**2. Contamination of bee products**

There are two ways of contamination of bee products with various chemicals; the indirect and the direct contamination. The indirect way reflects the transporting of toxic substances by foragers bees during the collection of nectar, honeydew, water, pollen and propolis. Many studies concern the contamination of hive products by agrochemicals and heavy metals, while few concern the presence of nitrofurans, toxins and PCBs in beehive products. The direct way, which is the most important, regards the contamination of bee products by acaricides, antibiotics and volatile pesticides caused by beekeeping practices.

#### **2.1 Indirect contamination of bee products**

Many researchers supported the theory that the transferring of pesticides from fields to beehive is prevented in various ways. The bees' death at the field, the lost of orientation of the foragers, the reluctance of guard bees to permit the entrance to foragers with contaminated nectar, the retaining of contaminated food in bees' stomach, the stopping of further elaboration of contaminated nectar by hive bees and the removal of affected bees from the hive are natural provisions against general contamination of honey (Johansen & Mayer, 1990; Atkins, 1992). Contrary to the above-mentioned cases, older studies reported that worker bees may carry high concentrations of pesticides into their beehive. In some cases the concentration of pesticides in the load was 25 times greater than the lethal dose of the bee (Jaycox, 1964).

#### **2.1.1 Pesticides**

Pesticides used on various crops are classified into groups based on their chemical structure (organophosphates, pyrethroids, organochlorines, carbamates, neonicotinoids etc.), mode of action (systemic, contact), target (insecticides, acaricides, herbicides, fungicides, bactericides, nematicides) and synthesis (synthetic or natural). The residues of pesticides detected in beehive products are classified in the groups of insecticides (organochlorines, organophosphates, carbamates and neonicotinoids), acaricides, fungicides and herbicides.

#### **2.1.1.1 Organochlorine pesticides (OCPs)**

This specific group of insecticides is considered particularly hazardous because of its ability to bioaccumulate into the food chain, to remain stable for many years and to move into the

Pesticide Residues in Bee Products 93

Nevertheless, the concentration of carbamate residues is low in pollen and honey, while no

Most studies reported on neonikotinoids insecticides, refer to the active ingredient imidacloprid. This substance was proved toxic to bees, but the concentrations of residues detected in honey were very low (0.001 mg kg-1 to 0.005 mg kg-1) (Bonmatin et al., 2003; Maus et al., 2003; Schmuck et al., 2001). In many cases residues did not exceed the limit of quantification (<0.002 mg kg-1) (Rogers & Kemp, 2003; Schöning & Schmuck, 2003; Stadler et al., 2003; Faucon et al., 2004). Detected residues of imidacloprid in pollen were 0.005 mg kg-1, while detection rate was 49.4% (Bonmatin et al., 2003). The hazard quotient (application rate in grams per hectare/LD50) of neonicotinoids is far below the trigger value of 50, but the most important is the chronic toxicity that they cause to bees. The long-term exposure to neonicotinoid after the behaviour of bees, reduce their reproduction capacity and lead of population decline. The low detectable concentrations in combination with the low toxicity of imidacloprid in humans are reassuring for consumer safety. On the contrary, the effects of imidacloprid residues on bees should be further explored. The high toxicity for bees makes neonicotinoid residues suspicious about the death of many forager bees collecting nectar from sunflower, corn and cotton crops. The implementation of neonicotinoid active substances in seed of plants like cotton, corn and sunflower led to a theory that this specific class of pesticides is responsible for the "colony collapse disorder" syndrome (CCD). The CCD is defined as the sudden depopulation of a beehive and the rapid collapse of the colony. The causes of this phenomenon are not clear yet. The suspicion is directed at the mite *Varroa destructor* Anderson & Trueman, while others blame the protozoan *Nosema ceranae*. Additionally, suspicion directed at poisoning of bees by neonicotinoid insecticides and at various forms of radiation (telephony, wireless networks etc.) as well. In fact, research on the toxicity of neonicotinoids to the bee, proved tolerance of the insect body at normal concentrations identified in honey, pollen and nectar (Schmuck et al., 2001; Faucon et al., 2004). More recent research is directed at the effect of imidaclorpid to the orientation

Fungicides are toxic substances that are used to kill or inhibit the growth of fungi that cause economic damage to crops and endanger the health of domestic animals or humans. Most fungicides are toxic to humans and can cause both acute and chronic problems if absorbed into food. Kubik et al. (1999; 2000) studied the possibility of contamination of beehive products with residues of chemicals used on apple and cherry trees. Vinclozolin, iprodione and thiophanate methyl residues were detected in honey and pollen collected from cherry flowers, while captan and difenuconazole were detected in beehive products collected from apple trees. Specifically, the average concentration levels of vinclozolin in honey were determined at 0.107 mg kg-1. A recent review of vinclozolin by the US Environmental Protection Agency has concluded that the chemical or its breakdown products are associated with the development of testicular tumors in rats. The mean concentrations of residues of other active compounds were 0.0006 mg kg-1, 0.009 mg kg-1, 0.023 mg kg-1 and 0.059 mg kg-1 for captan, difenuconazole, iprodione and methyl thiophanate respectively. In all cases, the concentration was lower in honey, than in stored

residues have been reported in other beehive products.

**2.1.1.4 Neonicotinoid pesticides** 

of bees (Bortolotti et al.; 2003).

**2.1.1.5 Fungicides** 

pollen (Kubic, 2000).

environment in every potential way (air, water, soil, biota). The case of bioaccumulation of DDT in the environment is the most characteristic, while the concentration detected in the higher levels of the food chain is 10,000,000 times greater than that detected into the water. In recent decades there had been many efforts worldwide to prevent the use of substances belonging to the group of persistent organic pollutants (POP), which includes many organochlorine compounds. The continuous transfer of semi-volatile compounds from tropical regions of the world to the colder poles is suspected for long-term effects on living beings (Carson R., 1962). Chlorinated hydrocarbons are detected in high concentrations in various products, because of their low rate of degradation. Wax is the beehive product more likely to be contaminated by organochlorine insecticides because of its strong lipophilic character. Moreover, OCPs were proved to remain stable during the conversion of old combs into new (Jimenez et al., 2005). The problem is magnified by the import of wax from continents where the use of chlorinated hydrocarbons is still permitted like Asia and Africa. The encouraging news is that the percentage of honey contaminated with chlorinated hydrocarbons dropped from 96.1% to 52.3% during the decades 1980 and 1990.

#### **2.1.1.2 Organophosphorus pesticides (OPPs)**

This specific class of pesticides is of relatively high toxicity for humans and was first studied and used as an asphyxiating gas during the Second World War. Organophosphorus compounds are not stable in the environment and are not bio-concentrated and this is probably the main reason why they were detected rarely and at lower concentrations into beehive products. Most of published information concerns the compound methyl parathion, which has been used in agricultural crops as preparation in the slow-release form of microcapsules. Residues of this chemical were detected in honey and pollen (Atkins & Kellum, 1984). The microcapsules stick on the dense coat of bees, transferred into their hive and stored along with pollen. Pollen is the main component of the diet of larvae. The presence of polluted pollen might cause poisoning and eventually death to brood of bees. In a survey conducted on pollen from France, residues of parathion and methyl parathion were found in 1.2% and 4.9% of the samples, respectively. The average concentration for parathion was 0.019 mg kg-1 and for parathion methyl was 0.025 mg kg-1 (Chauzat et al., 2006). Blasco et al. (2003) detected only heptenophos in 4% of honey samples that were analyzed, out of 23 organophosphorus pesticides that they researched. Heptenophos concentrations in this survey ranged from 0.08 mg kg-1 to 0.23 mg kg-1. Finally, Balayiannis and Balayiannis (2008) detected the organophosphorus compounds chlorfenvinphos, chlorpyriphos and phorate in honey originated from Greece, in concentrations ranged from 0.7 μg kg-1 to 0.89 μg kg-1.

#### **2.1.1.3 Carbamate pesticides**

Carbamate insecticides have a similar mode of action with organophosphates but their insecticidal activity is more selective and depends to a certain extent on the insect species. Some fungicides and herbicides belong to this family. These substances are highly volatile in the environment and in some cases they were detected in beehive products. Concentration of carbamate residues detected in pollen ranged from 0.126 mg kg-1 to 0.265 mg kg-1 for the active ingredient carbaryl, while the maximum concentration of carbofuran was 0.14 mg kg-1 (Chauzat et al., 2006). Concentration of carbaryl, carbofuran, pirimicarb and methiocarb residues, in most cases is considered low and does not exceed 0.071 mg kg-1. In only one Spanish honey the concentration of carbofuran was 0.645 mg kg-1 (Blasco et al., 2003).

environment in every potential way (air, water, soil, biota). The case of bioaccumulation of DDT in the environment is the most characteristic, while the concentration detected in the higher levels of the food chain is 10,000,000 times greater than that detected into the water. In recent decades there had been many efforts worldwide to prevent the use of substances belonging to the group of persistent organic pollutants (POP), which includes many organochlorine compounds. The continuous transfer of semi-volatile compounds from tropical regions of the world to the colder poles is suspected for long-term effects on living beings (Carson R., 1962). Chlorinated hydrocarbons are detected in high concentrations in various products, because of their low rate of degradation. Wax is the beehive product more likely to be contaminated by organochlorine insecticides because of its strong lipophilic character. Moreover, OCPs were proved to remain stable during the conversion of old combs into new (Jimenez et al., 2005). The problem is magnified by the import of wax from continents where the use of chlorinated hydrocarbons is still permitted like Asia and Africa. The encouraging news is that the percentage of honey contaminated with chlorinated

hydrocarbons dropped from 96.1% to 52.3% during the decades 1980 and 1990.

This specific class of pesticides is of relatively high toxicity for humans and was first studied and used as an asphyxiating gas during the Second World War. Organophosphorus compounds are not stable in the environment and are not bio-concentrated and this is probably the main reason why they were detected rarely and at lower concentrations into beehive products. Most of published information concerns the compound methyl parathion, which has been used in agricultural crops as preparation in the slow-release form of microcapsules. Residues of this chemical were detected in honey and pollen (Atkins & Kellum, 1984). The microcapsules stick on the dense coat of bees, transferred into their hive and stored along with pollen. Pollen is the main component of the diet of larvae. The presence of polluted pollen might cause poisoning and eventually death to brood of bees. In a survey conducted on pollen from France, residues of parathion and methyl parathion were found in 1.2% and 4.9% of the samples, respectively. The average concentration for parathion was 0.019 mg kg-1 and for parathion methyl was 0.025 mg kg-1 (Chauzat et al., 2006). Blasco et al. (2003) detected only heptenophos in 4% of honey samples that were analyzed, out of 23 organophosphorus pesticides that they researched. Heptenophos concentrations in this survey ranged from 0.08 mg kg-1 to 0.23 mg kg-1. Finally, Balayiannis and Balayiannis (2008) detected the organophosphorus compounds chlorfenvinphos, chlorpyriphos and phorate in honey originated from Greece, in concentrations ranged from

Carbamate insecticides have a similar mode of action with organophosphates but their insecticidal activity is more selective and depends to a certain extent on the insect species. Some fungicides and herbicides belong to this family. These substances are highly volatile in the environment and in some cases they were detected in beehive products. Concentration of carbamate residues detected in pollen ranged from 0.126 mg kg-1 to 0.265 mg kg-1 for the active ingredient carbaryl, while the maximum concentration of carbofuran was 0.14 mg kg-1 (Chauzat et al., 2006). Concentration of carbaryl, carbofuran, pirimicarb and methiocarb residues, in most cases is considered low and does not exceed 0.071 mg kg-1. In only one Spanish honey the concentration of carbofuran was 0.645 mg kg-1 (Blasco et al., 2003).

**2.1.1.2 Organophosphorus pesticides (OPPs)** 

0.7 μg kg-1 to 0.89 μg kg-1. **2.1.1.3 Carbamate pesticides**  Nevertheless, the concentration of carbamate residues is low in pollen and honey, while no residues have been reported in other beehive products.

#### **2.1.1.4 Neonicotinoid pesticides**

Most studies reported on neonikotinoids insecticides, refer to the active ingredient imidacloprid. This substance was proved toxic to bees, but the concentrations of residues detected in honey were very low (0.001 mg kg-1 to 0.005 mg kg-1) (Bonmatin et al., 2003; Maus et al., 2003; Schmuck et al., 2001). In many cases residues did not exceed the limit of quantification (<0.002 mg kg-1) (Rogers & Kemp, 2003; Schöning & Schmuck, 2003; Stadler et al., 2003; Faucon et al., 2004). Detected residues of imidacloprid in pollen were 0.005 mg kg-1, while detection rate was 49.4% (Bonmatin et al., 2003). The hazard quotient (application rate in grams per hectare/LD50) of neonicotinoids is far below the trigger value of 50, but the most important is the chronic toxicity that they cause to bees. The long-term exposure to neonicotinoid after the behaviour of bees, reduce their reproduction capacity and lead of population decline. The low detectable concentrations in combination with the low toxicity of imidacloprid in humans are reassuring for consumer safety. On the contrary, the effects of imidacloprid residues on bees should be further explored. The high toxicity for bees makes neonicotinoid residues suspicious about the death of many forager bees collecting nectar from sunflower, corn and cotton crops. The implementation of neonicotinoid active substances in seed of plants like cotton, corn and sunflower led to a theory that this specific class of pesticides is responsible for the "colony collapse disorder" syndrome (CCD). The CCD is defined as the sudden depopulation of a beehive and the rapid collapse of the colony. The causes of this phenomenon are not clear yet. The suspicion is directed at the mite *Varroa destructor* Anderson & Trueman, while others blame the protozoan *Nosema ceranae*. Additionally, suspicion directed at poisoning of bees by neonicotinoid insecticides and at various forms of radiation (telephony, wireless networks etc.) as well. In fact, research on the toxicity of neonicotinoids to the bee, proved tolerance of the insect body at normal concentrations identified in honey, pollen and nectar (Schmuck et al., 2001; Faucon et al., 2004). More recent research is directed at the effect of imidaclorpid to the orientation of bees (Bortolotti et al.; 2003).

#### **2.1.1.5 Fungicides**

Fungicides are toxic substances that are used to kill or inhibit the growth of fungi that cause economic damage to crops and endanger the health of domestic animals or humans. Most fungicides are toxic to humans and can cause both acute and chronic problems if absorbed into food. Kubik et al. (1999; 2000) studied the possibility of contamination of beehive products with residues of chemicals used on apple and cherry trees. Vinclozolin, iprodione and thiophanate methyl residues were detected in honey and pollen collected from cherry flowers, while captan and difenuconazole were detected in beehive products collected from apple trees. Specifically, the average concentration levels of vinclozolin in honey were determined at 0.107 mg kg-1. A recent review of vinclozolin by the US Environmental Protection Agency has concluded that the chemical or its breakdown products are associated with the development of testicular tumors in rats. The mean concentrations of residues of other active compounds were 0.0006 mg kg-1, 0.009 mg kg-1, 0.023 mg kg-1 and 0.059 mg kg-1 for captan, difenuconazole, iprodione and methyl thiophanate respectively. In all cases, the concentration was lower in honey, than in stored pollen (Kubic, 2000).

Pesticide Residues in Bee Products 95

because of the lack of approval for beekeeping use. Therefore, in this case the limit

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

corresponds to the Limit of Quantification, which is 0.01 mg kg-1.

development of resistance of varroa to amitraz (Eljen et al., 2000).

that it also controls the small hive beetle *Aethina tumida* Marey.

the application of the substance into the hive (Wilhelmina, 1992).

cholinergic synapses of the central nervous system.

authorization for beekeeping use.

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

Action: Substance with systemic action that causes death in insects and mites by affecting

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

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

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 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

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.

**2.2.1.2 Coumaphos** 
