterials and methods
determination range, the true PDCB content was determined by multiplying the measured PDCB value by a factor of 1.34. The detection limit of the method was 0.7 mg/kg.
All chemicals were of analytical grade.
RESULTS
The results, summarized in table 1show that a substantial num- ber of Swiss honeys are contaminated with PDCB. On average 30% of all Swiss honeys, produced between 1997 and 2001con- tained PDCB, 13% being above the STV of 10 µg/kg. On the other hand, the contamination rate of the imported honeys was much lower, on average 7%.
Table 2 summarizes the results of long-term monitoring of PDCB in Swiss beeswax. There is no trend of the annual Swiss values, and also of the average values, which vary from 2.5 to 10.9, and from 8.6 to 20.1mg/kg, respectively. The annual Swiss values are two-times lower than the average values. This is due to the fact that the annual Swiss values are corrected according to the production amount of the different manufacturers, which varies greatly (seeMaterials and methods). The individual values of the different manufacturers vary in a very broad range: min- imum 0.7, maximum 74.6 mg/kg; the average being 14.4 mg/kg.
In the model wax production experiment, the initial concentra- tion in old combs was determined to be 10.2 mg/kg (±1.0, range of duplicate), while 10.8 mg of PDCB per kg (±1.4, range of duplicate) was found in the new wax after melting of old combs.
DISCUSSION
The results show that a substantial amount of Swiss honey and wax is contaminated with PDCB. The contamination level of imported honeys was lower, but PDCB was found in some hon- eys originating from Europe, North America and Australia. The values found in Swiss honeys were similar to those found in pre- vious studies, reported in Germany at the beginning of the 1990s (Hamman et al., 1990; Wallner, 1992). Wax foundation and comb, contaminated by PDCB are possible contamination sources. Combs are treated with PDCB during storage. Old combs that might contain PDCB are generally recycled into foun- dation. The laboratory comb-melting experiment showed that PDCB is not removed from wax during the comb recycling process. On the other hand, most of the PDCB initially present evaporates from foundation upon aeration under laboratory conditions, but about 5–10% remained trapped (Wallner, 1992).
In Swiss beekeeping practice, one to two frames of comb foun- dation are placed in each beehive every year for comb renewal at least 4 weeks before the honey flow. In an experiment with thymol-contaminated foundation it was shown that within two weeks after placing contaminated foundation into the hive in spring, less than 1% of the initial concentration of thymol remained in the combs (Bogdanov et al., 1998b). PDCB, having a similar volatility to thymol, is expected to evaporate in a sim- ilar fashion. Thus, about 10 µg/kg could be present in the comb at the time of the honey flow.
The other contamination source is PDCB-treated honeycomb.
What is the concentration of PDCB in honeycomb treated for wax moth control? This will depend on the mode of use. Under laboratory conditions, aeration of 1kg comb foundation with 50 g of PDCB results in a concentration of about 4000 mg/kg wax after 2.5 months (Wallner, 1992). In Switzerland 100 g of PDCB balls are recommended for the fumigation of 1m3, a space where about 50 frames can be stored. Similar recommendations are given also in the USA (e.g. Tew, 1997). This means that under practical conditions a similar contamination of wax can be expected as the one measured under laboratory conditions. If one to 10% of the PDCB initially present remains in the comb, more than 40 000 µg/kg might theoretically be present in the comb during the time of the honey flow. The honey contami- nation is determined by the diffusion rate of PDCB from wax
into honey. For the volatile substance thymol the equilibrium concentration ratio between wax and honey is about 1000 : 1 (Bogdanov et al., 1998b). PDCB is slightly more volatile and less lipophilic than thymol, so that a distribution ratio between wax and honey similar to that of thymol can be assumed. Thus, about 40 µg/kg honey might result from contaminated comb, while no measurable contamination is expected to come from PDCB con- taminated foundation. This hypothesis is supported by the fact that in all cases where the honey residues exceeded the Swiss Tolerance Value MRL of 10 µg/kg, the beekeepers had used PDCB for wax moth control on their honeycomb. Although PDCB can partly evaporate from the honey surface it will remain in closed honey jars for a longer period of time (Wallner, 1992).
During normal honey processing, it is expected that only a small proportion of the PDCB might evaporate from honey.
Another substance, that can be used for wax moth control is naphthalene, which is more toxic than PDCB. Residues of this substance were found only in two Swiss honeys. No published reports of naphthalene residues in honey are available. Accord- ing to an official EU report, naphthalene is widely used in Turkey and residues are often found in Turkish honeys (European Com- mission, 2001).
Residues of PDCB or naphthalene can be avoided by carrying out alternative measures of wax moth control (Charriere &
Imdorf, 1999). The most important principals are: regular exchange of old comb with new; timely recycling of old comb into new beeswax; storage of comb under airy, light conditions or at temperatures below 12 °C and treatment with non-toxic substances, which do not produce residues, e.g. sulfur, acetic acid, formic acid, Bacillus thuringiensis.
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