Amitraz as a formamide insecticide and acaricide is used for the control of animal ectoparasites. It is one of the main and more commonly applied acaricides against
Amitraz is unstable and undergoes rapid biotransformation in the low pH environment of the hive. The metabolism of amitraz in bees has not been investigated (6). It is known that the amitraz molecule hydrolyses through the intermediate metabolites N-2,4-dimethylphenyl-N-methylformamidine (DPMF) and N-2,4-dimethylphenylformamide (DMF), to form the environmentally stable toxic compound 2,4-dimethylaniline (2,4-DMA) (2,10,12). The analysis of honey samples enriched with amitraz under laboratory conditions leads to the conclusion that the main amitraz metabolites in honey are DMF and DMPF with rather low concentrations of 2,4-DMA residues (15, 16).
The Commission Regulation (EU) No. 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits in food stuffs of animal origin defines amitraz marker residues as the sum of amitraz and all metabolites containing the 2,4-DMA moiety, expressed as amitraz. The maximum residue level (MRL) of amitraz in honey is 200 μg/kg (4). In order to harmonise the MRLs for certain substances including amitraz, the European Commission planned to transcribe the veterinary MRLs into pesticide MRL legislation (6).
The issue of honey contamination with amitraz is differently interpreted by different pieces of research. The results of certain investigations, particularly those published earlier, indicate a very low risk of honey pollution after treatment of honeybee colonies with amitraz (9, 17). In contradiction, the European Food Safety Authority (EFSA) report on pesticide residues in food indicates that amitraz residues are among the most common pesticides determined in honey in the EU (5, 7). It should be emphasised that so far there has been no research on the presence of amitraz metabolites in samples of honey, other bee products, wax, or the bees collected from bee colonies where drugs containing amitraz were applied experimentally by fumigation.
The study was designed to evaluate the concentration of amitraz marker residues in honey harvested after
All analysed honey samples from both groups (n = 54) were contaminated. At least one amitraz metabolite was found in each sample, however, no residues of parent amitraz were detected. The most frequently determined compound was 2,4 DMA, although around 80% of samples contained DMPF (Table 1). The mean concentrations of 2,4-DMA in honey samples collected in both groups one day after the fumigation were significantly higher than those of DMF and DMPF (P ≤ 0.05). Similar proportionality was observed for the amitraz metabolite profile in the centrifuged honey. After Apiwarol fumigation with the electric fumigator, DMF concentrations in centrifuged honey were lower than the LOQ (1 μg/kg), whilst DMPF and 2,4-DMA values ranged from 1.0 to 1.3 μg/kg (on average 1.13 μg/kg) and from 13.0 to 35.2 μg/kg (on average 23.2 μg/kg), respectively. After treatment of honeybee colonies with Apiwarol by smouldering tablet directly in hives, residues of DMF, DMPF, and 2,4-DMA in centrifuged honey reached mean levels of 2.3 μg/kg (from 1.9 to 2.6 μg/kg), 2.4 μg/kg (from 1.9 to 2.9 μg/kg), and 84.9 μg/kg (from 68.6 to 98.4 μg/kg), respectively.
The amitraz metabolite profile in honey samples, separately presented for each group
Group W – Apiwarol combustion in Wakont (n = 27) | Group B – Apiwarol combustion in hive (n = 27) | |||||||
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Concentration (μg/kg) | Concentration (μg/kg) | |||||||
Metabolite | percentage in samples containing residue | percentage of samples containing residue | ||||||
Min. | Max. | Mean | Min. | Max. | Mean | |||
DMF | 37.0% | 1.1 | 3.8 | 1.6 a | 66.6% | 1.0 | 29.9 | 4.6 a |
DMPF | 77.7% | 1.0 | 2.7 | 1.7 a | 81.4% | 1.0 | 16.4 | 4.1 a |
DMA | 92.5% | 10.0 | 60.5 | 26.6 b | 96.1% | 10.0 | 309.5 | 48.1 b |
a-b means with different small letters in the same column are significantly different (P ≤ 0.05)
The findings of total amitraz marker residue concentrations in honey samples, expressed according to the Commission Regulation (EU) No. 37/2010 (as sum of amitraz and all metabolites containing the 2,4-DMA moiety, expressed as amitraz), are presented in Table 2. For the group which was fumigated with the electric fumigator, amitraz concentration ranged between 5.2 and 146 μg/kg (mean 62.9 μg/kg) in the honey collected after the sequence of four treatments and from 34.5 to 87.5 μg/kg (mean 58.3 μg/kg) in extracted honey. The MRL was not exceeded in any of the honey samples taken from beehives fumigated indirectly with the Wakont fumigator. Amitraz residues in honey after tablet combustion directly in the hive ranged from 9.4 to 766 μg/kg after the sequence of four treatments and from 176 to 248 μg/kg after honey harvesting. The mean residue concentrations of amitraz were 109 and 214 μg/kg for these two periods, respectively. Quantities of amitraz exceeding the MRL were found in five honey samples: in three samples collected after the four Apiwarol applications and in two samples of the extracted honey (also after the four Apiwarol applications).
Average amitraz marker residue contents in honey samples ±SD (as sum of amitraz and all metabolites containing the 2,4-DMA moiety, expressed as amitraz, in μg/kg)
Groups | Group W – Apiwarol combustion in Wakont | Group B – Apiwarol combustion in hive | ||||||
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Consecutive treatment | 1st | 2nd | 3rd | 4th | 1st | 2nd | 3rd | 4th |
n = 6 | n = 6 | n = 6 | n = 6 | n = 6 | n = 6 | n = 6 | n = 6 | |
Samples from brood chamber | 71.7 ± 52.9 | 16.3 ± | 11.1 54.9 ± 2.1 | 56.3 ± 50.4 | 38.1 ± 14.3 | 41.1 ± | 31.1 91.5 ± 55.9 | 276.2 ± 288.9 |
Samples from super | 75.1 ± 18.9 | 38.4 ± 7.8 | 106.8 ± 0.8 | 72.2 ± 27.1 | 69.5 ± 9.9 | 85.1 ± | 26.8 53.9 ± 19.3 | 150.0 ± 70.1 |
In total | 73.4 ± 39.8 | 27.4 ± | 14.7 80.9 ± 26.0 | 64.3 ± 41.3 | 53.8 ± 20.0 | 63.1 ± | 36.4 72.7 ± 45.8 | 213.1 ± 219.5 |
Samples n = 3 in each from group centrifuged honey | 58.3 ± 21.98 means with different small letters in the same column are significantly different (P ≤ 0.05) | 214.4 ± 29.7 means with different small letters in the same column are significantly different (P ≤ 0.05) |
The average concentration of amitraz in honey of group B was significantly higher than that in honey of group W (P < 0.05). No significant effect on honey contamination deriving from the place of its exposure to smoke (combs of brood chambers and supers) was noted (P > 0.05). A wide range of amitraz concentration was proven both with regard to sampling sites and particular honeybee colonies. Treatment repetition with the Wakont did not significantly affect the rise of the concentration of amitraz metabolites in subsequent honey samples (P > 0.05). The four applications of Apiwarol directly inside the beehives caused a clear increase in amitraz marker residues in honey, although this rise was found to be statistically insignificant (P > 0.05).
Apiwarol fumigation of honeybee colonies is commonly used by Polish beekeepers to protect against
Amitraz metabolite concentrations in honey stored in hives during Apiwarol treatment were unknown prior to this research. Research on honey contamination with amitraz after administration to honeybee colonies of other veterinary medicinal products with an amitraz component is also limited. After two long-lasting treatments with Apivar (strips with 500 mg amitraz each, contact action, 42 days), no residue of the parent compound was detected in honey, regardless of the date of sampling, at the LOD 0.002 or at 0.01 mg/kg (9, 17). Different results were obtained after long-lasting Apivar application when the three metabolites of amitraz were measured in honey as well as amitraz itself (20). Ten days after the end of treatment, amitraz residues in honey from supers ranged from 0.06 to 0.09 mg/kg and 15 days after ranged from below LOQ (0.05 mg/kg) to 0.08 mg/kg. The exposure of bees to amitraz during treatment is disproportionately longer than during the Apiwarol method of application.
The breakdown of amitraz in honey spiked with amitraz and kept at room temperature has also been investigated (2, 10, 12, 15). Amitraz was found to be rapidly decomposed within 15 days. According to these authors, the main breakdown products of amitraz were 2,4-DMA, DMF, and DMPF, but the relative amounts of the degradation products were different. One author found relative amounts of DMPF, DMF, and DMA of 50, 25, and 25% (10), whereas another found 0%, 85%, and 15% (12), respectively. The amitraz metabolite profile analysed after 15 days, by Korta
Combustion of a single tablet with amitraz caused contamination of honey with amitraz metabolites. Rapid decomposition of amitraz and lack of parent residues would not mean that bee products are free from these toxic compounds, and it is pertinent that at least one amitraz metabolite was found in each analysed honey sample from this apiary trial. Concentration of amitraz residues is significantly higher when the Apiwarol tablet is administered directly into the hive. The manner of treatment (directly into the hive or introduced by the Wakont fumigator) has a significant impact on the concentration of amitraz residues when treatment is repeated several times.
The reports of EFSA confirm that amitraz residues in honey are a significant issue. It should be noted that amitraz residues in honey more likely originate from veterinary medicinal products used by beekeepers. According to the Commission Decision 2004/141/EC, amitraz is no longer approved as an active compound in plant protection products in the EU (3). Tests of beeswax and honeybee analysis also shed light on the issue of amitraz residues as a result of honeybee colony treatment (13, 19).
In conclusion, it seems necessary to review and update the knowledge relating to amitraz biotransformation in bees and the beehive environment.