# Honey as a Bioindicator: Pollution's Effects on Its Quality in Mining vs. Protected Sites
## 2 Materials and Methods
### 2.2.1. Phyxico-Chemical Analyses
#### 2.2.7 Heavy Metals (Pb, Cd, Fe, Cu, and Zn)  
A quantity of 10 g of each honey sample was accurately weighed and transferred into a porcelain crucible. The crucible was placed in a furnace at an initial temperature not exceeding 100 \({}^{\circ}\)C. The temperature was then increased at a maximum rate of 50 \({}^{\circ}\)C/hour until it reached 450 \({}^{\circ}\)C, where it was maintained for 24 h. After cooling, 5 mL of 6 M HCI was added to the crucible containing the ash to ensure full contact with the acid. The acid evaporated on a sand bath. The residue was dissolved in an exactly measured volume (10-25 mL) of 0.1 M HNO\({}_{3}\). A blank was prepared following the same procedure. The crucible was rotated to ensure that the entire residue encountered the acid, then covered and left to stand for 2 h. The solution was mixed and filtered into a plastic container. From the resulting mineralized solution, the metal content was determined. To calibrate the instrument, working standard solutions were prepared by diluting 1000 mg/L stock solutions for each element. The concentrations ranged from 0.5 to 5.0 \(\upmu\)g/L for Cd; 1.0 to 10.0 \(\upmu\)g/L for Pb; and 1.0 to 20.0 \(\upmu\)g/L for Cu, Zn, and Fe, depending on the linear range of each element. The standard solutions were prepared in the same matrix as the samples (0.1 M nitric acid) to ensure the comparability of the results. The solutions were analyzed by AAS using a Perkin Elmer AA700 spectrometer equipped with a graphite furnace HGA-700 and an AS 800 Autosampler (PerkinElmer, Waltham, MA, USA). The sample volume was automatically injected into the center of the graphite tube, which was then gradually heated using an electric current source. To prevent sample loss, the temperature was increased gradually according to a three-step program: 1. Drying the sample at a low temperature (100-200 \({}^{\circ}\)C) for a few seconds. 2. Pyrosis of the organic components at 500-1400 \({}^{\circ}\)C. 3. Atomization of the sample at 2500 \({}^{\circ}\)C. The absorption of light radiation by the metal atoms in the graphite tube cavity was measured. A narrow, tall, well-defined peak was recorded as the absorbance versus time. The measurements were made at element-specific wavelengths: Pb: 283.3 nm; Cd: 248.3 nm; Fe: 248.3 nm; Cu: 324.8 nm; and Zn: 213.9 nm. For each determination, a Zeeman background correction was used, along with the specific matrix modifiers NH\({}_{4}\)H\({}_{2}\)PO\({}_{4}\) and Mg(NO\({}_{3}\))\({}_{2}\)\(\cdot\)6H\({}_{2}\)O. A volume of 20 \(\upmu\)L from each sample was injected, comprising the standard solution, matrix modifier, blank, and sample. The calibration solutions were automatically prepared by the instrument, which performed the dilutions based on the programmed method.  
A minimum calibration curve linearity coefficient (R\({}^{2}\)) of 0.995 was required. All the measurements were performed in duplicate. The concentrations obtained were placed on the calibration curve, and the results were expressed in mg/kg, using the following formula--C [mg/kg] = (C\({}_{\mathrm{curve}}\) [mg/L] \(\times\) V\({}_{\mathrm{digestion}}\) [mL])/m\({}_{\mathrm{honey}}\) [g]--where C\({}_{\mathrm{curve}}\) is the concentration read from the instrument, V\({}_{\mathrm{digestion}}\) is the final volume after dilution, and m\({}_{\mathrm{honey}}\) is the mass of the honey sample. For concentrations exceeding the calibration range, the mineralized sample was diluted accordingly, and the final concentration was corrected using the appropriate dilution factor [42].