**2.1.2 Antibiotic residues due to agricultural use**

Antibiotics can find their ways to bee products not only from beekeepers but also from the environment. Bees collect and transfer readily in their hive bactericides that are used against *Erwinia amylovora*. Out of 166 Greek citrus honeys that had been analyzed the 146 of them were found having antibiotic residues of soulphonamides and streptomycine originating from the therapeutical products that had been used in citrus plants (Karampournioti, 2004). Similarly in South Germany 40 samples out of 183 (21%) were found having residues of that source (Wallner, 1998). Moreover, Brasse (2001) identified the antibiotic streptomycin in 27 out of 128 honey analyzed samples. Bees may also transfer antibiotics through water since sulphanimide and tetracyclines are used in drinking water from poultry farms, rabbit cages and other animals. The manure of pigs and cows treated with sulphonamides or sulphacompounds could also be the vector. Some herbicides products, like Asulan may be degradated to sulphanilamide and bees with nectar can transfer it into the hive (Bogdanov & Edder, 2004; Kaufmann & Kaenzig, 2004). Finally bees may rob honey from colonies of other apiary that had been treated by antibiotics and by this way can contaminate their product in detectable levels.

#### **2.2 Direct contamination of bee products**

Active substances used by beekeepers themselves are likely to contaminate bee products with undesirable residues. Acaricide and antibiotic preparations are used in order to control the mite *Varroa destructor*, American foulbrood, Nosemosis and other diseases. Moreover, several volatile insecticides were used in the warehouse, in order to fend lepidopteron *Galleria mellonella* Linnaeus, which is responsible for considerable damages to stored combs.

## **2.2.1 Acaricide residues**

The use of synthesized substances for crop protection and livestock is the easiest and most effective way for beekeepers to control mites. Acaricides like amitraz, cymiazole, bromopropylate, tau-fluvalinate, flumethrin, coumaphos and malathion have been used by beekeepers all over the world. Many preparations like Apistan (a.i. tau-fluvalinate), Perizin (a.i. coumaphos), CheckMite+ (a.i.coumaphos), Bayvarol (a.i. flumethrin) and Apiguard (a.i. thymol) gain approval in most European countries. There are substances like amitraz that got approval only in certain countries and others like malathion that have not been approved at all.

#### **2.2.1.1 Amitraz**

Structure: It belongs to the group of formamidines

Action: Non-systemic insecticide and acaricide, which causes stimulation of neuronal activity killing the target.

Preparation: The main commercial formulation is the Taktik, used in livestock and particularly horses and sheep. Other preparation used: Mitak and Bye Bye.

Ways to use in beekeeping: Fumigation, Spray.

Acceptable Daily Intake (ADI): 0.003 mg kg-1 body weight per day or 0.18 mg per person per day (EMEA, 1999).

MRL for honey: EU established maximum residue levels (MRL) for amitraz residues in honey. The MRL of amitraz established to 0.2 mg kg-1 including the parent compound and its metabolites containing 2,4-dimethylaniline moiety. It should be noted that despite the establishment of the MRL, amitraz residues in honey are not acceptable in some countries

Antibiotics can find their ways to bee products not only from beekeepers but also from the environment. Bees collect and transfer readily in their hive bactericides that are used against *Erwinia amylovora*. Out of 166 Greek citrus honeys that had been analyzed the 146 of them were found having antibiotic residues of soulphonamides and streptomycine originating from the therapeutical products that had been used in citrus plants (Karampournioti, 2004). Similarly in South Germany 40 samples out of 183 (21%) were found having residues of that source (Wallner, 1998). Moreover, Brasse (2001) identified the antibiotic streptomycin in 27 out of 128 honey analyzed samples. Bees may also transfer antibiotics through water since sulphanimide and tetracyclines are used in drinking water from poultry farms, rabbit cages and other animals. The manure of pigs and cows treated with sulphonamides or sulphacompounds could also be the vector. Some herbicides products, like Asulan may be degradated to sulphanilamide and bees with nectar can transfer it into the hive (Bogdanov & Edder, 2004; Kaufmann & Kaenzig, 2004). Finally bees may rob honey from colonies of other apiary that had been treated by antibiotics and by this way can contaminate their

Active substances used by beekeepers themselves are likely to contaminate bee products with undesirable residues. Acaricide and antibiotic preparations are used in order to control the mite *Varroa destructor*, American foulbrood, Nosemosis and other diseases. Moreover, several volatile insecticides were used in the warehouse, in order to fend lepidopteron *Galleria mellonella* Linnaeus, which is responsible for considerable damages to stored combs.

The use of synthesized substances for crop protection and livestock is the easiest and most effective way for beekeepers to control mites. Acaricides like amitraz, cymiazole, bromopropylate, tau-fluvalinate, flumethrin, coumaphos and malathion have been used by beekeepers all over the world. Many preparations like Apistan (a.i. tau-fluvalinate), Perizin (a.i. coumaphos), CheckMite+ (a.i.coumaphos), Bayvarol (a.i. flumethrin) and Apiguard (a.i. thymol) gain approval in most European countries. There are substances like amitraz that got approval only in certain countries and others like malathion that have not been

Action: Non-systemic insecticide and acaricide, which causes stimulation of neuronal

Preparation: The main commercial formulation is the Taktik, used in livestock and

Acceptable Daily Intake (ADI): 0.003 mg kg-1 body weight per day or 0.18 mg per person per

MRL for honey: EU established maximum residue levels (MRL) for amitraz residues in honey. The MRL of amitraz established to 0.2 mg kg-1 including the parent compound and its metabolites containing 2,4-dimethylaniline moiety. It should be noted that despite the establishment of the MRL, amitraz residues in honey are not acceptable in some countries

particularly horses and sheep. Other preparation used: Mitak and Bye Bye.

**2.1.2 Antibiotic residues due to agricultural use** 

product in detectable levels.

**2.2.1 Acaricide residues** 

approved at all. **2.2.1.1 Amitraz** 

activity killing the target.

day (EMEA, 1999).

**2.2 Direct contamination of bee products** 

Structure: It belongs to the group of formamidines

Ways to use in beekeeping: Fumigation, Spray.

because of the lack of approval for beekeeping use. Therefore, in this case the limit corresponds to the Limit of Quantification, which is 0.01 mg kg-1.

The use of active substance amitraz is widespread in several European countries and the United States. Moreover, the effectiveness of this substance against varroa is satisfactory. The residues of the active substance is not often detected because of the rapid degradation of amitraz, which takes place within three weeks in blossom honey and four weeks in honeydew honey. The difference in degradation interval was attributed to the lower pH of blossom honey, which accelerates the chemical reactions of decomposition (Corta et al., 1999). The active ingredient amitraz is usually detected in cases where the preharvest interval is very short. A study reports as final degradation product of amitraz in honey, the 2,4-dimethyl-aniline, which is classified as hazardous to public health (Taccheo et al., 1988a). Recently, several samples of pears found to contain significant concentrations of amitraz and its metabolites. This fact, as well as indications about carcinogenic effects of the substance, led to a series of inspections and repeated alerts reported on RASFF of EU (Rapid Alert System of Food And Feed). To date, no published RASFF on residues of amitraz in honey have been reported. Finally, a reference work published in USA, observed the development of resistance of varroa to amitraz (Eljen et al., 2000).

#### **2.2.1.2 Coumaphos**

Structure: It belongs to the group of organophosphorus insecticides-acaricides

Action: Substance with systemic action that causes death in insects and mites by affecting cholinergic synapses of the central nervous system.

Preparations: The active ingredient coumaphos prepared by Bayer as three different formulations; Perizin, CheckMite+ and Asuntol. The last is the only one without authorization for beekeeping use.

Ways to use in beekeeping: The active substance used as an aqueous solution or a controlled release film. Perizin is used as an aqueous solution applied as drops between the frames. Spraying or adding to food can also be used for the application of this preparation. Special mention should be made to the use of coumaphos in the form of controlled release strips (CheckMite+). Primarily, application of CheckMite+ took place in the U.S., by providing a limited number of films in beekeepers of every State (Sanford & Flottum, 1999). In Europe, CheckMite+ was granted authorization in 2006. The major advantage of this preparation is that it also controls the small hive beetle *Aethina tumida* Marey.

ADI: 0.25 mg kg-1 body weight per day or 15 mg per person per day (EMEA, 2001).

MRL for honey: the established MRL for coumaphos in the EU is 0.1 mg kg-1.

Coumaphos does not control mites exclusively through contact, like most acaricides used in beekeeping, but it has a systemic action as well. The advantage of this way of action is the greater efficacy, and the rapid dispersion throughout the whole area of the hive. However, the disadvantage of substances with systemic action like coumaphos is the great persistence. According to a study, bees produce wax with residues of coumaphos, even six months after the application of the substance into the hive (Wilhelmina, 1992).

The persistence and dispersion of coumaphos in the hive after the application of CheckMite+ was studied by Karazafiris et al. (2008). According to that study, concentration of coumaphos residues was great in honey frames, which were in contact with strips. In some cases, residues exceeded the value of the established MRL. Moreover, it was observed that the concentration of acaricide in honey was at the level of MRL even 103 days after the removal of the strips. Therefore, the exclusion of frames that are in contact with the strips

Pesticide Residues in Bee Products 97

of residues in the honeycombs (Tsigouri et al., 2004). Tau fluvalinate has been used by beekeepers for many decades. Nowadays, the beekeepers have stopped using it because the mites developed resistance towards this chemical. (Elzen et al., 2000; Milani et al., 1995;

Bromopropylate is one of the oldest compounds that had been used against Varroa under the commercial product FOLBEX-VA. Its use was totally abandoned in Switzerland in 1991. An analysis made 19 years later showed that bromopropylate was still in beeswax in high concentrations and scientists believe that more than 20 years will pass before it is expected

Action: bromopropylate is a broad spectrum, non-systemic insecticide-acaricide with high

Preparations: The name of the commercial preparation is Folbex VA, manufactured by Giba-

MRL for honey: no MRL exists for this chemical at E.U. Bromopropylate was used in crops such as pome fruits, stone fruits and plants of Solanacea family. The agricultural use has led to an establishment of MRL under the provisions of EU Regulation 396/2005. This new MRL corresponds to a concentration of 0.1 mg kg-1 and concerns the contamination of bee

Bromopropylate is not toxic to bees, while its metabolite 4,4-dibromobenzolic acid is likely to be detected. The use of bromopropylate was particularly widespread in Central Europe. The observation that bromopropylate degradation is slow and, therefore very stable in honey and wax, forced Europeans beekeepers to start using acaricides that are more environment and consumer friendly. According to a survey conducted by Taccheo et al. (1988b), concentration of bromopropylate residues was greater in honey from an uncapped comb than from a capped one. Moreover, the burning of fumigant strips in an empty floor above the hive reduced the concentration of residues in honey (Taccheo, 1988b). The use of bromopropylate has stopped in many countries and no samples with residues of

Action: malathion is broad spectrum, non-systemic insecticide-acaricide which acts through stomach. The way of action is similar to that of the organophosphate acaricide coumaphos. In addition, malathion oxidized to malaoxon, which is a substance with high toxicity to insects and mites. This chemical reaction does not occur in the body of mammals, limiting

Beekeeping use: Malathion has never had approval for beekeeping use. Despite this, many beekeepers use it as spraying material or as powdered sugar. Due to the high bee toxicity, malathion requires special attention during application. A slightly increased dose can be

Structure: It belongs to the group of chlorinated derivatives of benzene.

residual activity, which acts by contact and inhibits the synthesis of ATP.

Beekeeping use: The use of this preparation is in the form of fumigant. ADI: 0.03 mg kg-1 body weight per day or 1.8 mg per person per day.

bromopropylate were found in Greek honey (Karazafiris et al., 2007).

Structure: It belongs to the group of organophosphorus compounds.

products through the use of agricultural pesticides.

Thompson et al., 2003). **2.2.1.5 Bromopropylate** 

Geigy.

**2.2.1.6 Malathion** 

Preparations: Malathion

the toxicity of the acaricide for humans.

fatal for the colony, especially when used as a solution.

to fully disappear from beeswax.

could lead to a drastic reduction of residual coumaphos concentrations in the final product. On the contrary, it was observed that the concentration of coumaphos residues in honey chamber was significantly lower and in no case exceeded the MRL. Finally, the time between application of the preparation and the collection of honey affected the amount of residues. Gajduskova et al. (1990), studied the contamination of bee products under different methods of application, found that higher concentrations of coumaphos levels were recorded when the substance was added to the syrup rather than the usual method of dripping the chemical into the hive. Coumaphos proved very stable in honey, moved quickly to the wax because of its strong lipophilic character and remained there in significant concentrations even after melting of the wax (Krieger, 1991). Reports that mites became resistant to coumaphos have already been published (Maggi et al., 2009; Petis, 2004).

#### **2.2.1.3 Flumethrin**

Structure: It belongs to the group of pyrethroids.

Action: Non-systemic insecticide-acaricide which acts in contact through the stomach. As the majority of pyrethroid pesticides, flumethrin is characterized as a broad range pesticide presenting low toxicity to mammals. Through its action, flumethrin disrupts the functioning of the Na+ pump and therefore the equilibrium of Na+/K+ across the membrane.

Preparation: Bayvarol is one of the approved preparations for use in beekeeping. Production Company is the Bayer CropScience.

Beekeeping use: Flumethrin applied in the form of controlled release strips.

ADI: 1.8 mg kg-1 body weight per day or 108 mg per person per day (EMEA, 1998).

MRL for honey: The very low concentration required per hive and the low water solubility, are the main reasons why no detectable residues were detected in honey after the recommended use. That is the reason why no MRL has been established for this substance (EMEA, 1998). According to recent studies, mites became resistant to pyrethroids (Milani, 1995; Thompson, 2003).

#### **2.2.1.4 Tau fluvalinate**

Structure: It belongs to the group of pyrethroids.

Action: This is a broad range non-systemic insecticide-acaricide that acts by contact through stomach. The way of action of tau fluvalinate is similar to that of the flumethrin.

Preparation: There are three preparations used by beekeepers containing tau fluvalinate: Apistan, Mavrik and Klartan. Out of the three, only Apistan has an approval for beekeeping use.

Beekeeping use: The use of the authorized preparation is in the form of controlled release strips (Apistan).

ADI: 0.5 mg kg-1 body weight per day or 30 mg per person per day (EMEA, 1998).

MRL for honey: EU established no MRL for tau fluvalinate residues in honey, as the concentrations of detected residues were extremely low, based on the experimental results included in the file submitted (<0.01 mg kg-1) (EMEA, 1998).

The use of Apistan strips is likely to lead to accumulation of residues, if they have been left in the hive for more than 6 weeks. Balayiannis and Santas (1989) reported an increased persistence of residues in stored honey, compared with the honey in combs. This is apparently owed to the non-transfer of tau fluvalinate in wax. Tau fluvalinate is the most lipophilic of all compounds used in beekeeping. This property combined with the high stability of the substance in the wax contributes to the drastic increase in the concentration of residues in the honeycombs (Tsigouri et al., 2004). Tau fluvalinate has been used by beekeepers for many decades. Nowadays, the beekeepers have stopped using it because the mites developed resistance towards this chemical. (Elzen et al., 2000; Milani et al., 1995; Thompson et al., 2003).

#### **2.2.1.5 Bromopropylate**

96 Pesticides in the Modern World - Risks and Benefits

could lead to a drastic reduction of residual coumaphos concentrations in the final product. On the contrary, it was observed that the concentration of coumaphos residues in honey chamber was significantly lower and in no case exceeded the MRL. Finally, the time between application of the preparation and the collection of honey affected the amount of residues. Gajduskova et al. (1990), studied the contamination of bee products under different methods of application, found that higher concentrations of coumaphos levels were recorded when the substance was added to the syrup rather than the usual method of dripping the chemical into the hive. Coumaphos proved very stable in honey, moved quickly to the wax because of its strong lipophilic character and remained there in significant concentrations even after melting of the wax (Krieger, 1991). Reports that mites became resistant to coumaphos have already been published (Maggi et al., 2009; Petis, 2004).

Action: Non-systemic insecticide-acaricide which acts in contact through the stomach. As the majority of pyrethroid pesticides, flumethrin is characterized as a broad range pesticide presenting low toxicity to mammals. Through its action, flumethrin disrupts the functioning

Preparation: Bayvarol is one of the approved preparations for use in beekeeping. Production

MRL for honey: The very low concentration required per hive and the low water solubility, are the main reasons why no detectable residues were detected in honey after the recommended use. That is the reason why no MRL has been established for this substance (EMEA, 1998). According to recent studies, mites became resistant to pyrethroids (Milani,

Action: This is a broad range non-systemic insecticide-acaricide that acts by contact through

Preparation: There are three preparations used by beekeepers containing tau fluvalinate: Apistan, Mavrik and Klartan. Out of the three, only Apistan has an approval for beekeeping

Beekeeping use: The use of the authorized preparation is in the form of controlled release

MRL for honey: EU established no MRL for tau fluvalinate residues in honey, as the concentrations of detected residues were extremely low, based on the experimental results

The use of Apistan strips is likely to lead to accumulation of residues, if they have been left in the hive for more than 6 weeks. Balayiannis and Santas (1989) reported an increased persistence of residues in stored honey, compared with the honey in combs. This is apparently owed to the non-transfer of tau fluvalinate in wax. Tau fluvalinate is the most lipophilic of all compounds used in beekeeping. This property combined with the high stability of the substance in the wax contributes to the drastic increase in the concentration

stomach. The way of action of tau fluvalinate is similar to that of the flumethrin.

ADI: 0.5 mg kg-1 body weight per day or 30 mg per person per day (EMEA, 1998).

of the Na+ pump and therefore the equilibrium of Na+/K+ across the membrane.

Beekeeping use: Flumethrin applied in the form of controlled release strips. ADI: 1.8 mg kg-1 body weight per day or 108 mg per person per day (EMEA, 1998).

**2.2.1.3 Flumethrin** 

Structure: It belongs to the group of pyrethroids.

Structure: It belongs to the group of pyrethroids.

included in the file submitted (<0.01 mg kg-1) (EMEA, 1998).

Company is the Bayer CropScience.

1995; Thompson, 2003). **2.2.1.4 Tau fluvalinate** 

use.

strips (Apistan).

Bromopropylate is one of the oldest compounds that had been used against Varroa under the commercial product FOLBEX-VA. Its use was totally abandoned in Switzerland in 1991. An analysis made 19 years later showed that bromopropylate was still in beeswax in high concentrations and scientists believe that more than 20 years will pass before it is expected to fully disappear from beeswax.

Structure: It belongs to the group of chlorinated derivatives of benzene.

Action: bromopropylate is a broad spectrum, non-systemic insecticide-acaricide with high residual activity, which acts by contact and inhibits the synthesis of ATP.

Preparations: The name of the commercial preparation is Folbex VA, manufactured by Giba-Geigy.

Beekeeping use: The use of this preparation is in the form of fumigant.

ADI: 0.03 mg kg-1 body weight per day or 1.8 mg per person per day.

MRL for honey: no MRL exists for this chemical at E.U. Bromopropylate was used in crops such as pome fruits, stone fruits and plants of Solanacea family. The agricultural use has led to an establishment of MRL under the provisions of EU Regulation 396/2005. This new MRL corresponds to a concentration of 0.1 mg kg-1 and concerns the contamination of bee products through the use of agricultural pesticides.

Bromopropylate is not toxic to bees, while its metabolite 4,4-dibromobenzolic acid is likely to be detected. The use of bromopropylate was particularly widespread in Central Europe. The observation that bromopropylate degradation is slow and, therefore very stable in honey and wax, forced Europeans beekeepers to start using acaricides that are more environment and consumer friendly. According to a survey conducted by Taccheo et al. (1988b), concentration of bromopropylate residues was greater in honey from an uncapped comb than from a capped one. Moreover, the burning of fumigant strips in an empty floor above the hive reduced the concentration of residues in honey (Taccheo, 1988b). The use of bromopropylate has stopped in many countries and no samples with residues of bromopropylate were found in Greek honey (Karazafiris et al., 2007).

#### **2.2.1.6 Malathion**

Structure: It belongs to the group of organophosphorus compounds.

Action: malathion is broad spectrum, non-systemic insecticide-acaricide which acts through stomach. The way of action is similar to that of the organophosphate acaricide coumaphos. In addition, malathion oxidized to malaoxon, which is a substance with high toxicity to insects and mites. This chemical reaction does not occur in the body of mammals, limiting the toxicity of the acaricide for humans.

Preparations: Malathion

Beekeeping use: Malathion has never had approval for beekeeping use. Despite this, many beekeepers use it as spraying material or as powdered sugar. Due to the high bee toxicity, malathion requires special attention during application. A slightly increased dose can be fatal for the colony, especially when used as a solution.

Pesticide Residues in Bee Products 99

especially in foods may lead to the rapid emerge of resistant bacterial strains and consequently to the demand of new substances to replace the old. Moreover, the emergence of resistant bacteria involves the use of powerful antibiotics leading to serious consequences

Chloramphenicol is a potent antibiotic that has limited uses; it has been declared carcinogenic and causing fatal aplastic anemia, which makes it an unacceptable substance for use in production of food products where any residue may be found. Several reports document human fatalities resulting from ophthalmic preparations containing chloramphenicol, with exposure dozes that could be found in residues in food (Settepani, 1984). Chloramphenicol was detected in bee products, honey and royal jelly, imported from China and India. In 2002, alerts appeared from the U.S., Canada, and Europe that honey samples from China often contained traces of the antibiotic chloramphenicol with a range of 0.3 to 34 μg kg-1 (LOD= μg kg-1). Since China did not have stringent controls on veterinary use of various antibiotics, this drug had been used (along with streptomycin) by the Chinese beekeepers to control a bacterial epidemic that affected bee hives (Dharmananda, 2003). The EU, in an effort to protect consumers, banned the import of Chinese products of animal origin since 2004. Also in Switzerland, chloramphenicol residues detected in thirteen out of 75 (17%) of commercially obtained honey samples, ranged between 0.4 and 6.0 μg kg-1

Tetracycline is used by beekeepers in order to control AFB and EFB. Normally, it degrades in 6-10 weeks (Matsuka & Nakamura, 1990; Gilliam et al., 1979). In some cases tetracycline was detected in honey, even after three years, because of the high dose used by beekeepers (Shakaryan & Akopyan, 1973). Acidity, viscosity and organic acids of honey contribute to the stability of antibiotics (Gilliam et al., 1979). The treatment of the hive with antibiotics results in tetracycline residues in honey and wax (Gilliam et al., 1979; Corner & Gochnauer, 1971). In two studies of Shakaryan & Akopyan (1972 & 1973), 1.2% of the initial concentration of the antibiotic residues remained stable even after the heating of honey for three successive times in 90°C (30 minutes). Tetracyclines (tetracycline, oxytetracycline, chlortetracycline, doxycycline) have been found in honey in various countries. In a study conducted in Greece, tetracycline residues were found in 23% of the spring floral honey samples tested (Karazafiris et al., 2007). In another study, out of 251 greek honey samples, 29% were found contaminated with tetracycline residues ranged from 0.018 to 0.055 mg kg-1

The sulfonamides are analogues of para-aminobenzoic acid, which include sulfapyridine, sulfadimidine, sulfadiazine, sulfamethoxazole, sulfadimethoxin, sulfamethopyridazine, sulfadoxine, sulfamethoxypyridazine, sulfadoxine and sulfamethopyrazine. They are suspected to cause aplastic anemia, like chloramphenicol. It is the most stable antibiotic in honey (Bonvehi & Pajuelo, 1983). In the past, sulfathiazole was detected regularly in honey produced in the European countries. Beekeepers used sulfa-drugs in order to control AFB

in the normal flora of the human body.

**2.2.2.1 Chloramphenicol** 

(Ortelli et al., 2004). **2.2.2.2 Tetacyclines** 

(Saridaki-Papakonstadinou et al., 2006).

and EFB and in some cases Nosemosis.

**2.2.2.3 Sulfonamides** 

ADI: 0.03 mg kg-1 body weight per day or 1.8 mg per person per day.

MRL for honey: No MRLs have been established in honey, and therefore the threshold corresponding to the LOQ is 0.01 mg kg-1.

Two different studies were conducted by Thrasyvoulou et al. (1988) and Balayiannis et al. (1989) concerning the time of degradation of malathion in honey. Both two studies proved that the time of degradation of malathion is three months. In a survey conducted in 50 samples of Greek honey, 4% were found contaminated in concentrations that did not exceed 0.005 mg kg-1 (Thrasyvoulou et al., 1988). Futhermore, malathion was detected in 23 out of 593 honey samples analyzed in the laboratory of Apiculture-Sericulture, Aristotle University of Thessaloniki during the years 2003-2006 (Karazafiris et al., 2005). In Cuba, Pelayo et al. (1987) detected malathion in 12 out of 110 samples. The concentration of the active substance did not exceed 0.02 mg kg-1 in any case.

#### **2.2.1.7 Cymiazole**

Structure: It belongs to the group of iminophenyl thiazolidine.

Action: Cymiazole is another substance, like coumaphos, that used in beekeeping and has systemic action.

Preparation: The name of the commercial preparation is Apitol and is manufactured by Giba-Geigy.

Beekeeping use: Cymiazole can be applied in different ways.

ADI: 1 mg kg-1 body weight per day or 60 mg per person per day (EMEA, 1996).

MRL for honey: MRL that was established for cymiazole was 1 mg kg-1, but latest E.U. regulations established new MRL that corresponds to LOQ (0.01 mg kg-1).

#### **2.2.2 Antibiotic residues**

The term antibiotic originally refers to any agent with biological activity against living organisms; however, ''antibiotic" nowadays refers to substances with antibacterial, antifungal, or anti-parasitical activity. There are currently about 250 different substances registered for use in medicine and veterinary medicine (Kümmerer & Henninger, 2003).

Antibiotics such as tetracycline, chloramphenicol, sulfathiazole, streptomycin, tylosin, erythromycin etc, are commonly used by beekeepers, in order to control European Foulbrood Disease (EFB), American Foulbrood Disease (AFB) and Nosemosis caused by *Paenibacilus larvae larvae*, *Streptococcus pluton* bacteria and fungus of the genus *Nosema*, respectively. The use of antibiotics is not allowed in beekeeping since no MRLs have been set for honey. Some countries, like Switzerland, UK and Belgium, have established action limits for antibiotics in honey, which generally lie between 0.01 to 0.05 mg kg-1 for each antibiotic group. An action limit is the concentration of antibiotics in honey, above which the sample is considered non-compliant. The presence of antibiotic residues in honey and other hive products is not accepted in Europe for products imported from third countries. In case a product is found contaminated with antibiotics then it should be destroyed and the producer should be penalized. In the U.S.A., Canada and Argentina, preventive treatments with antibiotics are considered a routine procedure to control AFB. As a result, various strains of *P. larvae* have developed resistance to antibiotics, such as oxytetracycline (OTC). Such strains have been isolated in Argentina (Alippi, 2007) as well as in many areas of the U.S.A. (Miyagi et al., 2000). Generally, the presence of antibiotics in the environment

MRL for honey: No MRLs have been established in honey, and therefore the threshold

Two different studies were conducted by Thrasyvoulou et al. (1988) and Balayiannis et al. (1989) concerning the time of degradation of malathion in honey. Both two studies proved that the time of degradation of malathion is three months. In a survey conducted in 50 samples of Greek honey, 4% were found contaminated in concentrations that did not exceed 0.005 mg kg-1 (Thrasyvoulou et al., 1988). Futhermore, malathion was detected in 23 out of 593 honey samples analyzed in the laboratory of Apiculture-Sericulture, Aristotle University of Thessaloniki during the years 2003-2006 (Karazafiris et al., 2005). In Cuba, Pelayo et al. (1987) detected malathion in 12 out of 110 samples. The concentration of the active substance

Action: Cymiazole is another substance, like coumaphos, that used in beekeeping and has

Preparation: The name of the commercial preparation is Apitol and is manufactured by

MRL for honey: MRL that was established for cymiazole was 1 mg kg-1, but latest E.U.

The term antibiotic originally refers to any agent with biological activity against living organisms; however, ''antibiotic" nowadays refers to substances with antibacterial, antifungal, or anti-parasitical activity. There are currently about 250 different substances registered for use in medicine and veterinary medicine (Kümmerer & Henninger, 2003). Antibiotics such as tetracycline, chloramphenicol, sulfathiazole, streptomycin, tylosin, erythromycin etc, are commonly used by beekeepers, in order to control European Foulbrood Disease (EFB), American Foulbrood Disease (AFB) and Nosemosis caused by *Paenibacilus larvae larvae*, *Streptococcus pluton* bacteria and fungus of the genus *Nosema*, respectively. The use of antibiotics is not allowed in beekeeping since no MRLs have been set for honey. Some countries, like Switzerland, UK and Belgium, have established action limits for antibiotics in honey, which generally lie between 0.01 to 0.05 mg kg-1 for each antibiotic group. An action limit is the concentration of antibiotics in honey, above which the sample is considered non-compliant. The presence of antibiotic residues in honey and other hive products is not accepted in Europe for products imported from third countries. In case a product is found contaminated with antibiotics then it should be destroyed and the producer should be penalized. In the U.S.A., Canada and Argentina, preventive treatments with antibiotics are considered a routine procedure to control AFB. As a result, various strains of *P. larvae* have developed resistance to antibiotics, such as oxytetracycline (OTC). Such strains have been isolated in Argentina (Alippi, 2007) as well as in many areas of the U.S.A. (Miyagi et al., 2000). Generally, the presence of antibiotics in the environment

ADI: 1 mg kg-1 body weight per day or 60 mg per person per day (EMEA, 1996).

regulations established new MRL that corresponds to LOQ (0.01 mg kg-1).

ADI: 0.03 mg kg-1 body weight per day or 1.8 mg per person per day.

Structure: It belongs to the group of iminophenyl thiazolidine.

Beekeeping use: Cymiazole can be applied in different ways.

corresponding to the LOQ is 0.01 mg kg-1.

did not exceed 0.02 mg kg-1 in any case.

**2.2.1.7 Cymiazole** 

systemic action.

**2.2.2 Antibiotic residues** 

Giba-Geigy.

especially in foods may lead to the rapid emerge of resistant bacterial strains and consequently to the demand of new substances to replace the old. Moreover, the emergence of resistant bacteria involves the use of powerful antibiotics leading to serious consequences in the normal flora of the human body.

#### **2.2.2.1 Chloramphenicol**

Chloramphenicol is a potent antibiotic that has limited uses; it has been declared carcinogenic and causing fatal aplastic anemia, which makes it an unacceptable substance for use in production of food products where any residue may be found. Several reports document human fatalities resulting from ophthalmic preparations containing chloramphenicol, with exposure dozes that could be found in residues in food (Settepani, 1984). Chloramphenicol was detected in bee products, honey and royal jelly, imported from China and India. In 2002, alerts appeared from the U.S., Canada, and Europe that honey samples from China often contained traces of the antibiotic chloramphenicol with a range of 0.3 to 34 μg kg-1 (LOD= μg kg-1). Since China did not have stringent controls on veterinary use of various antibiotics, this drug had been used (along with streptomycin) by the Chinese beekeepers to control a bacterial epidemic that affected bee hives (Dharmananda, 2003). The EU, in an effort to protect consumers, banned the import of Chinese products of animal origin since 2004. Also in Switzerland, chloramphenicol residues detected in thirteen out of 75 (17%) of commercially obtained honey samples, ranged between 0.4 and 6.0 μg kg-1 (Ortelli et al., 2004).

#### **2.2.2.2 Tetacyclines**

Tetracycline is used by beekeepers in order to control AFB and EFB. Normally, it degrades in 6-10 weeks (Matsuka & Nakamura, 1990; Gilliam et al., 1979). In some cases tetracycline was detected in honey, even after three years, because of the high dose used by beekeepers (Shakaryan & Akopyan, 1973). Acidity, viscosity and organic acids of honey contribute to the stability of antibiotics (Gilliam et al., 1979). The treatment of the hive with antibiotics results in tetracycline residues in honey and wax (Gilliam et al., 1979; Corner & Gochnauer, 1971). In two studies of Shakaryan & Akopyan (1972 & 1973), 1.2% of the initial concentration of the antibiotic residues remained stable even after the heating of honey for three successive times in 90°C (30 minutes). Tetracyclines (tetracycline, oxytetracycline, chlortetracycline, doxycycline) have been found in honey in various countries. In a study conducted in Greece, tetracycline residues were found in 23% of the spring floral honey samples tested (Karazafiris et al., 2007). In another study, out of 251 greek honey samples, 29% were found contaminated with tetracycline residues ranged from 0.018 to 0.055 mg kg-1 (Saridaki-Papakonstadinou et al., 2006).

#### **2.2.2.3 Sulfonamides**

The sulfonamides are analogues of para-aminobenzoic acid, which include sulfapyridine, sulfadimidine, sulfadiazine, sulfamethoxazole, sulfadimethoxin, sulfamethopyridazine, sulfadoxine, sulfamethoxypyridazine, sulfadoxine and sulfamethopyrazine. They are suspected to cause aplastic anemia, like chloramphenicol. It is the most stable antibiotic in honey (Bonvehi & Pajuelo, 1983). In the past, sulfathiazole was detected regularly in honey produced in the European countries. Beekeepers used sulfa-drugs in order to control AFB and EFB and in some cases Nosemosis.

Pesticide Residues in Bee Products 101

The greater wax moth *Galleria mellonella* is a serious pest of stored combs and weak colonies. Adult female wax moths enter hives and lay their eggs on wax combs or in small crevices between wooden parts of the hives not easily accessible to honey bees. After few days the larvae hatch and begin feeding on bees-wax, pollen, cast larval skins and other remains in cells. This devastating activity of wax moths leads to great financial losses every year in the

Strong colonies are the best control against the wax moth in the field. In comb storage chests, technical, physical, biological and chemical methods have been used to control the pest. The most effective method to avoid the destruction of combs from wax moth is their continuous maintenance in temperatures of the refrigerator, or their passing from the freezer for a short time. Cantwell and Smith (1970) confirmed that temperature lower than - 18 °C destroys all stages of the wax moth insect (egg, immature forms and adult). Although this treatment requires expensive facilities, it is successfully applied nowadays protecting

In addition, biological and environment-friendly control method were developed such us the male sterile technique with gamma-rays (Jafari et al., 2010), the trapping of moths by using pheromone (Flint & Merkle, 1983) and the use of the bacterium *Bacillus thuringiensis* that kills the wax moth larvae when it ingests the spores (Burges & Bailey, 1968; Burges

Chemical methods, includes substances that are considered friendly to environment like methyl salicylate, clove oil, formic acid, sulphur, acetic acid, basil oil and other have been used (Wilson, 1965; Williams, 1980; Owayss & Abd-Elgayed, 2007). Most of these compounds are dangerous for bee brood and human health, while they require repeated application and may react and destroy the metal parts of the combs. Besides these, 1,2-dibromo-ethane (DBE), 1,4 dichloro-benzene (p-DCB), naphthalene had been used for many years in different countries

DBE is a manufactured chemical. In nature, it is produced in small amounts in the sea water, where it is formed, probably by algae and kelp. It is dissolved in water and by this way it can stay in groundwater and in soil for a long time. In air it breaks down quickly. This substance has been used as a pesticide in soil, and on citrus, vegetables, and grain crops. EPA has banned most of these uses since 1984. The same organization has also set a limit of

The compound p-DCB is one of the three di-chloro-benzene isomers (1,2-DCB, 1,3-DCB and 1,4-DCB), which is commonly used as a space deodorant i toilets and for moth control. It is a volatile colorless to white crystalline material with a mothball-like, penetrating odor and it

Naphthalene is a white solid substance that evaporates easily. Its major use is in the manufacture of polyvinyl chloride (PVC) plastics and it is also used in moth repellents and toilet deodorant blocks. That use of naphthalene accounted for 73% and 60% of commercial demand for naphthalene in Japan and the United States, respectively in 1999, (ATSDR,

No MRL's in honey for the above three compounds were defined until 2005 when the European regulation 396/2005 EC set the limit at 10 μg kg-1 for substances for which no MRL had been established. This limit for p-DCB was also the Swiss Tolerance Limit (STL)

the honeycombs from the wax moth without contaminating the beehive products.

even though their use causes significant contamination of bee products.

0.05 μg.cm-3 of 1,2-dibromo-ethane in drinking water (ATSDR, 1992).

is commercially, the most important isomer (ATSDR, 2006).

**2.2.3 Residues of volatile insecticides in bee products** 

field of beekeeping.

1997; Charriere & Imdorf, 2004).

2005).

In 2002, sulfa drugs were detected in 3 out of 91 samples of honey collected from the Belgian market. Moreover, 12 out of 203 honey samples collected in 2003 were contaminated by residues of sulfonamides (Reybroeck et al., 2004).

#### **2.2.2.4 Streptomycin**

The problem with streptomycin is that it may cause ototoxicity and nephrotoxicity. It is considered more dangerous than oxytetracycline and less hazardous than sulfathiazole and chloramphenicol regarding side effects. According to the Food Standards Agency of UK, an Indian honey was found to be contaminated by streptomycin in 2003 (Mayande, 2007).
