Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v65i4.3394Sociobiology 65(4): 727-736 (October, 2018) Special Issue Honey from Stingless Bee as Indicator of Contamination with Metals Introduction Bees can carry contaminants from the environment to the beehive and consequently to honey, due to their foraging activity. When foraging areas for bees are polluted, several undesirable chemicals may be introduced into honey through nectar, pollen or sugary exudates from plants growing on contaminated soil and/or absorbing contaminated water. Additionally, elements in the atmosphere also are important source of contaminants that may mix with the resources collected by bees (Porrini et al., 2003; Stecka et al., 2014; Di et al., 2016). Honey can be contaminated with inorganic chemical elements during raw material collection by bees or in the honey extraction process. Moreover, different weather conditions, seasons and honey botanical origin are variables that affect of metal contents in bee products. As bee products are the final stage of a bioaccumulation process, chemical study on honey Abstract Melipona scutellaris Latreille (Apidae, Meliponini) is one of the main species of stingless bees used in beekeeping in the Northeast of Brazil. We examined the honey of M. scutellaris as an indicator to evaluate the levels of metals at sampling sites subject to a broad spectrum of environmental pollutants. The collections were carried out in the urban-industrial area of Salvador, Bahia and the metropolitan region. Samples (n = 58) were submitted to the nitroperchloric digestion procedure. We used the inductively coupled plasma optical emission spectrometry technique (ICP OES) to determine the concentration of metals (Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb and Zn) in the samples. The studied metals were detected among the samples, which presented tolerable levels according to current Brazilian legislation and recommendations from the World Health Organization (WHO), except for Cr, which presented mean values higher than the threshold for all sampling sites. The detection of the analyzed metals indicates that the honey of M. scutellaris is a useful tool to evaluate the presence of environmental contaminants; therefore, it can be considered a good indicator of environmental contamination for monitoring a particular region and preventing issues due to the release of metals into the environment. Sociobiology An international journal on social insects AS Nascimento1, ED Chambó2, DJ Oliveira1, BR Andrade1, JS Bonsucesso1, CAL Carvalho1 Article History Edited by Cândida Aguiar, UEFS, Brazil Received 30 April 2018 Initial acceptance 13 June 2018 Final acceptance 17 August 2018 Publication date 11 October 2018 Keywords Meliponini, Melipona scutellaris, bioindicators, environmental pollution, ICP OES. Corresponding author Andreia Santos do Nascimento Universidade Federal do Recôncavo da Bahia Centro de Ciências Agrárias, Ambientais e Biológicas Rua Rui Barbosa nº 710, Centro CEP 44380-000, Cruz das Almas-BA, Brasil. E-Mail: asndea@gmail.com can provide useful information on the environmental quality of the area where bees feed (Pisani et al., 2008; Stecka et al., 2014; Silici et al., 2016). Plants can accumulate metals in their tissues due to their ability to adapt to various chemical conditions in the environment. Therefore, some species are considered accumulators of metals found in the soil, water and air (Malavolta, 1994). The geographic and botanical origin, as well as anthropogenic factors near colonies are determinant for the presence of high concentrations of metals in bee products (Bogdanov et al., 2007; Silici et al., 2016). In this sense, the pollen analysis of honey in bee products is very important because it helps a better understanding of the levels of metals in samples from different sites. The presence of toxic metals in honey can threaten human health (Ru et al., 2013). Additionally, it can serve as an indicator of environmental pollution. Thus, many studies 1 - Universidade Federal do Recôncavo da Bahia, Cruz das Almas, Brazil 2 - Universidade Federal do Amazonas, Benjamin Constant, Brazil RESEARCH ARTICLE - BEES AS Nascimento et al. – Stingless Bee honey as Indicator of Contamination728 have investigated metal concentration in the body of bees and in their products (Porrini et al., 2003; Batista et al., 2012; Pohl et al., 2012; Bastías et al., 2013; Aghamirlou et al., 2015; Nascimento et al., 2015; Martin et al., 2016; Steen et al., 2016; Zarić et al., 2016; Bonsucesso et al., 2018). However, our study is the first carried out in an industrial urban area using honey of Melipona scutellaris Latreille, a stingless bee, as an indicator of environmental quality. Bees react to changes in the environment they inhabit, especially in relation to amounts of toxic metals in the soil, air and plants. This characterizes bees as reliable indicators of environmental quality, allowing their use in biomonitoring (Zhelyazkova, 2012). Stingless bees (Meliponini) have potential for use as indicators of environmental contamination with toxic metals (Nascimento et al., 2015). These bees have an atrophied sting, which facilitates management (Camargo et al., 2017), and are commonly reared in an urban environment, where there are many anthropogenic activities near the beehives, thus the bees are more exposed to loads of pollutants, especially in large urban centers. M. scutellaris (Apidae, Meliponini) is one of the main species used in the beekeeping activity in northeastern Brazil (Villas-Bôas, 2012). It was the most frequent species in the region evaluated in this study and presented a satisfactory honey production, allowing its use for this research. This study used honey from M. scutellaris as an indicator to evaluate the levels of metals at sampling sites subject to a broad spectrum of environmental pollutants. Material and Methods Study site The collections were carried out in urban-industrial area of Salvador and metropolitan region, Bahia, Brazil (Fig 1). The selected meliponaries were installed in urban (sites A-D), semi-rural (site E) and rural environments (site F), near the petrochemical Complex of Camaçari (Site E-F), sanitary landfill of Salvador and highways Cia-Aeroporto (site A-D), Base Naval Road and BA 093 (site A) (Table 1). The colonies were exposed to a very high range of pollutants that may be present in the atmosphere, soil and water. One month before sample collection, we prepared the colonies used in the experiment, leaving these colonies with empty honey pots, which allowed collection of honey stored by bees during the sampling period. Fig 1. Geographical location of sampling sites in Salvador and the metropolitan region, state of Bahia, Brazil. Sociobiology 65(4): 727-736 (October, 2018) Special Issue 729 A meliponary installed in a non-urban area was used to collect control samples, located in Baía do Iguape, Cachoeira, Bahia. The Marine Extractive Reserve of the Baía do Iguape (currently known as Iguape) is a federal conservation unit in Brazil categorized as an extractive reserve (RESEX) in the state of Bahia. Sites Municipality Environment Anthropogenic influence Meliponary A Salvador 12º51’32.4’’ S 038º27’9.90’’ W Urban area Near (~ 600 m distance) highway Base Naval, with intense traffic of vehicles Meliponary B Salvador 12º49’58.7’’ S 038º22’27.4’’ W Urban area Region of the Industrial Center of Aratu (CIA), highway CIA-Aeroporto, with intense traffic of vehicles Meliponary C Salvador 12º51’28.3’’ S 038º21’54.3’’ W Urban area Region of the Industrial Center of Aratu (CIA), highway CIA-Aeroporto, with intense traffic of vehicles, near a landfill and unpaved road Meliponary D Lauro de Freitas 12º50’38.1’’ S 038º21’12.1’’ W Urban area Metropolitan Region of Salvador, Bahia. unpaved road, near (~ 800 m distance) an indian reserve Meliponary E Simões Filho 12º43’55.5’’ S 038º23’51.6’’ W Semi-rural Metropolitan Region of Salvador, Bahia. Farm located approximately 300 m from highway BA 093, with intense traffic of vehicles, near the petrochemical complex of Camaçari, Bahia Meliponary F Dias D’Ávila 12º32’28.0’’ S 038º21’42.3’’ W Rural Metropolitan Region of Salvador, Bahia. Farm located approximately 8 km from highway BA 093 with intense traffic of vehicles Meliponary G Cachoeira 12º38’25.3’’ S 38º51’42.0’’ W Non-urban area Federal conservation unit of Brazil. Marine Extractive Reserve of Baía do Iguape (Control) Table 1. Description of sampling sites in Salvador and the metropolitan region, Bahia, Brazil. Thermo Scientific iCAP 6000 Series, Model 6300 Duo. Table 2 presents the analysis conditions of ICP OES. Accuracy of the analytical method was evaluated in terms of repeatability of experimental results of real samples (triplicate) and expressed as standard deviation of the mean. Accuracy was verified by calibration (using standard solutions). Pollen analysis To determine the botanical origin of honey, samples were prepared according to the methodology described by Jones and Bryant Jr. (2004) and later submitted to the acetolysis process of Erdtman (1960). The resulting pellet was mounted on slides for microscopy, followed by the identification and counting of the pollen grains that compose the pollen spectrum of the sample. The pollen types were identified using specialized literature, such as Barth (1989), Roubik and Moreno (1991), Punt et al. (2007) and consulting database and images of the Palinoteca of the Universidade Federal do Recôncavo da Bahia, Brazil. Frequency class of each pollen type was determined according to Louveaux et al. (1978), classified as: Predominant Pollen ( PP- > 45% of total grains), Secondary Pollen (SP-16 to 45%), Important Minor Pollen (IMP – 3 to 15%) and Minor Pollen (MP - < 3%). Sampling The samples (n = 58), composed each of approximately 250 g of honey, were collected directly from the colonies of M. scutellaris with disposable syringes, placed in properly identified sterile plastic containers. The sampling period was one year, between August 2014 and July 2015. Preparation of samples We used the method of nitro-perchloric digestion proposed by Malavolta et al. (1989) in the preparation of samples to identify the metals. We used 2 g of each honey sample, the evaluations of each sample being performed in triplicate. All the glassware used was placed in a 10% nitric acid solution (HNO3) for 24 h for decontamination, after which all the material was washed with ultrapure water (18.2 MΩ.cm). We used a standard solution (blank solution) containing only acids, which was submitted to the same procedures for the digestion of honey samples. For the analyses, reagents of certified analytical grade were used. Determination of metal concentration Metals cadmium (Cd), copper (Cu), lead (Pb), cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni) and zinc (Zn) were selected for this study because of their importance as environmental contaminants. To determine the concentration of metals in the samples, the Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) technique was used in the ICP (Spectrometer) AS Nascimento et al. – Stingless Bee honey as Indicator of Contamination730 M et al Sa m pl in g si te s B ra zi lia n L eg is la tio n* W H O * (m g/ kg /d ay ) A (n =9 ) B (n =8 ) C (n =1 0) D (n =1 2) E (n =9 ) F (n =1 0) G (n =1 0) C on tr ol M ea n± SD M ea n± SD M ea n± SD M ea n± SD M ea n± SD M ea n± SD M ea n± SD C da 0. 00 10 ±0 .0 03 0