Minerals content in Basilicata region (southern Italy) honeys from areas with different anthropic impact

Summary The aim of this study was to examine the minerals content (toxic elements, macro-elements and trace elements) of Basilicata region (southern Italy) honeys and compare the mineral proﬁle of honeys classiﬁed as rural, industrial and urban according to anthropic characteristics of geographical origin. Overall, Ca, Na, Mg, Fe, Al, Zn and Mn were the most abundant elements detected in honeys, with average contents exceeding 1 ppm, whereas heavy metals content was lower than the maximum limit established for honey. Statistical analysis revealed signiﬁcant diﬀerences ( P < 0.05) among honeys as function of anthropic characteristics of geographical origin, with the exception for Se, Co and Ag content. Industrial honeys were characterised by the highest Zn, Cr, Sn, Cd and Pb content, urban honeys showed the highest As, Fe, Ni, Mn, Na, Mg and Ca content, whereas rural honeys showed the highest Cu, Al and Ba content ( P < 0.05). The ﬁndings of this study highlighted that honeys mineral proﬁle is closely related to diﬀerent content of elements in environment, which is aﬀected by anthropogenic activities.


Introduction
Honey is a natural product of honey bees, closely linked to the territory, characterised by high concentration of sugars, water, minerals, proteins, vitamins, organic acids, flavonoids, phenolic acids and enzymes.It is strongly influenced by both natural and anthropogenic factors which vary based on its botanical and geographical origins (Perna et al., 2012).
In fact, it is considered as the result of a bioaccumulation process of many metals and it plays an important role as environmental bio-indicator (Tuzen et al., 2007;Rashed et al., 2009;Perna et al., 2014).Although honey contains 0.02-1.03% of minerals, these play a fundamental role in determining its quality.In fact, some macro (Ca, K, Mg, Na) and microminerals (Co, Cr, Cu, Fe, Mn, Ni, Se, Zn) have important functions in several biochemical processes as constituents of many bioactive compounds (Tuzen et al., 2007;Perna et al., 2014), whereas others (As, Cd, Hg, Pb) are considered toxic for human metabolism (Yarsan et al., 2007;Altundag et al., 2015).
Minerals presence and content in honey reflects the area surrounding the apiary; the foraging activity of the bees extends for about 10 km 2, and when the bees collect nectar or pollen, these elements are transferred, contributing to the levels in the honey.This is why apiaries located in proximity to polluted areas can help in monitoring heavy metals from various sources.Basilicata honey production is heavily based on the availability of rural and native bush vegetation; in fact, Basilicata region is characterised by areas with different climatic characteristics and a high diversity of botanical species collected by bees.Moreover, current trend is the production of urban honey as an artisanal product; in urban environments, there is an abundant presence of resources for bees; however, the close proximity to human activities influences the presence of particular trace elements and contaminants in honey.To date, several studies (Aldgini et al., 2019;Hungerford et al., 2020) detected in honey and its products different mineral profiles according to the anthropic characteristics of the geographical origin (urban, rural and industrial), highlighting the role of honey as indicator of anthropic impact and for quality control.Thus, the aim of this study was to examine and compare the mineral profile of honeys from Basilicata region (southern Italy) areas characterised by different anthropic impact.

Samples
During 2019, sixty honey samples were collected directly from beekeepers in different Basilicata region areas characterised by different anthropic impact (rural, industrial, and urban).In particular, 20 rural honeys were collected in agricultural and forestry areas, 20 urban honeys were collected in metropolitan areas, and 20 industrial honeys were collected in areas with manufacturing industries, such as metallurgical and chemical industries (Fig. 1).Honey samples were obtained from apiary located at a maximum distance of 5 km from the area of interest.All samples were subjected to melissopalynological analyses and classified as multifloral honeys.Two hundred and fifty grams of each honey sample was placed into clean glass bottles and stored at room temperature in the dark until analysed.

Metals analysis
Trace elements (Ag, Al, As, Ba, Be, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, Sn, Sr, V and Zn) in honeys were analysed in triplicate by inductively coupled plasmamass spectrometry (ICP-QMS; Elan DRC II, Perkin-Elmer SCIEX, CT, USA) as reported by Perna et al., (2014), and the results were expressed as µg metal per kg honey.Minerals (Ca, Mg and Na) were analysed in triplicate by inductively coupled plasma-optical emission spectrometry (ICP-OES; model iCAP 6000, Thermo Scientific, Cambridge, UK) as reported by Perna et al., (2012), and the results were expressed as mg metal per kg honey.

Statistical analysis
Statistical analysis was performed using the general linear model (GLM) procedure of statistical analysis system (SAS Institute, 1996) using a monofactorial model.Differences among metal content in different geographical areas were analysed using Student's ttest, and differences between means at the 95% (P < 0.05) confidence level were considered statistically significant.Pearson's correlation test was conducted to determine the linear correlation between the variables.

Results and discussion
Trace elements, macro-elements and heavy metals in southern Italy honeys In Table 1, are reported the results (mean, median, standard deviation, minimum and maximum values) of mineral content detected in 60 studied honey samples.Mineral profile included toxic elements (Al, As, Ba, Be, Cd, Pb), macro-elements (Ca, Mg, Na) and trace elements (Ag, Co, Cr, Cu, Fe, Mo, Mn, Ni, Se, Sn, Sr, V and Zn).The findings highlighted a certain variability in elements composition of honey samples; this is due to different botanical and geographical origin, as reported by several authors (Rashed & Soltan, 2004;Fern andez-Torres et al., 2005;Pisani et al., 2008).Ca, Na, Mg, Fe, Al, Zn and Mn were the most abundant elements, with average contents exceeding 1 mg kg À1 .In particular, among macro-elements, Na content was the highest (49.15 mg kg À1 ), followed by Ca (44.09 mg kg À1 ), and Mg (19.30 mg kg À1 ).Na content detected in studied honeys was in line with what found in Croatian honeys (44.04 ppm; Biland zi c et al., 2017), whereas it resulted lower than that detected in Italian (96 mg kg À1 ; Conti, 2000;Pisani et al., 2008), and Malaysian honeys (358.64 mg kg À1 ; Chua et al., 2012).Na plays a key role in maintaining optimal blood pressure, kidney, nerve and muscle functions (Hall, 2003;Sobotka et al., 2008).Ca content detected in studied honeys was lower than that detected in Croatian honeys (111 mg kg À1 kg À1 ; Downey et al., 2005).The variations in the Ca content were certainly due to the different pedo-climatic characteristics of the geographical origin of honeys (Bogdanov et al., 2007;Perna et al., 2014).Ca is an essential mineral with important functions in the cardiac, nervous and musculoskeletal system, and it is also essential for bone and tooth formation, mineral homeostasis, as well as acting as a cofactor for many enzymes (Theobald, 2005;Huskisson et al., 2007;Morgan, 2008).Mg content in studied honeys ranged from 9.00 to 38.89 mg kg À1 (Table 1).Mg is an essential element for all living organisms: acts as a cofactor for up to 300 enzymes, most of which are involved in antioxidant reactions; it is involved in the structure of proteins, lipids and carbohydrates; Mg deficiency in humans can cause muscle spasms and cardiovascular diseases such as hypertension (Durlach et al., 1998;Huskisson et al., 2007).Mn values ranged from 113.06 to 9021.14 µg kg À1 , with a average value of 1603.06 µg kg À1 (Table 1).Mn, naturally present in many types of rock and soil, is an element with low toxicity and high biological function (Yazbeck, 2011).The Mn average value detected in this study was in line with what detected by Pisani et al., (2008) in honeys from Siena County (Italy), but it was much lower than that found in honeys from other Italian areas (about 3 mg kg À1 ; Conti, 2000;Bontempo et al., 2017) et al., 2015).On the contrary, Mn content in studied honeys was higher than that found in Turkish (45.60 µg kg À1 ; Altun et al., 2017), Argentine (700 µg kg À1 ; Conti et al., 2014) and Irish honeys (40 µg kg À1 ; Downey et al., 2005).Al, third on the list of most abundant elements in the earth's crust, has a negative toxicological impact on the central nervous, skeletal and hematopoietic systems; in addition, its ions can hinder various metabolic processes due to competitive reactions with ions, as Ca, Mg, and Fe, F (Yokel et al., 2008).In this study, Al content ranged from 630.26 to 3844.39 µg kg À1 , with a average value of 1395.73 µg kg À1 (Table 1); it was in line with that found by Czipa et al., (2015) in Croatian honeys (1.03 mg kg À1 ), but was lower than that reported by Di Bella et al., (2015) in Italian honeys (5.67 mg kg À1 ).Bees during foraging activity are exposed to Al from several sources; in addition, a further exposure to Al can be due to secondary sources, such as metal containers used for storage during harvesting and handling processes; in fact, Al contamination is usually accompanied by Cr contamination from the same source (Pisani et al., 2008).Fe is an essential element for the production of red blood cells, in fact a deficiency causes anaemia; moreover, it mediates electron transfer in the catalysis of enzymatic reactions which is toxic because it catalyse the conversion of hydrogen peroxide into free radicals (Andrews, 1999).Fe content in studied honeys ranged from 477.38 to 3954.53 µg kg À1 (Table 1).The Fe average content found in this study was lower than that detected in Italian honeys and, in particular, in Siena Country (Toscana Region) honeys (3.07 mg kg À1 ; Pisani et al., 2008) and Lazio Region honeys (4.50 mg kg À1 ; Conti, 2000), and in French honeys (11.03 mg kg À1 ; Devillers et al., 2002), whereas it was higher than that found in Romanian honeys (22.70 µg kg À1 ; Oroian et al., 2016).After Fe, Zn is the most abundant transition metal in organisms (NRC, 2000).It is the unique metal which arises in all enzyme tribes and is considered an antioxidant because is found in nearly 100 specific enzymes (Broadley et al., 2007).Zn content varied between 503.67 and 1971.91 µg kg À1 , and its average content (1081.13µg kg À1 ) was markedly lower than that found in an our previous study on honeys from the same geographical area (Perna et al., 2012), whereas was slightly lower than that detected in central Italy honeys, and in particular in Siena Country (1.82 mg kg À1 , Pisani et al., 2008) and Lazio Region honeys (3.1 mg kg À1 ; Conti, 2000).Zn is an essential element for humans, but high levels in the diet can cause anaemia and decreased of Cu and Fe absorption (Roohani et al., 2013).Overall, the trace elements content detected in studied honeys was very low and the value was comparable to content found in honeys from uncontaminated areas (Tuzen et al., 2007).Cu is one trace heavy element essential for life; its Recommended Dietary Allowance (RDA) for adult is 1.0 mg/day, whereas the Provisional Maximum Tolerable Daily Intake (PMTDI) is 0.5 mg/kg of body weight, an excess of Cu can cause disease of Wilson (WHO, 1982).The Cu average content detected in studied honeys (236.65 µg kg À1 ) was higher compared to found in Turkish honeys (68.50 µg kg À1 ; Altun et al., 2017), whereas it was lower than that detected in Argentine honeys (Conti et al., 2014).Co value ranged between 0.82 and 14.99 µg kg À1 ; it is an integral part of vitamin B12 and is therefore essential for the functioning of the cell (Yamada, 2013).In this study, Ni content (32.89 µg kg À1 ) was markedly lower than that found in Turkish honeys (Y€ ucel & Sultanoglu, 2013).At low concentrations, Ni is necessary for plants; however at high concentrations, it is toxic (Seenivasan et al., 2008).Se is an important micronutrient; in particular for immune system and thyroid function, it also plays an essential role in antioxidant metabolism (Triggiani et al., 2009) In the literature, there are few data on the Mo, Ag, Ba, Be, Sn and V concentration in honey; however, the contents detected in this study were lower than that found in Australian (Hungerford et al., 2020) and in Greece honeys (Karabagias et al., 2017a,b).Pb, Cd and As are highly toxic elements, and their presence in honey indicates environmental pollution (Bogdanov et al., 2007)  International Journal of Food Science and Technology 2021 ound in Mediterranean honeys (Tuzen et al., 2007;Silici et al., 2008;Y€ ucel & Sultanoglu, 2013).Cd content found in this study in honeys varied between 0.40 and 9.36 µg kg À1 (Table 1), with an average content (3.31 µg kg À1 ) lower than that found in honeys collected in different Italian areas (Perna et al., 2012;Quinto et al., 2016;Conti et al., 2018), and clearly lower than that reported for honeys collected in six continents (Solayman et al., 2016).Furthermore, Pb and Cd content detected in this study was markedly lower than those found in an our previous study in the same area (Basilicata region, 0.25 mg kg À1 ; Perna et al., 2014); this could be explained by the use of automotive engine catalysts over the past 10 years.Pearson's correlation analysis was applied to examine the relationship between metal concentrations in honey samples (Table 2).Overall, in agreement with what reported by many authors in literature (Bogdanov et al., 2007;Sarker et al., 2015;Quinto et al., 2016), statistically significant correlations were observed between metals, indicating that the levels of the elements are closely interlinked with one another.In particular, some significant positive correlations were detected between elements with affinity in the periodic table, such as Ba and Sr (r = 0.911; P < 0.001), Mg and Ca (r = 0.871; P < 0.001), and this could be due to a similar absorption mechanism soil-plants-honey.A strong positive correlation was observed between the Mn and Cu contents (r = 0.868; P < 0.001), and this could be due to same influential anthropic factors (Esmaeili et al., 2014).The other significant positive correlations found between metals with same anthropic origin were Cd and Pb (r = 1.000;P < 0.001), Cd and Zn (r = 0.986; P < 0.001) and Pb and Zn (r = 0.986; P < 0.001).Thus, the results of Pearson's correlations can only be explained by the mutual affinity of metals and/or anthropic factors.
Comparison of mineral profile in honeys from urban, industrial and rural area  the bees' forage area affected the chemical composition of the hive products.Statistical analysis revealed significant differences among honeys from different areas, with the exception for Se, Co and Ag content (P > 0.05).Significant differences between urban and rural honeys were found for all studied elements, with the exception for As, Cr, Be and Sr content; no significant difference was also detected between urban and industrial honeys for Al, V, Cu, Zn, Be, and Sn content; in addition, no significant difference was found between rural and industrial honeys for Na, Mg, Ca, Sn, Cu, Mn, and Ni content.In this study, industrial honeys showed higher Cr, Zn, Cd and Pb levels compared to honeys from other studied areas.Several studies (Bratu & Georgescu, 2005;Perna et al., 2012Perna et al., , 2014) ) reported higher concentrations of toxic trace elements or heavy metal in honeys from industrial areas.Furthermore, as we expected, Pd and Cd content was lower in rural honeys (Table 3).Thus, we can say that the differences in toxic elements content among honeys from studied areas are due to environmental or geographical factors, to which the anthropogenic source must be added.In fact, traffic-related pollution and chemical-intensive agriculture can contribute to the increasing levels of these elements in honey (Street et al., 2009;Silici et al., 2013).The Cr presence detected in all honey samples may be due to both the contact of stainless steel surfaces during harvesting, processing and/or preparation, in fact honey due to its acidity is corrosive (Ajibola et al., 2012), and to other contamination sources as stationary point sources, coal, and oil combustion.In this study, Zn level was higher in industrial and urban honeys (1369.15 and 1137.41 µg kg À1 , respectively) than in rural honeys (866.55 µg kg À1 ; P < 0.05), whereas Al, Cu, V, Ba and Sr content was higher in rural honeys compared to honeys from other studied areas (P < 0.05).The highest Na, Mg, Ca, Ni, Fe, and Mn content was found in urban honeys (P < 0.05).Certainly the beekeeping practices, honey processing, and environment contribute to the diversified mineral content in honey (Anklam, 1998;Pohl, 2009).However, in addition to these factors, the different mineral profile of the studied honeys was clearly explained by the different content of elements in the soil of studied areas, which is due to anthropogenic activities, such as agricultural practices, proximity to industries, waste dumps, and urban centres.

Conclusions
The present study furnished a detailed picture of mineral profile of honeys collected in areas of Basilicata region (southern Italy) characterised by different anthropic impact.All chemical elements detected in this study can be both from natural sources (soil, plants) and anthropogenic sources.Overall, in studied honeys, Ca, Na, Mg, Fe, Al, Zn, and Mn were the most abundant elements, with average contents exceeding 1 ppm, whereas the toxic elements content was really low and the variations reflect the anthropic characteristics of considered areas.Moreover, several differences of mineral profile was detected among honeys collected in area with different anthropic impact, with the exception for Se, Co and Ag content.In particular, industrial honeys were characterised by the highest Zn, Cr, Sn, Cd and Pb content, urban honeys showed the highest As, Fe, Ni, Mn, Na, Mg and Ca content, whereas rural honeys showed the highest Cu, Al, and Ba content.The findings of this study highlighted that the mineral profile of honeys is closely related to the degree of trace element contamination of the environment.These results are useful for both evaluate the nutritional quality of honey and greater consumers' confidence in purchasing and consuming this product.

Table 2
Pearson correlation coefficients between metals in the investigated honeys À1) established by the EC Reg.2015/1005.Moreover, the Pb average content was lower than that f-

Table 3
Trace elements, macro-elements, and heavy metals in Basilicata region (southern Italy) honeys classified according to the antropic characteristics of the origin area(rural, industrial, and urban) a,b,c Means within a row with different superscripts differ (P < 0.05).© 2021 The Authors.International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF).International Journal of Food Science and Technology 2021