Assessment of spatial and temporal variations in trace element concentrations using honeybees (Apis mellifera) as bioindicators

Sampling regions

There were five different sampling regions. Each region had at least one apiary from which the samples were taken. At each apiary at least two colonies were sampled.

Belgrade (BG)

Belgrade is the capital of Serbia. There were two apiaries in the city of Belgrade, both in the urban center (Fig. 1). One was located at the Faculty of Veterinary Medicine—University of Belgrade, while the other was at the Faculty of Agriculture—University of Belgrade.

Sampling

During 2014 samples from Mesić were taken in July from two colonies in one apiary. In Belgrade samples were taken from two colonies at each of the two apiaries in July, while in September samples were taken from one colony at each apiary. In TPP region samples were taken from one colony at apiaries located in Kostolac and Drmno, and from two colonies at apiary in Stari Kostolac in July and from one colony at each of the three apiaries in September. In Pančevo both in July and September samples were taken from two colonies at both apiaries. In Pavliš in July and September samples were taken from two colonies at one apiary. Samples were not taken from location MS in September (Table 1).

For the 2015 sampling campaign two locations were chosen: Pančevo and Pavliš. These locations were chosen to represent two locations with different human activities having different environmental impacts. Pančevo represented an industrial city with petrochemical industry and oil refinery, and Pavliš represented a suburban area not influenced by industrial or traffic pollution. In Pančevo there were two apiaries and honeybees were sampled from one colony at each apiary in Jun, July and September. In Pavliš there was one apiary, where samples were taken from two colonies in June, July, August and September (Table 1).

The samples were taken on the same day from each apiary. Forager bees were collected directly from the outer forage frame, containing bees but no brood (Van Der Steen, De Kraker & Grotenhuis, 2012). It is documented that adult forager honeybees, specialized in gathering of nectar and pollen, are located at the outer frame of the hive (Bilalov et al., 2015). About 100 of worker bees, weighing aproximatelly10g, were taken and transferred directly into sterile plastic containers. The bees were killed in the laboratory by freezing at −21 ± 3 °C. Considering that there are approximately 20 to 80 thousand worker bees in each colony, our experiment did not influence the survival of the bee colony.

Sample preparation and analysis

Samples of honeybees were measured and dried in the oven at 60 °C until constant mass (cca. 96h). Aliquot of each dried sample around 0.5000 g was measured and digested according to the US EPA SW-846 Method 3052, in closed Teflon vessels, under high pressure, with 7 ml of concentrated HNO₃ (p.a.) and 2 ml of concentrated H₂O₂ (p.a.). Digestion was performed in a closed microwave digestion system (ETHOS 1, Advanced Microwave Digestion System, Milestone, Italy) by gradually heating the samples up to 200 °C (15 min), followed by another 15 min at the same temperature. Each sample was cooled, transferred to a 25-mL volumetric flask and diluted to 25 mL with deionized water.

The concentrations of As, Cs, Hg, Mo, Sb, Se, U and V were determined by the inductively coupled plasma mass spectrometry (ICP-MS) using Agilent 7500ce instrument equipped with Octopole Reaction System (ORS) in FullQuant mode. The measurements for every sample were done in three replicates and the average values were calculated for every sample. Calibration of ICP-MS was performed using Multielement Standard Solution IV (Fluka) with six standard solutions. Standard solutions and blanks were prepared in 2% HNO₃. Tuning solution containing 1 µg l⁻¹ Li, Mg, Co, Y, Ce and Tl (Agilent, Santa Clara, CA, USA) was used for the instrument optimization. Accuracy was in the range 94–108% of the certified reference material SLRS-6 (river water certified reference material for trace metals and other constituents, National Research Council of Canada).

LOD were 0.015 mg kg⁻¹, 0.002 mg kg⁻¹, 0.01 mg kg⁻¹, 0.07 mg kg⁻¹, 0.01 mg kg⁻¹, 0.083 mg kg⁻¹, 0.001 mg kg⁻¹, and 0.001 mg kg⁻¹ for As, Cs, Hg, Mo, Sb, Se, U and V respectively.

Data analysis

Average values and standard deviations were calculated for every region. Normality of the data was checked using Kolmogorov–Smirnov test. For some elements, the data was not normally distributed. Such data was log-transformed prior to further statistical analysis. Statistically significant differences between locations, year or month of sampling were determined using one-way ANOVA, followed by Tukey’s post hoc test (differences were considered significant if p ≤ 0.05). For statistical analysis, in samples where the elements concentrations were below LOD the used values were one half of LOD.

Spatial variations

Higher Sb concentrations in urban regions has been reported in earlier studies (Manta et al., 2002; Yongming et al., 2006). Concentrations of Sb in urban environments has been linked to intense traffic, specifically to the use of brakes (Thorpe & Harrison, 2008). Antimony was detected in brake dust, since manufacturers use Sb₂S₂ instead of asbestos in brake linings (Jang & Kim, 2000; Von Uexküll et al., 2005).

Considering that the results showed higher concentrations of U, Se and V in the area impacted by thermal power plants Kostolac A and Kostolac B, it can be assumed that the elements originate from coal combustion occurring in these power plants. It was already pointed out that these power plants are sources of high concentrations of Al, Cr and Fe found in the region (Zarić et al., 2016; Zarić et al., 2018). Earlier studies showed that U comes from the combustion of coal in thermal power plants (Sengupta & Agrahari, 2017).The same was observed for coal fired power plants in Serbia (Životić et al., 2008; Ćujić et al., 2015). The study by Životić et al. (2008) also stated that non-volatile elements like U are mostly retained in solid combustion waste. The TPP region also contains an ash disposal site, which implicates that higher concentrations of U detected in honeybees from this region are the result of coal combustion in thermal power plants Kostolac A and Kostolac B. Since the concentrations of Se and V in TPP region are higher only in comparison to MS it can be suggested that these elements do not originate only from coal combustion.

Temporal variations

In BG higher concentrations of Sb and U in July compared to September of 2014 is most likely due to ecology of the honeybees and plant lifecycle. In July more plants bloom and honeybees more frequently fly out of the hive to collect pollen and nectar (Zarić et al., 2016). With the reduction of amount of pollen and nectar in the autumn (September) brood rearing is decreased, and with it the proportion of older bees in the colony. This leads to larger amount of younger bees in the sample that have been less exposed to environmental pollution (Kauffeld, 1980).

Higher concentrations in September compared to July in 2014 in PA and PV for As, Sb, and Hg, respectively, can be explained by extreme weather conditions during the months of intense bee activity. During 2014 PA and PV had extreme rain and biggest floods in the last 100 years (Tošić, Unkašević & Putniković, 2017; Zarić et al., 2017). Earlier studies concluded that wet weather has an impact on the reduction of metal concentrations in honeybees (Lambert et al., 2012; Satta et al., 2012).

Although for most of the samples taken in 2014 there were no statistically significant differences between the months of the sampling it can be observed that the concentrations are higher in July, which was explained by the ecology of honeybees and life cycle of plants (Table S1).

To better study temporal variation in metal concentrations, two locations, PA and PV, were selected out of the five previously sampled. At these locations samples were taken in June, July, August and September 2015. ANOVA statistical analysis was performed on these samples. Each location was tested separately, as well as with the other location to check whether there were significant differences between different months of sampling. The results showed no statistically significant differences whatsoever. Standard deviations are high due to the nature of honeybees (Formicki et al., 2013; Zarić et al., 2016). This could be the reason behind the lack of statistically significant differences, although differences in concentrations between months can be observed in Table 3.

Also, metal concentrations in 2014 and 2015 were compared to check if there are differences between the years of sampling. Statistically significant differences were observed; Hg, Mo and V have higher concentration in 2014. It should be mentioned that Hg was below the LOD for all of the samples in 2015, except for location PV in June, so this affected the findings. In an earlier study it was concluded that metal concentration in honeybees from Serbia are in decline throughout the years (Zarić et al., 2017). The present study confirms previous conclusions.

Comparison with literature data

For the analyzed elements available literature data together with data from this study is presented in Table 5. For Cs and U there is no literature data available. To the best of our knowledge this is the first time that concentrations of Cs and U in bodies of honeybees are reported.

Range and average concentrations of most analyzed trace elements are in the ranges reported in the literature. Only Sb average concentrations in this study are considerably lower than those reported in two studies by Van der Steen et al. (2012) and Van der Steen et al. (2016), although they have the same range.

Factors influencing metal accumulation by honeybees

There are a number of factors that could influence the level of metal accumulation by honeybees. The most important factor is certainly the levels of metals present in the environment surrounding the honeybee colony. But regardless of levels of metals there factors including health of the colony and individual bees and their age that can also influence metal accumulation.

Healthy colonies with healthy individual bees are more likely to accumulate larger amounts of metals. This is because bees in healthy colonies are more vital. More bees go foraging and consequently more metals are accumulated. Honeybee health can be altered in different ways. Pathogens are one of the most common reasons for the decline of health of individual bees, as well as whole bee colonies (Potts et al., 2010). Another reason behind unhealthy bees and colonies can be the influence of agrochemicals, mainly pesticides. Even sub-lethal exposure to pesticides can have a negative effect on bees. It can affect their communication, as well as their lifecycle. Some pesticides can cause the shortening the life of bees up to 4 days (Wu, Anelli & Sheppard, 2011). This can influence the accumulation of metals, since these bees are a shorter time exposed to metals.

Age of the individual bees that were sampled can also have an important effect on concentrations of metals present in the honeybees. Younger bees that have not been outside of the hive are exposed to pollutants through food brought into the hive by older bees or by impact of immediate surrounding of the hive. Forager bees are bees that are 23–38 days old (Huang & Robinson, 1996). These are the bees that fly out of the hive and are able to accumulate metal pollution directly from the environment. This is the reason why mostly these bees are chosen for bioindicator studies.

Potential effects of studied elements on human health

As can have an acute and chronic poisoning effect on humans. Acute poisoning leads to muscular pain, nausea and vomiting, abdominal pain, diarrhea, and different vascular problems that can cause circulatory collapse and kidney damage (Saha et al., 1999). Chronic exposure leads to skin lesions, and damage of internal organs, the respiratory, digestive, circulatory, neural, and renal systems. Cancer is the most significant consequence of chronic arsenic poisoning (Ng, Wang & Shraim, 2003).

Stable cesium 133 is found to be relatively safe. Some signs of mild toxicity are hypotension, gastrointestinal distress, numbness of the lips. Although it was thought that cancer cells are vulnerable to Cs there is no evidence to support this claim (Melnikov & Zanoni, 2010). Radiocesium 137 due to its emitting of beta and gama radiation is extremely hazardous even without taking it in to the body. Radioactive Cs is associated with Thyroid Cancer (Sangvanich et al., 2010).

Hg can have different effects depending on the exposure route. Inhalation of Hg vapor interstitial pneumonitis, corrosive bronchitis and may affect the nervous system. Ingestion of Hg leads to corrosive ulceration, necrosis of the gastrointestinal tract and bleeding followed by gastrointestinal damage and renal failure(Goyer & Clarkson, 2001). Methyl mercury causes neurotoxic effects in adults and toxicity to the fetuses of mothers exposed to methyl mercury during pregnancy (Bakir et al., 1973).

Mo is an essential element. The deficiency of molybdenum can cause mouth and gum disorders, hypouricemia, hyperoxypurinemia, mental disturbances, and coma. Although essential Mo can also be toxic effecting appetite, slowing growth, and causing diarrhea and anemia (Nielsen, 1996).

Many compounds containing Sb are gastrointestinal irritants that can cause nausea, vomiting, abdominal colic and diarrhea. It can also have an effect on respiratory and cardiovascular system, where it can cause myocardial damage, heart failure and cardiac arrest (Winship, 1987).

Deficiency of Se can cause Keshan disease, which is an endemic cardiomyopathy. When intake exceeds excretory capacity selenium can become toxic. Toxic effects include skin eruptions and lesions, diseased nails, and can cause different neurological symptoms (Goyer & Clarkson, 2001).

Some U salts will cause skin burns. The soluble uranium can cause acute renal damage and renal failure (Goyer & Clarkson, 2001).

Although V is thought to be an essential element no deficiency disease has been discovered. The toxicity of vanadium increases with its valence. V is mostly toxic to lungs, causing bronchitis and bronchopneumonia, but it can also cause gastrointestinal distress (Barceloux, 1999).