Cien. Inv. Agr. 33(1): 50-58. 2006 www.riar.puc.

cl
RESEARCH PAPER

Heavy metals and other trace elements contents in Chilean honey1

C. Fredes2 and G. Montenegro
Departamento de Ciencias Vegetales, Facultad de Agronoma e Ingeniera Forestal Pontificia Universidad Catlica de Chile Casilla 306-22, Santiago, Chile Abstract
C. Fredes and G. Montenegro. 2006. Heavy metal and other trace elements contents in Chilean

honey. Cien. Inv. Agr. (in English) 33(1):50-58. Forty seven honeys collected between IV and X Administrative Region of Chile, were studied to determine the presence of the following 11 trace elements: Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Sr and Zn. Two different methods based on different types of organic matter decomposition procedures were used. The first method consisted of acid digestion and the second one of a high temperature dry oxidation. Element determination was performed by inductively coupled plasma optical emission spectrometry (ICP-OES). In spite of the fact that both methods detected the 11 trace elements, the second method had more resolution than the first one, with the exception of Cd, Pb, Ni and Zn. The elements with the highest frequency were Al, Fe, Mn, and Zn. The high Al content was found to be related to beehives located in trumaos soils in Southern Chile and also with honeys stored in aluminum containers. On the other hand the high Fe, Mn and Zn contents were found to be related to hualputra (Lotus uliginosus) honeys and with multiflora honeys collected in the V and X Administrative Regions of Chile. Cadmium and Pb, the two most dangerous heavy metals for human health were found in 14.9% and 34.0% respectively of the studied samples. Their maximum residues limit was below to the European Standards.

Key words: Al, electrical conductivity, heavy metals, honey, ICP-OES, pH, Sr.

Introduction The current international honey market trend, regarding quality is every day more demanding. Hence, it is necessary to promote all feasible activities in order to produce residue free honey (McKee, 2003). The detection of compounds, not previously found in honey imports by the European Union has generated increased control of residues that might be present in honeys (Danty, 2003). Consequently, National Beekeeping Network together with Pontificia Universidad Catlica de Chile and the Instituto

Nacional de Normalizacin (National Institute of the Norm) are working to update the standards that can guarantee the origin and safety of Chilean honey. Since honey is a nutritional resource that depends on biotic and abiotic factors around the beehives, the presence of heavy metals could be related to its geographical and botanical origin. Several authors have indicated that bees and their products may be used as biological indicators of the environmental pollution present in the area where they fly (Fernndez et al. 1994; Sanna et al., 2000; Buldini et al., 2001; Bogdanov et al., 2003; Porrini et al., 2003; Celli and Maccagnani, 2003). However, Jones (1987)

Received 28 January 2005; Accepted 14 October 2005 1 This work is part of the MS thesis presented at the FAIF PUC as part of the requests for the Masters Degree in Plant Sciences. 2 Corresponding author: cpfredes@uc.cl

VOL 33 No1 JANUARY - APRIL 2006.

51

indicates that there is no correlation between the content of elements such as Ag, Cd and Pb present in the soil and in the honey. Furthermore, the exact way in which honey reflects the mineral components of, plants visited by bees, soil, or atmosphere is unknown. Additionally, honey is not the most sensitive tool to evaluate environmental contamination with heavy metals, due to the low concentration present in this product and the great variability caused by factors like origin source, floral density, season of the year and rainfall, among others. Therefore it is important to take in account the type of equipment used to produce honey as well as the quality of the equipment used to store honey after harvesting as possible sources of honey contamination with heavy metals. Contact with stainless steel surfaces during harvesting, processing and/or preparation of honey for the market, can generate high Cr content, due to the corrosive effect of honey acidity. Likewise, storing honey in galvanized containers can be a source of Zn contamination (Gonzlez et al., 2000; Bogdanov et al., 2003). The international information about the usefulness of honey as a geographical and botanical bioindicator of contamination with metals is controversial and limited. In Chile, there is not an official method to detect trace elements in honey and thus the information regarding metal content in Chilean honey is lacking. The aim of the present work were a) to determine the amount of heavy metals, b) to determine pH, and electric conductivity (E.C) of honey samples of known botanical origin, collected between the IV and X Administrative Region of Chile, by two analytical methods of determination. Materials and Methods Geographical and botanical honey characterization Honey samples obtained from the IV and the X Administrative Region of Chile of known botanical origin have been widely studied (Montenegro et al., 1989; Montenegro et al. 1992; Montenegro et al., 2002; Montenegro et

al., 2003). Samples for this work were taken from storage containers of harvested honey. The original samples were collected between 2001 and 2003. The botanical origin of the honey was obtained by melisso-palynologycal analysis, using Mauricios method (1975) with 95% confidence level. The geographical origin was assigned by the Administrative Region from where the beehives were located as indicated in Table 1. Trace element determination Two analytical methods were evaluated for the analysis of the honey samples. Method 1: Acid digestion (AD). Samples were prepared according to 920,180 method of the A.O.A.C (2000b). Five mL of 75% HNO3 were added to each 2 g sample of honey within a porcelain crucible. Then, the acid was evaporated on an electrically heated metal sheet at 100-120 C, then it was gauged to 10 mL with distilled water (dilution 1:5). Method 2: High temperature dry oxidation (HTDO). This method was adapted from 985,01 method of the A.O.A.C (2000a). Samples were prepared according to 920,180 method of the A.O.A.C (2000b). Each 5 g honey sample was dried in a porcelain crucible at 100 C to dry weight. Then the samples were heated to 600 C for 16 h (adapted from 920,181 method of the A.O.A.C (2000b). Three mL of HNO3 were added, followed by acid evaporation on an electrically heated metal sheet at 100-120 C. Then, 5 mL of HCl were added and gauged to 10 mL with distilled water (dilution 1:2). Triplicate samples were analyzed. Two blanks were made for each determination. A commercial honey sample, of a precisely known composition was used as control for all analysis procedures. For each honey sample of Al, Cd, Co, Cu, Cr, Sr, Fe, Mn, Ni, Pb and Zn concentrations were determined. A VARIAN (Liberty RL Sequential ICP-OES) inductively coupled plasma optical emission spectrometer was used.

52

CIENCIA E INVESTIGACION AGRARIA

Table 1. Sample number, botanical and geographical origin of the 47 honey samples analyzed. Samples collected between IV and X Administrative Regions during 2001 to 2003. N Botanical origin Geographical origin Place 23 26 28 32 33 7 8 10 11 12 16 17 71 91 92 93 21 22 35 76 74 47 52 60 174 223 237 81 84 85 87 156 157 181 182 94 97 99 100 113 118 119 120 121 140 150 153 Multiflora Villaseca Multiflora Las Breas Multiflora El Bolsico Multiflora Valle del Limar Multiflora Pedregal Siete camisas Putaendo Multiflora La Vega Siete camisas Las Palmas Quillay Colliguay Multiflora Chincolco Multiflora Panquehue Quillay Longotoma Multiflora Peralillo Multiflora Manquehua Multiflora Manquehua Multiflora Matancilla Multiflora Curic Multiflora San Clemente Multiflora Curepto Multiflora Santa Brbara Multiflora Santa Brbara Hualputra Hualapulli Hualputra Villarrica Multiflora Afunalhue Multiflora Gorbea Multiflora Malloco Multiflora Hualapulli Multiflora Raln Ulmo Chaitn Ulmo Multiflora Puyehue Hualputra Ulmo Multiflora Pumillahue Ulmo Patagonia Multiflora Melipilla Multiflora Multiflora Alhue Multiflora Quincanque Multiflora Monte Las Mercedes Multiflora Multiflora Melipilla Quillay Melipilla Quillay Melipilla Multiflora Alhue Multiflora Melipilla Quillay Quelentaro Alto Administrative Region IV IV IV IV IV V V V V V V V VI VI VI VI VII VII VII VIII VIII IX IX IX IX IX IX X X X X X X X X RM RM RM RM RM RM RM RM RM RM RM RM

Measurement wavelengths were: Al (237.312 nm), Cd (228.802 nm), Co (238.892 nm), Cu (224.700 nm), Cr (206.149 nm), Sr (407.771 nm), Fe (259.940 nm), Mn (257.610 nm), Ni (221.647 nm), Pb (220.353 nm) and Zn (206.200 nm). For the calibration curve of the equipment, a standard ICP multi-element MERCK IV solution with 1000 mgL-1: Ag, Al, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, In, K, Li, Mg, Mn, Na, Ni, Pb, Sr, Tl, Zn was used. The concentrations employed to construct the standard curve were 1, 2, 3, 4, and 5 mgL-1. Detection limits for each method (AD and HTDO) and elements in ugg-1 were: Al 0.083 and 0.001, Cd 0.003 and 0.002, Co below the detection limit (BDL) and 0.0001, Cu BDL and 0.015, Cr BDL and 0.005, Sr 0.003 and BDL; Fe 0.016 and 0.008, Mn 0.01 and 0.0003, Ni 0.01 and 0.01, Pb BDL and BDL and Zn 0.007 and BDL. E.C and pH determination These variables were determined using the 962,19 method A.O.A.C (2.000b) where 10 g of honey were dissolved in 75 mL of distilled water. Ash determination This variable was calculated adapting 920,181 method A.O.A.C (2000b), increasing combustion time to 16 h. Impurities determination Each 10 g sample of honey was dissolved in 75 mL of distilled water and kept in an agitator at 5,000 rpm during 15 min. Then the solution was passed through a Whatman N 1 filter paper and dried at 50 C. Data analysis For comparison between methods, a simple lineal regression was performed between the elements obtained using method 1 (AD) and method 2 (HTDO). The gradient and intercept were established for each regression equation, and it was statistically demonstrated if these were equivalent to one and cero, respectively (null hypothesis). A 95% confidence level was used.

VOL 33 No1 JANUARY - APRIL 2006.

53

To reduce the size of the data matrix obtained, multivariate statistical analysis such as Principal Components Analysis (PCA) was performed. SAS and NTsys software were used. Results and discussion Trace elements in honey The number of positive responses, defined as concentrations higher than the different detection limits (Devillers et al., 2002), for each trace element and method of determination in the 47 honey samples analyzed are showed in Table 2.

Table 2. Positive responses in a total of 47 honey samples, according to the analytical acid digestion (AD) method and high temperature dry oxidation (HTDO) method for 11 detected elements. Samples collected between IV and X Administrative Regions during 2001 to 2003. Element Al Cd Co Cr Cu Fe Mn Ni Pb Sr Zn Positive responses ( N) AD method 45 7 9 14 11 46 41 26 16 31 44 HTDO method 47 1 29 21 47 47 47 20 1 46 38

Table 3. Concentration mean, standard deviation and range for 11 trace elements analyzed by acid digestion (AD) method -1 and high temperature dry oxidation (HTDO) method in mg kg (wet wt). Samples collected between IV and X Administrative Regions during 2001 to 2003.
Element Mean Min Max Mean Min Max Cd Co Cr Cu Fe Mn Ni Pb Sr Al Zn AD method 0.010.01 0.050.13 0.070.29 0.080.30 1.451.36 0.530.63 0.170.26 0.020.03 2,395,26 1.942.84 0.661.01 0.01 0.05 0.01 0.03 0.60 0.03 1.92 0.06 2.00 0.10 6.36 0.01 3.14 0.01 1.04 0.01 0.11 0.10 0,01 23,06 0.25 14.25 0.01 4.73

HTDO method 0.090.17 0.070.30 0.910.66 3.131.44 1.261.29 0.170.36 0.01 0.62 0.01 1.98 0.09 4.32 0.78 7.66 0.02 6.97 0.01 1.48 2.365.33 4.536.15 1.111.13 0.04 22.06 0.04 22.06 0.19 4.93

- non detected

Table 3 indicates the main trace elements found in the honey samples analyzed: Al, Fe, Mn and Zn. There were differences in the number of positive responses between methods 1 and 2 (Table 2). These differences can be explained due to the fact of greater concentration of honey in the samples analyzed with Method 2 (dilution 1:2) versus Method 1 (dilution 1:5). This fact could allow the detection of lower concentrations of elements in the honey using Method 2, except for Cd, Pb, Ni and Zn. For instance when using method 1 we found that Cd y Pb were in 14.9 and 34.0%, respectively of the studied samples. However, when using method 2 the percentage for both elements was 2.1%. This may be due to losses experienced by these elements through volatilization during drying and ash production when using Method 2 (Case and Jones, 1990). The decrease of positive responses for Ni and Zn,

were due to heterogeneity that was seen in the resulting ashes, implying an incomplete destruction of organic matter which would prevent a complete recovery of the elements from the resulting matrix. Hence, this method requires further and critical analysis of the procedure using control samples. USDA database (2005) of nutrients, reports characteristic levels for some trace elements in honey like Fe (0.42 mgkg-1), Mn (0.8 mgkg-1), Zn (2.2 mgkg-1) and Cu (0.36 mgkg-1). pH, E.C and impurities in honey Table 4 shows pH, electrical conductivity and impurities percentage of the 47 honey samples analyzed. E.C was considerably lower than that established by the Codex Alimentarius (< 0.8 mScm -1 ) and the pH was within the accepted range for honey according to the same standards (3.5 5.5).

54

CIENCIA E INVESTIGACION AGRARIA

Table 4. Electrical conductivity, pH and impurities percentage for 47 honey samples collected between IV and X Administrative Regions during 2001 to 2003. Variable Mean Min Max E.C (mScm-1) 0.380.16 0.11 0.97 pH 4.230.29 3.79 5.08 Impurities (%) 3.311.67 1.94 11.15

Impurities percentage was higher than the established standards (<0,1%), which could be due to the presence of wax in the honey samples analyzed, which were taken directly from the storage metal barrels and did not undergo any filtering process. It was found that samples that had high ash percentages manifested the highest electrical

conductivity with a logarithmic increase as shown in Figure 1. On the other hand, there was no clear relationship between electrical conductivity and total heavy metal content and other trace elements measured. E.C is related to the presence of soluble ions and probably heavy metals and trace elements that are chelated with organic compounds of the honeys can not conduct electricity. Therefore, they would not necessarily have a direct relation with E.C, since they change the proportions of metalligand. The relationship between ash percentage and electrical conductivity are important because they permit an indirect estimate of mineral content. E.C measurement will replace, in the short term, ash content as a certified parameter for honey quality (Bogdanov, 1997).

0.45 0.40 0.35 0.30

CE (mScm-1)

0.25 0.20 0.15 0.10 0.05 0.00 0.00 y = 0.1439Ln(x) + 0.5944 R2 = 0.8972

0.05

0.10

0.15

0.20

0.25

0.30

Ash (%)

Figure 1. Regression equation between electrical conductivity (E.C) and ash percentage of honeys analyzed. Samples collected between IV and X Administrative Regions during 2001 to 2003.

Methods regression. Regression equations (y = ax+b) for Method 1 (AD) and the Method 2 (HTDO) for the elements analyzed are showed in Table 5, where Cr had a well adjusted regression curve. The regression curves for the other elements analyzed did not have good adjustments, possibly due to interference of masses in the resultant matrixes in both methods and to the heterogeneity of the honey samples. Principal components analysis. The multivariable analysis performed from the results of Method

1 (AD), indicated 11 principal components that correspond to the 11 variables analyzed, which explain the 100% data variability, on which were based the maps of Figure 2 and 3. Distribution along PC1 would be mainly dependent on the concentrations of Fe, Mn, Cu and Zn, considered essential elements or micronutrients for superior plants (Adriano, 2001), while distribution along PC2 would be mainly dependent on Cd and Pb concentrations,

VOL 33 No1 JANUARY - APRIL 2006.

55

considered non essential elements for plants (Gonzlez, 1994; Adriano, 2001).
Table 5. Regression equation (y = ax+b) between acid digestion (AD) method and high temperature dry oxidation (HTDO) method for 47 honey samples collected between IV and X Administrative Regions during 2001 to 2003. Element Al Co Cr Cu Fe Mn Ni Sr Zn Regression and determination coefficient y = 2.0807x + 0.484 y = 0.9142x + 0.0475 y = 1.0311x + 0.0007 y = 1.6787x + 0.7793 y = 0.8064x + 1.9592 y = 1.6618x + 0.3906 y = 0.7885x + 0.0365 y = 0.8273x + 0.378 y = 0.9572x + 0.4809 R =0.92 2 R =0.45 2 R =0.99 2 R =0.59 2 R =0.58 2 R =0.67 2 R =0.34 2 R =0.66 2 R =0.74
2

The factorial map PC1-PC2 explains 43.7% of total data variability (28.6% and 15.1%, respectively). A main honey group close to the axis of interception of both components, which is characterized by a low content of heavy metals and trace elements is showed in Figure 2. Honey

samples 21, 81, 35 and 84, had the highest Cd content (0.05 mgkg-1 for honey samples 81 and 84) and Pb content (0.1 mgkg-1 for honey samples 81 and 35). These honey samples come from beehives that are close to roads and highways. Regarding honey samples with high micronutrient content seen along the PC1 gradient, honey sample 223 had the highest Mn content (3.1 mgkg-1); sample 8, the highest Cu and Zn contents (2.0 and 4.7 mg kg-1, respectively). In spite of honey sample 150 had the highest Fe (6,4 mgkg-1) content, it appears closer to the interception axis of both components, due to the lowest Mn and Zn contents (0.8 and 1.2 mgkg-1) and the non Cu detection. Regarding the botanical origin of this honey group, hualputra specie (uniflora honey 47 and 52) prevails, which could be considered a wetland plant, characterized by growing in soil with sediment and having important Zn accumulations (Schonoor, 1997). The rest of the samples correspond to multiflora honeys (8, 223, 16 and 174), with important participation of herbaceous plants. With respect to geographical origin, these six honey samples were collected in the V (8 and 16) and X Administrative Regions (47, 52, 223, and 174).

Figure 2. Factorial map PC1-PC2 for 47 honeys analyzed (numbers indicate the number of each honey sample). Samples collected between IV and X Administrative Regions during 2001 to 2003. PC: Principal component.

To complete the results analysis, a second factorial PC3-PC5 map was made (Figure 3). Distribution along PC3 would be mainly dependent on the Ni, Al, and Cr concentrations, which could be related to the type of storage container used after honey harvest. Distribution

along PC5 would be mainly dependent on the Sr concentrations, a trace element found in most honey samples analyzed. In this work, this element was found in higher concentration than those concentrations detected in a Spanish research (Terrab et al., 2004). The PC3-PC5

56

CIENCIA E INVESTIGACION AGRARIA

factorial map explains 24.3% of the total data variability (14.3% and 9.4%, respectively). A honey group close to the interception axis of PC3 and PC5 that were characterized by honeys having low Ni, Al, Cr and Sr content is showed in Figure 3. Honey 74 had the greatest Al content (14.3 mg kg-1), higher than those described for France (Devillers et al., 2002). Similar results high Al concentrations- were found in honey samples 47, 76 and 52 with contents of 9.1, 14.3, and 9.1 mgkg-1, respectively. These honey samples were obtained from the center-south region of Chile (VIII to X Administrative Regions), that has trumaos soil rich in Al with soft textures whose particles could deposit on

flowers or reach the beehives. This could explain the presence of this element in the honey, but the higher content could be due to the use of aluminum containers. Furthermore these honey samples had lower pH values (4.2) than the pH mean found in this study. Honey 74 and 76 had the lowest pH than the other samples analyzed (3.8 and 3.9 respectively). Even though to date no maximum limits for Al has been established internationally, it would be important to report this type of information to the beekeepers, in order to handle better the possible sources of contamination with this element, since it is possible relation with Alzheimers disease (Flaten, 2001).

Figure 3. Factorial map PC3-PC5 for 47 honeys analyzed (numbers indicate the number of each honey sample). Samples collected between IV and X Administrative Regions during 2001 to 2003. PC: Principal component.

In conclusion, Cd and Pb levels in the 47 uniflora and multiflora honey samples collected between the IV and X Administrative Region of Chile were found to be below the maximum values allowed according to the current European standards. The acid digestion (AD) method should be preferred for Cd, Ni, Pb and Zn, while for the other elements the high temperature dry oxidation (HTDO) method would be better. It would be interesting to analyze a larger number of samples and continue performing this type of research analyzing the metal content in different parts of reproductive structures of

plants that use beehoneys for pollination, in known contaminated areas. Resumen Para la determinacin de 11 elementos traza (Al, Cd, Co, Cu, Cr, Sr, Fe, Mn, Ni, Pb y Zn) en 47 mieles colectadas entre la IV y X Regin de Chile se adaptaron y evaluaron dos mtodos de determinacin basados en diferentes tipos de destruccin de la materia orgnica: digestin hmeda cida y oxidacin seca a alta temperatura. La determinacin de los elementos, para ambos mtodos de anlisis, se hizo mediante espectrometra de emisin ptica de plasma inductivamente acoplado (ICP-OES).

VOL 33 No1 JANUARY - APRIL 2006.

57

Si bien ambos mtodos permitieron la determinacin de los 11 elementos, el mtodo 2 present mayor sensibilidad, salvo para el Cd, Pb, Ni y Zn. Los elementos que se encontraron con mayor frecuencia en las mieles analizadas fueron Al, Fe, Mn y Zn. Los mayores contenidos de Al se asociaron a mieles provenientes de colmenares cercanos a suelos trumaos del sur de Chile y al almacenaje de la miel en envases de Al. Los mayores contenidos de Fe, Mn y Zn se presentaron en mieles provenientes de hualputra (Lotus uliginosus) y poliflorales de la V y X regin. Cadmio y Pb, considerados como los metales pesados con mayor impacto sobre la salud humana, se encontraron en un 14,9% y 34,0% de las muestras analizados y bajo los lmites mximos permitidos de acuerdo a los estndares europeos. Palabras clave: Al, conductividad elctrica, ICP-OES, Metales pesados, mieles, pH, Sr. Acknowledgements Projects FONDEF D03-1045 and FONDECYT 1060535 and beekeepers of National Beekeeping Network Inc.
References Adriano D.C. 2001. Trace Elements in Terrestrial Environments. Biogeochemistry, Bioavailability and Risk of Metals. Second Edition. SpringerVerlag, N.Y., USA. 867 pp. AOAC Official Methods of Analysis. 2000a. Plants. In: Horwitz, W. (ed.). Official Methods of Analysis of AOAC International. Maryland, USA. 1(3): 4-5. AOAC Official Methods of Analysis. 2000b. Sugars and sugar products. In: Horwitz, W. (ed.). Official Methods of Analysis of AOAC International. Maryland, USA. 2(44): 22-33. Bogdanov, S. 1997. Harmonized Methods of the International Honey Comission. Swiss Bee Research Centre. Bern, Switzerland. 61 pp. Bogdanov, S., A. Imdorf, J. Charriere, P. Fluri, and V. Kilchenmann. 2003. The contaminants of the bee colony. Swiss Bee Research Centre. Bern, Switzerland. 12 pp. Buldini, P.L., S. Cavalli, A. Mevoli, and J.L. Sharma. 2001. Ion chromatographic and voltammetric determination of heavy and transition metals in honey. Food Chemistry 73: 487-495.

Case, W., and J. Jones. 1990. Sampling, handling and analyzing plant tissue samples. p. 389-427. In: Westerman, R.L (ed.) Soil Testing and Plant Analysis. Third Edition. Soil Science Society of America, Inc. Madison Wisconsin, USA. Celli, G., and B. Maccagnani. 2003. Honey bees as biondicators of environmental pulltion. Bulletin of Insectology 56: 137-139. Danty, J. 2003. El mercado de la miel. Oficina de Estudios y Polticas Agrarias (ODEPA), Gobierno de Chile, Santiago, Chile. 11 pp. Devillers, J., J. Dore, M. Marenco, F. Poirier-Duchene, N. Galand, and C. Viel. 2002. Chemometrical analysis of 18 metallic and nonmetallic elements found in honey sold in France. J. Agric. Food. Chem. 50: 5998-6007. Fernndez, M.C., E. Subr y A. Ortiz. 1994. La miel, indicador ambiental. Prcticas ecolgicas para una agricultura de calidad. I Congreso de la Sociedad Espaola de Agricultura Ecolgica (Proceedings). Toledo, Espaa. p. 37-46. Flaten, T.P. 2001. Aluminium as a risk factor in Alzheimers disease, with emphasis on drinking water. Brain Res. Bull. 55: 187-196. Gonzlez, S. 1994. Geoqumica de metales pesados en Chile. VI Simposio sobre contaminacin ambiental. Universidad de Chile. Santiago, Chile. p. 10-44. Gonzlez, A., J. Gmez, R. Garca-Villanova, T. Rivas, R. Ardanuy, and J. Snchez. 2000. Geographical discrimination of honeys by using mineral composition and common chemical quality parameters. Journal of the Science of Food and Agriculture 80: 157-165. Jones, K. C. 1987. Honey as an indicator of heavy metal contamination. Water, Air and Soil Pollution 33: 179-189. Montenegro G., M. Schuck, A.M. Mujica y S. Teillier. 1989. Flora utilizada por abejas melferas (Apis mellifera) como fuente de polen en Paine, Regin Metropolitana, Chile. Ciencia e Investigacin Agraria16:47-53. Montenegro G., M. Gmez y G. Avila. 1992. Importancia relativa de especies cuyo polen es utilizado por Apis mellifera en el rea de la Reserva Nacional Los Ruiles, VII Regin de Chile. Acta Botnica Malacitana.17:167-174. Montenegro G, R. Pizarro, G. vila, M. Gmez, F. Bas, L. Olivares, M. Villena, G. Rizzardini, C. Ros, L. Gonzlez y A.M. Mujica. 2002. Certificacin de origen botnico de las mieles chilenas. VIII Congreso Latinoamericano y II Congreso Colombiano de Botnica. Cartagena de Indias, Colombia. 110-118. Montenegro, G., R. Pizarro, G. Avila, R. Castro, C.

58

CIENCIA E INVESTIGACION AGRARIA

Ros, O. Muoz, F. Bas, and M. Gmez. 2003. Origen botnico y propiedades qumicas de las mieles de la Regin Mediterrnea rida de Chile. Ciencia e Investigacin Agraria. 30:161174. McKee, B. 2003. Prevention of residues in honey: A future perspective. Apiacta 38: 173-177. Mauricio, A. 1975. Microscopy of honey. p. 240257. In: E. Crane (ed.) Honey: A Comprehensive Survey. London, England. Porrini, C., A. Sabatini, S. Girotti, S. Ghini, P. Medrzycki, F. Grillenzoni, L. Bortolotti, E. Gattavecchia and G. Celli. 2003. Honey bees and bee products as monitors of the environmental contamination. Apiacta 38: 63-70.

Sanna, G., M. Pilo, P. Piu, A, Tapparo, and R. Seeber. 2000. Determination of heavy metals in honey by anodic stripping voltammetry at microelectrodes. Analytica Chimica Acta 415: 165-173. Schonoor, J. 1997. Phytoremediation. Ground-Water Remediation Technologies Analysis Center, Pittsburgh, USA. 37 pp. Terrab, A., F. Recamales, M. Gonzlez-Miret, and F. Heredia. 2004. Contribution to the study of avocado honeys by their mineral contents using inductively coupled plasma optical emission spectrometry. Food Chemistry. 92(2): 305-309. USDA. 2005. Nutrient Data Laboratory. . United States Department of Agriculture, Agricultural Research Service. USA.