**7.1. Pesticides and antibiotics**

do Norte, and the Rio Grande do Sul) and showed that there was absence of coliforms in these samples [9, 61–63], that is, the harvesting, the management, and the processing of the samples

The detection of *Salmonella* spp. should be performed using the International Standard ISO 6579:2002 [64]. The genus *Salmonella* includes several pathogenic serotypes, which can cause from gastroenteritis (fever, diarrhea, and abdominal cramps) to serious systemic infections (enteric fevers), like *Salmonella* typhi that causes typhoid fever. However, gastroenteritis is the most common form of salmonellosis and the major mode of transmission is by means of contaminated food. Some studies showed that there was an absence of *Salmonella* species in

The enumeration of yeasts and molds, in its turn, is performed using the colony‐count technique according to ISO 21527‐2:2008, which specifies a method for the enumeration of viable xerophilic molds and osmophilic yeasts in products that have a water activity less than or equal to 0.95 [66]. Luiz et al. [65] evaluated Brazilian honey samples produced in several cities of the state of Minas Gerais (Southeast region), and the yeast and mold counts varied from

by Schlabitz et al. [63] with honey samples from state of Rio Grande do Sul (South region of Brazil), the majority of samples (10 samples) were within specifications, since the enumera-

produced in the state of Ceará (Northeast region of Brazil) were evaluated by Santos and Oliveira [61]. The authors showed that the majority of samples were within specifications,

CFU/g.

Although not required by Brazilian law, the detection of *Clostridium* spp. (spore‐forming bacteria) also is important, since honey samples may be contaminated with spores of *Clostridium botulinum*, the etiological agent of botulism (potentially fatal disease). While the ingestion of these spores is considered harmless to healthy adults, the spores may germinate in the gut of infants under 6 months of age, multiply and produce botulinum toxins. This would not occur in children older than about 6 months and adults due to natural defenses that develop over time [55]. Ragazani et al. [68] evaluated honey samples from different regions of Brazil (states of São Paulo, Minas Gerais, Goiás, Ceará, Mato Grosso, and Santa Catarina) and isolated *C. botulinum* from 7% of the samples. In other studies, Schlabitz et al. [63] and Luiz et al. [65] showed that there was an absence of sulfite‐reducing clostridia in Brazilian honey

and 6.1 x 10<sup>2</sup>

samples from states of Rio Grande do Sul and Minas Gerais, respectively.

**7. Contaminants analysis: metals, pesticides, analysis, and results**

Honey is traditionally consumed by humans for being considered a product of natural origin and healthy. However, honey and other bee products can also be a source of toxic substances,

CFU/g, that is, all samples were according to Brazilian law. In another study

CFU/g. However, two samples had

CFU/g. Only one sample had a

CFU/g, respectively. Several honey samples

to 8.0 x 10<sup>1</sup>

were made as recommended in order to obtain a good quality of honey.

Brazilian honey samples from different regions of Brazil [9, 61, 63, 65].

<10.0 to 3.3 x 10<sup>1</sup>

158 Honey Analysis

tion of yeasts and molds varied from <1.0 x 10<sup>1</sup>

count above 100 CFU/g, since it had 1.8 × 10<sup>2</sup>

since yeast and mold counts varied from < 10.0 to 6.0 x 10<sup>1</sup>

values above 100 CFU/g:1.3 x 10<sup>2</sup>

The presence of contaminants in bee products decreases its quality and it may carry serious health hazards, consequently, being a public health problem. Widely used in agricultural practices, pesticide residues have been shown to cause genetic mutations and cellular degradation and the presence of antibiotics might increase resistant human or animal's pathogen [71].

The pesticide residues may originate from the treatment of beehives with acaricides and organophosphorus pesticides (OPPs) in the control of Varroa jacobsoni and Ascosphaera apis. Indirect honey contamination can occur during pesticide application in agriculture also for wax moth and small hive beetle control. Pesticide application in crops can contaminate soil, air, water, and the flowers from which bees collect nectar for honey production [72, 73].

Another source of contamination are the antibiotics such as tetracyclines, streptomycin, sulfonamides, and chloramphenicol used for the treatment of bee disease, migration from wax to honey, and also of some infestations such as Varroa destructor, Acarapis wood, and Paenibacillus larvae [69, 72].

The determination of pesticide in food due to the low concentration, the distinct chemical properties, and the matrices complexity, requires sample preparation, purification, identification, and quantification of compounds. Therefore, honey is a complex matrix and this implies the need for effective clean‐up treatment before the analysis. Among the extraction methods commonly used in honey analysis are the typical clean‐up/extraction procedures, such as liquid‐liquid extraction (LLE) or solid‐phase extraction (SPE); however, they have the disadvantages of being expensive and using large amounts of organic solvents, which are generally toxic for the technician and can contaminate the environment and usually enable the extraction of analytes belonging to only one chemical class [32, 70]. Additionally, there are other extraction techniques, which have been employed to reduce a number of reagents and time spends on sample preparation, for example, supercritical fluid extraction (SFE), matrix solid phase dispersion (MSPD), solid phase microextraction (SPME), and stir bar sorptive extraction (SBSE). Besides the extraction and purification procedures, the choice of the separation/detection approach is of fundamental importance. The step of identification and quantification of pesticide residues in honey is based mainly on gas chromatography (GC) or high‐performance liquid chromatography (HPLC) techniques, both coupled with tandem mass spectrometric detection have shown great success in the multiresidue analysis of antibiotics and pesticides in honey [71].

Rissato et al. [74] confirmed 48 pesticides of different classes (organohalogen, organophosphorus, organonitrogen, and pyrethroids) in low levels in Brazilian honey samples (Bauru, São Paulo, Brazil) by gas chromatography‐mass spectrometry (GC‐MS/MS). Nevertheless, malathion residues were detected in all the samples, in a high concentration, and it was attributed to pesticide application for dengue vector control in the area. A study realized by De Pinho et al. [73] showed that of the 11 honey samples from eight regions of the state of Minas Gerais (Brazil) analyzed only two presented chlorpyrifos and k‐cyhalothrin residues using liquid‐ liquid extraction with low‐temperature purification for pesticide residue analysis by gas chromatography. However, the concentrations obtained were below the maximum residue levels (MRLs) established for pesticides in foods products. The presence of these compounds was confirmed by mass spectrometry (GC‐MS).

Additionally, Orso et al. [75] developed and validated a method for the simultaneous determination of 79 pesticides and 13 antibiotics for 43 honey samples from different regions of Rio Grande do Sul State, Brazil, among them are monofloral and multifloral honey. The pesticides and antibiotic residues were extracted using a water‐acetonitrile followed by a cleanup with dispersive solid phase (d‐SPE) and analyzed by UHPLC‐MS/MS. The results of the analysis demonstrated that 50% of the samples presented residues of one or more analytes in the samples. The maximum residue limit was not exceeded in any sample. Residues of insecticides and acaricides, fungicides, antimicrobials, and herbicide were found at concentrations below the MRLs, according to the limits established by National Program for Honey Residues Control established by the Brazilian Ministry of Agriculture (Brazil) for honey. Second, the authors, the residues found in honey samples are due to the proximity of the beehives with soybean, corn, or wheat crops, considering that bee realizes the pollination process, reaching large distances to collect nectar, water, and pollen of flowers.

#### **7.2. Heavy metals**

The bees are exposed to metals contained in pollen or nectar, it can to accumulate them and finally into the honey produced from it [76]. A number of different minerals and heavy metals in honey are largely dependent on the soil composition, as well as various types of floral plants [77]. Additionally, metal pollutants are discharged into the air, water, and soil through mining, agriculture practice, waste dump, coal burning, hydraulic fracturing to extract gas and oil, and industrial and municipal waste production. Agroecosystems fertilized with manures and biosolids can become contaminated with metals, and repeated fungicide application can cause the buildup of metals [78].

Trace metals such as sodium, potassium, calcium, iron, zinc, and copper can be considered essential for the biological metabolism of living organisms, when present in optimum concentrations are helpful. Other metals such as lead, cadmium, mercury, and aluminum are classified as microcontaminants of the environment, toxic or nonessential to living organisms, and at high concentrations can be even lethal, due to the inability of the heavy metal to be metabolized by the body, leading to accumulation in human or animal soft tissues without being fully inactivated or destroyed [77, 79]. In addition, the problems caused by heavy metals include headaches, metabolic abnormalities, respiratory disorders, nausea, vomiting, damage to the brain, kidney, nervous system, and red blood cells [77].

mass spectrometric detection have shown great success in the multiresidue analysis of antibi-

Rissato et al. [74] confirmed 48 pesticides of different classes (organohalogen, organophosphorus, organonitrogen, and pyrethroids) in low levels in Brazilian honey samples (Bauru, São Paulo, Brazil) by gas chromatography‐mass spectrometry (GC‐MS/MS). Nevertheless, malathion residues were detected in all the samples, in a high concentration, and it was attributed to pesticide application for dengue vector control in the area. A study realized by De Pinho et al. [73] showed that of the 11 honey samples from eight regions of the state of Minas Gerais (Brazil) analyzed only two presented chlorpyrifos and k‐cyhalothrin residues using liquid‐ liquid extraction with low‐temperature purification for pesticide residue analysis by gas chromatography. However, the concentrations obtained were below the maximum residue levels (MRLs) established for pesticides in foods products. The presence of these compounds was

Additionally, Orso et al. [75] developed and validated a method for the simultaneous determination of 79 pesticides and 13 antibiotics for 43 honey samples from different regions of Rio Grande do Sul State, Brazil, among them are monofloral and multifloral honey. The pesticides and antibiotic residues were extracted using a water‐acetonitrile followed by a cleanup with dispersive solid phase (d‐SPE) and analyzed by UHPLC‐MS/MS. The results of the analysis demonstrated that 50% of the samples presented residues of one or more analytes in the samples. The maximum residue limit was not exceeded in any sample. Residues of insecticides and acaricides, fungicides, antimicrobials, and herbicide were found at concentrations below the MRLs, according to the limits established by National Program for Honey Residues Control established by the Brazilian Ministry of Agriculture (Brazil) for honey. Second, the authors, the residues found in honey samples are due to the proximity of the beehives with soybean, corn, or wheat crops, considering that bee realizes the pollination process, reaching large distances to collect nectar, water,

The bees are exposed to metals contained in pollen or nectar, it can to accumulate them and finally into the honey produced from it [76]. A number of different minerals and heavy metals in honey are largely dependent on the soil composition, as well as various types of floral plants [77]. Additionally, metal pollutants are discharged into the air, water, and soil through mining, agriculture practice, waste dump, coal burning, hydraulic fracturing to extract gas and oil, and industrial and municipal waste production. Agroecosystems fertilized with manures and biosolids can become contaminated with metals, and repeated fungicide appli-

Trace metals such as sodium, potassium, calcium, iron, zinc, and copper can be considered essential for the biological metabolism of living organisms, when present in optimum concentrations are helpful. Other metals such as lead, cadmium, mercury, and aluminum are classified as microcontaminants of the environment, toxic or nonessential to living organisms,

otics and pesticides in honey [71].

160 Honey Analysis

confirmed by mass spectrometry (GC‐MS).

cation can cause the buildup of metals [78].

and pollen of flowers.

**7.2. Heavy metals**

The methods used to determine the chemical elements in honey are based on spectroscopy or spectrometry techniques (including flame emission photometry or spectrometry (FES), inductively coupled plasma optical emission spectrometry (ICP‐OES), inductively coupled plasma mass spectrometry (ICP‐MS), flame atomic absorption spectrometry (FAAS), electrothermal atomic absorption spectrometry (ET‐AAS), graphite furnace atomic absorption spectrometry (GF‐AAS), hydride generation‐atomic fluorescence spectrometry (HG‐AAS), ion chromatography EDTA titration) [77].

De Andrade et al. [80] determined the trace elements, Pb, Cd, and Cr in 52 honey samples from eight different regions from the state of Paraná (Brazil), using slurry sampling and graphite furnace electrothermal atomic absorption spectrometry. The mean concentration of the elements followed the order Pb > Cr > Cd, but the study concluded that honey samples from Paraná have food security, as regular consumption of this product does not put risks to human health in terms of intake of this metallic species. Furthermore, Batista et al. [81] determined 42 chemical elements (toxic and essential elements) in Brazilian honey samples collected in different cities of Brazil (poli, orange, and sugarcane flowers) by the inductively coupled plasma mass spectrometry method. The authors observed that in general Brazilian honey presented higher mean concentrations for Ni, Mg, and Al and lower mean concentrations of Pb, Cd, and Cu. The mean values found for P, Zn, Mn, and Fe were very similar to those found in honey samples from other countries.

Thus, the presence of pesticides and antibiotic residues and trace metals in honey is of interest for quality control and also as a bioindicator of environmental contamination. Therefore, these analyses are important to determine the nutritional value and also the potential effect of honey on human well‐being, and they can be called upon to ensure the general safety and purity of honey.
