Ecotoxicol. Environ. Contam., v. 8, n.

2, 2013, 31-36
doi: 10.5132/eec.2013.02.005

Detection of metals by differential pulse anodic stripping

voltammetry (DPASV) in pollen collected from a fragment of the
atlantic forest in Piracicaba/SP
T.A. da Silveira1; D.F.D. Araujo1; L.C. Marchini1; A.C.C.C. Moreti2 & R.A. Olinda3

1
Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, ESALQ/USP. Departamento de Entomologia e
Acarologia, Laboratório de Insetos Úteis.
2
Instituto de Zootecnia, Secretaria de Agricultura e Abastecimento
3
Universidade Estadual da Paraíba, Centro de Ciências e Tecnologia – CCT/UEPB

(Received September 14, 2012; Accept July 26, 2013)

Abstract

Detection techniques of trace elements have high costs for routine analysis while differential pulse anodic stripping voltammetry
(DPASV) has low operational costs and few structural requirements. This technique was considered a good alternative for
pollen analysis. In the analyses, Zinc, Copper and Lead showed concentrations higher than the threshold established for
human daily intake of food; however, Cadmium concentrations did not exceed the limits. The high concentrations found ​​may
be explained by the geographical locality of the apiary, which is located near the main road to access the city and a water and
sewage treatment plant. In addition, other anthropogenic processes, namely agricultural, industrial and residential activities
may have influenced. The technique of voltammetry can be considered a good alternative for pollen routine analysis, providing
accuracy at lower costs. The study provides information about the contamination of the matrix of bee pollen.
Keywords: bee, biomonitoring, detection limits, trace elements.

INTRODUCTION worker bees through nectar and saliva substances, collected at

the hive entrance. Although pollen has well-defined rules as
Interest in consumption of safe products has been to their physical-chemical compositions, the presence of trace
increasing in the past years worldwide. People have become metals (heavy metals) in pollen is another factor has greatly
more concerned about the health conditions and well-being, concerned inspecting agencies. Produced in areas near urban
seeking food with no or minimal concentrations of chemical environments to facilitate the activity, pollen may be subject
additives (preservatives, acidulants, among others). to contamination sources (roads, farms, buildings, etc.) that go
unnoticed by beekeepers (Pohl et al., 2009).
In the group of natural foods, safety plays a key role in
their marketing and acceptance in the market, receiving a Bees move around an approximately 10 km radius to
great deal of attention from inspecting agencies. Over the search for food source quality, and during those trips, they
years, bee products (pollen, royal jelly, bee venom) as well as come into contact with many contamination sources. Heavy
honey have gained a significant market share in the sector of metals in the atmosphere can be deposited on flowers, water,
natural products. air, soil and hairs of bee body, and thus, be transported the
According to Ordinance No. 3 of 2001 of the Brazilian hives along with pollen grains (Porrini et al., 2003).
Ministry of Agriculture and Supply (Brasil 2001), pollen is It is known that trace metals such as sodium, potassium,
defined as the result of agglutination of flower pollen, made by calcium, iron, zinc and copper can be considered essential for

*Corresponding author: Talita Antonia da Silveira; e-mail: tali_aband@hotmail.com

32 Ecotoxicol. Environ. Contam., v. 8, n. 2, 2013 Silveira et al.

the biological metabolism of living organisms. Other metals 30 minutes. Afterwards, the material was cooled to room
such as lead, cadmium, mercury, aluminum are classified as temperature, then 2 mL of nitric acid (HNO3) and 3 mL of
micro-contaminants of the environment, toxic or non-essential concentrated perchloric acid (HClO4, Sigma-Aldrich®) were
to living organisms and at high concentrations, can be even added to submit to reflux for 60 minutes once again. Thereafter,
lethal (Oga et al., 2008) the condenser was removed for the elimination of acids and
Detection techniques of trace metals generally have high concentration of the sample. The concentrate was diluted in
costs to be used in routine analysis (Tonietto et al., 2012). ultrapure water (Mohm 18.2 cm) in a 25 mL volumetric flask.
However, differential pulse anodic stripping voltammetry The blank solution containing only acid used in the digestion
(DPASV), has low operating costs and few structural underwent a similar procedure (Pereira, 2010).
requirements, besides having a scanning of “ppt” (parts per
trillion) (Rodrigues 1998). Voltammetry
Therefore, this study aimed to apply the voltammetry The trace metals (Cu, Pb, Cd and Zn) were quantified in a
technique as an alternative to measure trace metals in pollen voltammetric analyzer 797 VA ComputraceMetrohm© using
for in routine analysis, providing accuracy at lower costs electrochemical measures. The differential pulse voltammetry
over other methods. The present study aimed to also provide is an electroanalytical technique based on phenomena
information about the contamination of the matrix of pollen. occurring at the interface between the electrode surface and
the thin layer of solution adjacent to the surface. The method
MATERIALS AND METHODS is considered dynamic, because the electrochemical cell is
operated in the presence of electrical current (i > 0), which,
The experiment was conducted in a fragment of the Atlantic in turn, is measured by the controlled application of potential
forest located at the Department of Entomology and Acarology (Skoog et al., 1992) Therefore, information on the analyte
at College of Agriculture “Luiz de Queiroz”, campus ESALQ/ (aqueous solution containing ions) is obtained by measuring
USP, in the municipality of Piracicaba, in São Paulo state, the magnitude of the electrical current that appears in the
Brazil. The site was chosen for having a 7-km radius and its working electrode by applying a voltage between a working
proximity to agricultural, industrial and urban areas, a sewage and an auxiliary electrode. The registration of the current
and water treatment plant of the Piracicamirim River and main potential may be related to the amount of analyte found in the
road to access the city. Thus, the bee products generated could electrode interface (Montenegro et al., 1991).
be exposed to contamination sources. The equipment is coupled to a computer with the 797 VA
In the beehive, we kept five colonies of Africanized honey Computrace software version 1.3.1 Metrohm® to record and
bees (Apis mellifera L) Langstroth-type boxes. The pollen was read the measures. To determine zinc (mg Kg-1), cadmium
sampled during one year in each of the four seasons (summer, (µg Kg-1), lead (mg Kg-1) and copper (mg Kg-1), we used the
autumn, winter and spring) with two collections, one at the technique of differential pulse anodic stripping voltammetry
beginning and another at the end of the season (A and B winter; (DPASV) (Taddia et al., 2004).
C and D spring; E and F summer and G and H autumn). Were The standard addition method was used for the determination
installed a front-type trap to obtain at least 50 g of pollen per of the metals. The reagents and standard solutions used
beehive per season. The beehives were kept under conditions were from Sigma-Aldrich® and QuimLabSpecSol®. The
to favor pollen collection, standardization of the bee population solutions were prepared with ultrapure water (Mohm 18.2
and percentage of pollen retained in the traps. cm) and all flasks were kept in HNO3 at 10% for 24 hours
for decontamination. Prior to use, all material was rinsed with
Samples ultrapure water. The parameters and conditions of the analyses
are shown in Table 1.
Pollen loads in the traps were removed every collection
day, cleaned by grooming, weighed and stored in 250 mL
Statistical Analyses
plastic bottles, sealed and stored in a freezer at - 26°C until
analysis. In all analyses, we used samples in a fresh base. The data for the Zn, Cu, Pb and Cd were analyzed by
analysis of variance (ANOVA). We checked the ANOVA
Analysis of minerals and trace metals: Zn, Cu, Cd and Pb assumptions using the optimal transformation family of Box-
Cox (Box et. al., 1964) and the Hartley (1950) test was used
to verify the homogeneity variances. The experimental design
Sample digestion
was completely randomized and the treatments comprised
We weighed approximately 0.5 g of the pollen sample different pollen types. Once the ANOVA assumptions
in a 250 mL flat-bottomed flask and 5 mL of concentrated were verified, we applied the F test (p < 0.05) to investigate
nitric acid (HNO3, Sigma-Aldrich®) was added, with a reflux possible differences between the levels of the Pollen factor.
condenser connected to the flask mouth. The flaks were kept We proceeded with the analysis by performing test of means
on a plate heated at 120°C for the mixture remain in reflux comparisons, two by two, in the Tukey test (p < 0.05). In order
Detection of metals by differential pulse... Ecotoxicol. Environ. Contam., v. 8, n. 2, 2013 33

Table 1 – Parameters and conditions used in the polarographic cell for the high compared to the recommended daily intake for adults of
determination of Zn2+, Cd2+, Pb2+ and Cu2+ by DPASV
about 15 mg Kg-1 (Franco 1999). Serra-Bonvehí et al. (1997)
Voltametric Parameters Unity Zn2+, Cd2+, Pb2+and Cu2+ found different results for pollens collected in Spain where
the Zn contents did not exceed a maximum of 81.1 mg Kg-1
Dilluted sample volume mL 1 and minimum of 18.8 mg Kg-1, which are similar to results for
other honey products in that country. In Australia, Somerville
Ultrapure water volume mL 10
& Nikol (2002) found Zn contents similar those in this study,
Electrolytes
which may be linked to the dominant Eucalyptus species in
Brazil that has great Zn contents. Morgano et .al., (2012)
KCl 1.5 mol L-1 and Acetate found Zn contents that ranged from 41.4-65.3 mg Kg-1, and
mL 2
of Sodium 0.5 mol L-1 only one sample presented a lower content (5.1 mg Kg-1).

Working electrode HMDE

Intoxication cases may occur either through ingestion of
contaminated food and beverages, by exposure to dust and fumes
No. of drops 4 with high zinc concentrations or skin contact with zinc salts. Zinc
No. of additions 2 becomes dangerous when combined with other metals such as
cadmium and lead (Rocha et al., 1985) (Table 2).
No. of replications 3
The results show that only Cadmium levels in spring
Reference electrode Ag/AgCl (KCl 3 mol L -1) exceeded the limit recommended for daily intake (Table 2).
Auxiliar electrode Platinum FAO (2005) established a provisionally tolerable weekly
intake of 7μg Kg-1 of body mass index. Considering the
Shake speed 2000 RPM threshold for a 70 Kg person, the cadmium concentration found
Purge time (N2ultrapure) s 300 did not exceed 490 µg Kg-1. In the other seasons, cadmium
showed concentrations below tolerable limits. Morgano et
Additional purge time s 10
al., (2010) apud Leita, reported high Cd contents (near 50 m)
Deposition potential V -1.15 in urban centers with high traffic intensity that surpassed the
Deposition time s 90
concentration of 2.1 mg Kg-1 in the pollen collected by bees in
this region. Morgado et al., (2010) found Cd in 79% of the
Equilibrium time s 10
samples, which was greater than in this study (<0.001-0.233
Pulse amplitude mV 0.05 mg Kg-1) compared to samples collected in Italy (<0.015-0.090
mg Kg-1, wet weight), in the United Kingdom (<0.07-0.13 mg
Initial potential V -1.15
Kg-1) and Spain (0.07-0.10 mg Kg-1) (Maff 1995).
Zn 2+
V -0.98
Cadmium is listed in 8th place as hazardous substances
Cd2+ V -0.61 (Salgado 2008) Cadmium in the environment is absorbed and
retained by aquatic and terrestrial plants, by animals that feed
Pb2+ V - 0.38
on these plants and ultimately by humans, as the last link of
Cu2+ V -0.16
the food chain (Ramos 2005).
Lead concentrations exceeded the tolerable limits in all
to verify if the seasons can be classified using the concentrations samples (Table 2). The weekly tolerable intake is 25μg Kg-1 of
of heavy metals (Zn, Cu, Pb and Cd), we used the canonical body mass (WHO 2005), which is equivalent to 3.57 μg Kg-1 of
discriminant analysis. Data were analyzed using the computer body weight per day. It is worth noting that decree No. 685 of
program (SAS/Stat 9.1.3 Service Pack 2, 2003). August 27, 1998, of the National Agency of Health Vigilance

RESULTS AND DISCUSSION Table 2 - Mean values ​​(1) of trace elements zinc (mg Kg-1), copper (mg Kg-1),
lead (mg Kg-1) and cadmium (µg Kg-1) in pollen loads collected in hives of
Apis mellifera in the municipality of Piracicaba, São Paulo state, Brazil, in
The analysis of Zn, Cu, Pb and Cd showed different four seasons between 2010-2011.
profiles in each season. Thus, a number of variables such as
Season Metals
climate, season and botanical origin must be considered when
Zn 2+ Cd2+ Pb2+ Cu2+
using bees or bee products to monitor trace metals in the
Winter 193.63±21.42 A 115.80±50.29BC 29.620±2.38 A 85.66 ±21.95A
environment (Bogdanov 2006; Embrapa Clima Temperado Winter 259.52±57.93A 231.04±55.54 B 27.320±3.85A 65.16±7.58 AB
2008). The mineral composition depends not only on the Spring 231.04±50.81 A 565.54±85.34 A 32.020±6.04 A 53.36±6.87 AB
botanical origin, but on the growth conditions such as soil Spring 212.22±34.39 A 245.08±17.18 B 25.340±1.89 A 44.98±4.50 B
type and geographical origin (Serra-Bonvehí et al., 1997). Summer 223.30±34.86 A 195.00±21.45 B 25.440±3.90 A 37.32 ±4.72B
Summer 229.38±50.29 A 159.22±41.20BC 26.200±3.82 A 55.52±7.05 AB
The average zinc concentrations found in pollen collected Autumn 187.72±14.46 A 199.90±35.74 B 26.400 ±4.60A 38.66±6.38 B
in the four seasons showed no significant differences in the Autumn 124.58±14.58AB 112.60±9.56 BC 23.320 ±3.84A 36.70 ±5.67B
variance analysis. In this study, the maximum Zn content was Means followed by the same letters do not differ statistically (p< 0.05) in the
259.52 mg Kg-1 and minimum 124.58 mg Kg-1, which are very Tukey test. (1)
34 Ecotoxicol. Environ. Contam., v. 8, n. 2, 2013 Silveira et al.

(Brazil), established lead threshold for several foodstuffs, Pb, Zn and Cd reached 200% greater in urban pollen compared
however, there is no reference to levels ​​in bee products. Thus, to fresh pollen, depending on the species.
lead concentration in soils varies from region to region, so in Will hardly reported cases of intoxication with ingestion of
regions close to roads with heavy traffic and industries, lead pollen once the maximum daily consumption of this product
levels are much higher than in isolated areas (Larini 1993). in humans is 25 g that hardly reach the maximum limit of
The high level of lead in spring may be explained by a higher daily ingestion of metals studied (Zn, Cd, Pb and Cu).
occurrence of wind in this season, which would transport the
metal over long distances.
Canonical Discriminant Analysis
Lead showed a high concentration in the first sampling
of spring when cadmium concentrations were also high. In the set of samples evaluated, including heavy metals
Morgano et al., (2010) conducted studies in São Paulo State, concentration (Zn, Cd, Pb and Cu) and the seasons when the
Brazil, and found pollen with higher Cd and Pb contents in the samplings were made, we attempted to determine whether the
spring in the month of October, and Pb was present in 63% seasons would discriminate among one another.
of the samples. Similarly to results found for Cd contents, Evaluating the sample dispersion and the ellipses formation
São Paulo State showed the highest Pb contents (<0.01-0.44 in some groups (Fig. 1), there is a separation on one of the
mg Kg-1). The same condition was reported in studies carried canonical axes which represents the first sampling in spring
out on pollens in Europe (<0.02-0.329 mg Kg-1 in Italy; 0.3- that was the only sampling to distant from the others. In order
1.5 mg Kg-1, in the United Kingdom and 0.1-0.14 mg Kg-1 in to verify if the seasons can be classified by concentrations
Spain) (Maff 1995). of heavy metals (Zn, Cu, Pb and Cd), we used a canonical
discriminant analysis based on Mahalanobis distance, taking
The study of metal content of Swiss honeys and honeydew
into account the correlations between the variables with which
in contaminated areas (large cities, roads, incineration areas) different patterns can be identified and analyzed. Therefore, it
showed a positive relationship between Pb and Cd in honey was possible to determine similarity between the samples of
and their concentrations in the environment. The authors winter, summer, autumn and spring 2, considering correlations
also noted that while Pb concentration increases in double, in the data set.
Cd concentrations did not change. This is because cadmium
These results for the metals analyzed in pollen corroborate
reaches the soil and contamination occurs in the soil, while Pb
data found by (Bromenshenk et al., 1984; Kovacheva et al.,
contamination occurs in the air. The cited authors noted that
2000; Lozak et al., 2002; Atrouse et al., 2004; Rissato et al.,
metal concentrations in honey are directly related to botanical
2006 & Sobukola et al., 2008), where accidental contamination
resources, considering, therefore, honey as a good indicator of
may occur due to environmental pollution caused by pesticides
Pb concentrations in various regions of a contaminated area
and fertilizers in water and soil, or air caused by heavy traffic
within a radius of 2 Km (Bogdanov 1986).
of vehicles emitting pollutants.
The daily intake threshold for copper established by the Thus, concentrations of trace metals in honey bee products
Brazilian legislation for adults is 900 μg (Brasil 2005). In reflect the profile of metal of a given region, which may be a
healthy adults and not occupationally exposed to copper, useful tool to assess contamination levels of the environment.
the main pathway for the copper ingestion is oral. Chronic Therefore, determining trace metals concentrations is of
intoxication in humans is uncommon and is usually associated great importance especially for quality control of honey bee
with cases of accidental ingestion (Lima et al., 2001). products, since higher levels of metals are undesirable because
Like other metals, copper is dispersed in the environment of their toxicity (Bogdanov et al. 2003).
by wind, rain and water streams. Water is an important source
of copper exposure, especially in households where pipes are
made of copper. Copper concentration in food generally varies
from 0.2 to 44 µg g-1 of dry weight (Lima et al., 2001).
Copper showed an average of 85.6 mg Kg-1 in the first
sampling in winter and 36.7 mg Kg-1 in the second sampling in
fall. Morgano et al., (2012) found concentrations between 10-
18 mg Kg-1 for the São Paulo State. This high contamination
level can be associated to the proximity of the area with the
water and sewage treatment plant, since copper sulphate is
used to inhibit algae growth in tanks of the treatment plant
(Ramos 2005).
Kalbande et al., (2008) evaluated pollen grains from
different species under different conditions of exposition to the
environment and observed that there is a significant difference
between the fresh pollen and pollen from urban centers in
terms of metal contents in the pollen. The concentrations of Figure 1 – Discriminant functions of seasons and metal concentrations.
Detection of metals by differential pulse... Ecotoxicol. Environ. Contam., v. 8, n. 2, 2013 35

The technique of voltammetry to measure trace elements in HARTLEY, H.O. 1950. The use of range in analysis of variance.
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