Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v65i2.2078Sociobiology 65(2): 259-270 (June, 2018) Pollen storage by stingless bees as an environmental marker for metal contamination: spatial and temporal distribution of metal elements Introduction Since the middle of the 20th century, growing industrialization, urbanization, transportation and agriculture has led to overall ecosystem contamination and major modifications to landscape structure and composition. The presence of metals in the environment can be the result of external sources such as industrial smelter pollution, emissions from ferrous metallurgy, external mining activities and busy highway traffic (Bilandžić et al., 2011; Lambert et al., 2012). Mining activities represent a major source of environmental contamination by metal residues (Freedman & Hutchinson, 1981; Perugini et al., 2011). In the Brazilian state of Minas Gerais, mine exploitation in the Iron Quadrangle reached its peak towards the middle of last century and the activity is still intense (Meneses et al., 2011). One of the consequences of this industry is the daily release of huge amounts of dust into the atmosphere (Meneses et al., 2011). Abstract Since the middle of the 20th century, human activities have led to overall ecosystem contamination and to major modifications in landscape structure and composition. Mining activities represent a major source of environmental contamination by metal residues. The objective of our study was to evaluate the presence of heavy metals and other elements on stingless bee pollen, and compare them to samples of Suspended Particulate Material (SPM) in five points a Mineral Province, in Brazil. More than 50 elements were identified by ICP-OES and ICP-MS, after microwave digestion. Overall, we found a strong relation among elements present on pollen and SPM. Samples from the four areas exhibited higher levels of minerals compared to the reference site. Mineral levels varied widely within the two seasonal periods. Some elements, like Pb, Cd, As, Cu, Zn, and Fe were found at levels considered potentially toxic to human health. Pollen stored by stingless bees was a successful bioindicator, and demonstrated the value of quantitative ecological information for detecting air pollution. Sociobiology An international journal on social insects NO Nascimento1, HA Nalini2, F Ataide2, AT Abreu2, Y Antonini1 Article History Edited by Celso Martins, UFPB, Brazil Received 13 November 2017 Initial acceptance 08 December 2017 Final acceptance 21 January 2018 Publication date 09 July 2018 Keywords Bee; Pollen; suspended particulate material; mining activities; Iron Quadrangle. Corresponding author Yasmine Antonini Universidade Federal de Ouro Preto Departamento de Biodiversidade Evolução e Meio Ambiente Instituto de Ciências Exatas e Biológicas (ICEB) Campus Morro do Cruzeiro, s/nº, Bauxita Ouro Preto, Minas Gerais, Brasil. E-Mail: antonini.y@gmail.com The rocks (or soils) of Iron Quadrangle naturally have high levels of metals that can be considered toxic for human health (Rapini et al., 2008; Azevedo et al., 2012; Messias et al., 2013; Carvalho-Filho et al., 2010). On the other hand, these regions shelter a relatively high variety of very poorly studied metallophilic plants, living in a harsh environment with high concentrations of metals in the soil and subject to atmospheric deposition of those toxic elements, due to the mining activities. Studies carried by Valim (2012) and Baêta (2012) for example, found that relevant quantities of elements such as Al, Ba, Ca, Fe, K, Na, Mg, Mn, S, Sr, and Zn are deposited annually on the soil surface and vegetation via wet and dry deposition. According to these authors, this deposition depends on the geochemical nature of the environments that may be affected by weathering and anthropic activities, being sources of input of both water and nutrients and even pollutants for plants and animals. 1 - Universidade Federal de Ouro Preto, Departamento de Biodiversidade Evolução e Meio Ambiente, Instituto de Ciências Exatas e Biológicas, Ouro Preto, Minas Gerais, Brazil 2 - Universidade Federal de Ouro Preto, Departamento de Geologia, Escola de Minas, Ouro Preto, Minas Gerais, Brazil RESEARCH ARTICLE - BEES mailto:antonini.y@gmail.com NO Nascimento, HA Nalini, F Ataide, AT Abreu, Y Antonini – Stingless bees as an environmental marker for metal contamination260 Due to their enormous diversity (species richness and abundance) and role in ecosystem functioning, the use of insects as bioindicators has been increasing (Mcgeoch, 2007; Lambert et al., 2012). The functions and use of bioindicators are better depicted if we take a look at the bees. The idea of employing bees in environmental monitoring is not a new one. Since 1970, honeybees have been increasingly employed to monitor heavy metals in territorial and urban areas, pesticides in rural areas and radio nuclides (see Pohl, 2009 for a revision; Oliveira et al., 2017). Besides honeybees, their products have been employed in studies aiming to determine levels of metal pollution in different ecosystems (agricultural, urban and industrial; e.g. Bratu & Beorgescu, 2005; Bilandžić et al., 2011; Morgano et al., 2012; Formick et al., 2013; Lambert et al., 2012; Leita et al., 1996). In many countries, there are no reports regarding the use of stingless bees to measure pollen contamination by toxic elements or as environmental markers in mining areas. Apparently, mines, steelworks, industrial and urban areas, and highways in or near bee foraging areas can result in an increase in the concentrations of certain metals (e.g., Al, Ba, Ca, Cd, Cu, Mg, Mn, Ni, Pb, Pd or Zn) in bee matrices (honey and pollen) due to pollution from chemical wastes and exhaust fumes (Wayne, 1983; Bilandžić et al., 2011; Formick et al., 2013). The fundamental difference between heavy metals and other pollutants, like pesticides, is their mode of introduction and their environmental fate. Pesticides are scattered both in time and space and, depending on the type of chemical compound, are degraded by various environmental processes over longer or shorter periods of time. Heavy metals, on the other hand, are emitted in a continuous manner by various natural and anthropic sources and, since they are not degraded, are continuously kept “in play”, thus entering into physical and biological cycles (Matina et al., 2016). In this scenario, we propose the use of stingless bees as active environmental markers in mining areas; they have been mostly used in standard exploration procedures of mining companies as efficient mineral prospectors. Stingless bees, like other bee species, are exposed to atmospheric pollutants during their foraging activities. Their hairy bodies easily hold residues, and they may be exposed to contaminants via contaminated food resources such as nectar, pollen or water (Pohl. 2009). Heavy metals present in the atmosphere can be deposited on these hairy bodies and be brought back to the hive with pollen, or they may be absorbed together with nectar, water or honeydew (Caroli et al., 1999; Matei et al., 2004; Rodriguez Garcia et al., 2006; Bogdanov, 2006). Furthermore, almost all environments (soil, vegetation, water and air) are sampled by stingless bees, and thus provide numerous indicators (through foraging) for each season. Finally, a variety of potentially contaminated materials are brought into the hive (nectar, pollen, honeydew, propolis and water) and stored accordingly. A number of variables need to be considered when using bees, or beehive products such as pollen, to monitor heavy metals in the environment. These include flight distance, proximity to the contamination source, weather (rain and wind can clean the atmosphere or transfer heavy metals to other environmental sectors), season (nectar flow, which is usually greater in spring than in summer and autumn, could dilute pollutants) and botanical origin of the honey (nectar of flowers with an open morphology and honeydew are much more exposed to pollutants). The present study reports the first effort to use stingless bees as ecological indicators. Our purpose was to investigate whether stingless bees are an effective environmental marker of contamination for monitoring environment pollution caused by mining activities through the analysis of pollen matrices over two climatic periods (dry and wet season) by comparing their contaminant content with suspended particulate matter (SPM). The study areas were located in mining sites that are susceptible to various levels of contamination due to different kinds of minerals present in the rocks. Materials and methods Sampling sites and biological matrices Sampling of pollen matrices of Tetragonisca angustula (Latreille) beehives and suspended particulate matter (SPM) were performed from February to April (wet period) and August to September (dry period) of 2013 in five areas of the Iron Quadrangle, Minas Gerais, Brazil (Figure 1). We avoid sampling during periods of intense rainfall, in wet period. Suspended particulate matter (SPM) is here considered as finely divided solids that may be dispersed through the air from mining activities. This area is known worldwide as one of the most important iron and gold producers of the world consequently undergo environmental contamination (CPRM 2014, Figueiredo et al., 2000). The substrates in this region are rich in iron, manganese and aluminum, and have given rise to the development of Rupestrian Fields (Campos Rupestres), also called Ferruginous Rupestrian Fields, ferruginous rocky outcrops, or “canga” vegetation (Schaefer et al., 2016; Silveira et al, 2016). Four sites were located in mining sites of iron (SM), limestone (BM), gravel (IM) and soapstone (VM) mines, and one control site (CT). Besides the past intensive mining activity in a radius of 3 km, the CT site is today a Natural Protected area, covered by forest. Three samples in each period (dry and wet) were taken from the bee hives of Tetragosnica angustula (3 colonies in each sampling site, totaling 90 samples) installed one km distance to the main mining pit. The distance of 1km is twice a maximum distance that T. angustula workers can fly (Nogueira-Neto, 1997). The samples were collected from the same colonies throughout the survey, with the exception that dead colonies were replaced in order to maintain the number of hives sampled. Pollen was collected directly from uncapped pollen combs in order to assure fresh pollen was collected. Sociobiology 65(2): 259-270 (June, 2018) 261 These field-collected pools were immediately placed on ice and then stored at 22ºC until analysis. Colonies used for the experiment were obtained from natural areas, outside of Iron Quadrangle. with a final volume of 50 mL. Chemical element content of the solutions was measured by ICP-OES (optical emission spectrometry with inductively coupled plasma) and ICP-MS (mass spectrometry with inductively coupled plasma). Instruments and equipment Analyses were carried out using a Microwave Milestone Ethos s1, ICP-MS (model Agilent 7700x) and ICP- OES (model Agilent 725). Analyses employed the following procedure: optimization of the instrument, calibration with standard solutions, analysis of the sample blank consisting of 2% ultra-pure nitric acid and analysis of the reference materials (after every 10 samples). The analyses were performed at the Laboratory of Geochemistry of the Geological Department of the Federal University of Ouro Preto (Brazil). Assurance of analytical quality Method precision was evaluated by recovery tests conducted at two concentration levels covering the concentration ranges in the samples. These solutions were analyzed after every ten samples and the coefficient of variation was determined for 3 repetitions due to the heterogeneity of the pollen samples. Because of the unavailability of certified reference material for bee pollen, a certified reference material (Apple leaf, Nist SRM 1515) was evaluated to assess method accuracy for the recovery of elements. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated as suggested by Mermet and Poussel (1995): LOD = (3 RSDxBEC)/100 and LOQ = 5 x LOD, where RSD is the relative standard deviation of blank samples and BEC is the background equivalent concentration, determined experimentally (n = 10). Limits of quantification (LOQ) are provided in Table 1. Fig 1. Sampling areas of the Iron Quadrangle, Minas Gerais, Brazil. BM (Bemil), CT (Control area), IM (Irmãos Machado), SM(SAFM), VM (Viamar). Sample preparation A simple system made from a high-density polyethylene (HDPE) bottle connected to a HDPE funnel was used for total SPM collection, following Azimi et al. (2003) (Suplementary Material Figure 1). These collectors were arranged in a north- south-east-west direction, relative to the bee’s nest with a collector next to it in a central position. The five collectors per sampling area were arranged distant about 50 meters from the central. Collection bottles were filled in advance with 100 mL of a solution of 10% nitric acid (65% Suprapur nitric acid, Merck) and Mili-Q water in order to dissolve particulate matter and to avoid trace metal adsorption by bottle walls. The collection bottles were filled with concentrated nitric acid after the sampling period (7 days) to reach a final pH of 1, and samples were kept in a dark room at 5°C for one month in order to dissolve most of the particles. After storage for one- month, samples were filtered under a class 100 laminar hood with 0.45 μm porosity filters (Sartorius, cellulose nitrate). The sub-samples obtained were kept at 5 °C until analysis. Materials were washed following the procedures of Azimi et al. (2003). Sample preparation was performed according to previously described methods (Miller & Miller, 2005; Vinas et al., 2000). Triplicates of 0.5 g of each samples of pollen were incinerated in a microwave oven (Step1: Ramp time - 10 min until 200°C; Step 2: Hold time - 15 min at 200 ºC; Step 3: Cooling - 30 min) after acid digestion with 7 mL 65% m/m nitric acid (Suprapur, Merck, Darmstadt, Germany) and 1 mL 30% m/m hydrogen peroxide (Suprapur, Merck). All solutions were prepared with deionized water obtained by passing distilled water through a Millipore Milli-Q water purification system (Waters Corporation, Milford, MA, USA) Table 1. Limits of Quantification (LOQ) for major, minor and trace elements determined by ICP-OES and ICP-MS. Element SPM (µg/L) Pollen (*µg/kg; **mg/Kg) Al 8.920 3.674** As 104.000 0.0328* Ba 0.480 0.121* Cd 6.640 0.00733* Co 26.300 0.00766* Cr 17.100 0.142* Cu 5.370 0.896** Fe 7.040 3.068** Mn 2.280 0.382** Ni 37.000 0.214* Pb 181.000 0.0639* Sb 0.0296* Y 2.740 0.044** Zn 6.210 0.634** NO Nascimento, HA Nalini, F Ataide, AT Abreu, Y Antonini – Stingless bees as an environmental marker for metal contamination262 Statistical analysis In total, 54 pollen and 60 SPM samples were analysed. For the statistical analyses, the site SM had to be excluded – the colonies perished due to high levels of dust deposited outside the nests. The statistical parameters for concentrations (mean, median and standard deviation) were calculated from all the analyzed samples of each matrix, and not only from samples for which residues were detected or quantified. When a compound was not quantified (< LOQ), the concentration used for statistical analysis was LOQ/2 (Hewett & Ganser, 2007). Statistical analyses were performed only for pollen and SPM residues that were detected or quantified at least once. Comparisons of the means of element concentrations in pollen between climatic periods (dry and wet) were performed using the Friedman test (non-parametric test for paired samples). The effects of climatic period and site location on metal concentrations were evaluated using a general linear mixed effect model (GLM). To be valid, a linear mixed effects model must exhibit independent and normally distributed residuals, which were assessed for each model through diagnostic plots using R-packages (plots of deviance residuals versus fitted values and normal quartile plot of Pearson residuals; plots not shown). Contrast analyses comparing metal concentrations among sites using metal as the explanatory variable were then been performed using R software (R Development Core Team 2010). AGLM analysis was used to test if the atmospheric deposition (SPM), as explanatory variable, was the primary factor for metal concentrations in pollen. Principal component analysis (PCA) was used to investigate the multivariate structure of the dataset and to highlight possible trends among the data. Principal component analysis reduces the number of dimensions of a dataset by determining a linear combination of initial variables that maximizes the information content of a group of data. This is achieved by decomposition of the correlation matrix into eigenvalues and eigenvectors, with the eigenvectors representing the linear combination of coefficients and the corresponding eigenvalues representing the variance described by each linear combination. Since the eigenvalues are in decreasing order, the first linear components account for the largest amount of variance. Once derived, the principal components (PCs) can be used for further analyses to visualize groupings of the data, to verify the presence of outlier values and to determine the variables that discriminate among groups. For all these statistical tests, a significant effect was considered to have a type-I error risk of p < 0.05. Results Forty-seven chemical elements were detected and quantified from the pollen samples and 34 elements from the SPM samples. Mean contents, standard deviations and concentration ranges obtained for the elements of the 54 dehydrated bee pollen samples and 60 SPM samples from the four sites in the Iron Quadrangle are shown in Tables 2 and 3, respectively. Sampling sites CT IM VM BM Element Average±SD Min/Max Al 78.18±11.69 470±381 318.7±114.5 140.5±27.4 68.3/101 102/836 207/461 116/179 As 24.01±10.12 104.99±9.88 118.5±71.7 38.68±11.54 11.87/35.33 94.51/114.14 57.2/242.7 29.69/55.85 Ba 6552*±206 11590±4287 2690±681 461.1*±65.2 6315/6692 6095/15686 1821/3431 415/507.2 Cd