I N V E S T I G A T I O N O F T H E USE OF HONEY BEES AND HONEY BEE

PRODUCTS TO ASSESS HEAVY METALS CONTAMINATION

L. LEITAI, * G. MUHLBACHOVA 2, S. CESCO 1, R. BARBATrINI 3 and C. MONDINI 4

l Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Via delle Scienze 208, 1-33100 Udine,
Italy; 2 Research Institute of Crop Production, Drnovska 507,CZ-16105 Prague, Czech Republic;
3 Dipartimento di Biologia Applicata alla Difesa delle Piante, Via delle Scienze 208, 1-33100 Udine,
Italy; 4 Istituto Sperimentale per la Nutrizione delle Piante, section of Gorizia, Via Trieste 23,
1-34170 Gorizia, Italy

Abstract. Experiment was carried out using 12 colonies of honey bees bred in hives located near an
extraurban crossroad. We analyzed the Pb, Cd and Zn deposited on the bee's surfaces and the heavy
metal accumulation in the foragers, dead bees, honey products and some environmental markers
during nine weeks of the experiment. Results showed a large amount of Zn and Cd on the bee's
surface as a consequence of atmospheric fallout, whereas Pb seems to be either water-extractable
and/or likely accumulated in the body of the insect. Dead bees expelled from the hives displayed a
progressive accumulation of all heavy metals during the experimental period. Royal jelly and honey
contained large amounts of heavy metals. In particular, we found a linear relationship between Cd in
the honey and that found in flowers of Trifoliumpratense L. Results obtained suggested that honey bee
products and the examined environmental markers may be considered useful parameters to assess the
presence of environmental contaminants, whereas the measurements of heavy metals in the dead bees
may be considered a suitable tool also to verify a possible dynamics of accumulation of pollutants.

I. Introduction

T h e approach to ecological thought and research proposed by Tansley (1935) sug-

gesting that the living systems are in a stable state o f dynamic equilibrium, led us
to evaluate the pollution o f an ecosystem as a stress imposed. Therefore, anthro-
pogenic activities are responsible for disturbing onvironmental equilibria, almost
through qualitative and quantitative modifications o f animal and plant species
enables to be vectors o f toxic molecules or accumulate them in target organs and
products.
Surveying the environmental pollution status is increasing b e c o m i n g task o f
biological monitors, i.e., animal and plant organisms able to provide useful infor-
mation. The use o f biomonitors of the environment contamination, such as honey
bee, over chemical or physical detectors, would appear to have potential in this
respect (Jones, 1987). Bees, foragers in particular, readily inhabit all environmental
compartments (soil, vegetation, air and water) and are manageable, social insects
generating a wide amount o f material for sampling and analysis (Crane, 1975).
Bees actively forage the area around the hive and the n u m b e r o f foragers per hive
is quite great. In fact, in a single hive there is an average o f 104 foragers and each

* To whom correspondence should be addressed.

Environmental Monitoring and Assessment 43" 1-9, 1996.

t~) 1996 Kluwer Academic Publishers. Printed in the Netherlands.
2 L. LEITA ET AL.

of them works daily about 103 flowers, providing a largely representative sampling
strategy characterized by about 107 sampling points per day. An average sample of
pollutants can be obtained with honey bees from an area of more than 7 km 2 (Celli,
1984; Bromenshenk and Carlson, 1985). Pollutants may affect honey bee colonies
by several ways, e.g., contamination of the body, especially as concern pollutants
which are likely to be encountered either in a gaseous form or a water-soluble form.
Most research work has emphasized deleterious impacts to bees rather than the
use of bees as chemical monitors. Because bees have low tolerance to many toxic
chemicals (Wallwolk-Barber et aL, 1982), they provide a potentially sensitive indi-
cation of pollutant-induced harm. In addition, pollination service and bee products
can be affected by environmental contamination. The potential use of bee products,
such as honey, indicating level of environmental contamination has received some
attention (Barbina et al., 1993; Bromenshenk and Carlson, 1985; Bromenshenk
et al., 1988). However, there is still a lack of reliable information from which to
assess the usefulness of honey as environmental monitor (Cesco et al., 1994). For
example, the extent to which honey 'reflects' the elemental composition of foraged
plants, of soil or of atmospheric deposition is unclear (Jones, 1987).
According with Freedman and Hutchinson (1981 ) there are six major categories
of sources of environmental metal contamination: natural sources, metal contain-
ing agricultural amendments, disposal of wastes from mines or mills, industrial
emissions, emissions from municipal utilities, and emission from moving sources,
principally automobiles. In particular, petroleum fuels when combusted are a source
of heavy metals, as are fuel additives, particularly tetraethyl Pb. Antiwear protec-
tants incorporated in lubricants often contain, among metals, Cd, Pb and Zn, which
are released to the environment. A network of hives established near a crossroad
may provide a constant monitoring system that requires very little maintenance
and which may provide information on the emission of Pb, Zn and Cd from a point
source.
The aim of this work is to study the heavy metal accumulation in honey bee
and honey bee products collected from hives located next to a crossroad in order
to assess the response of honey bee to environmental pollution by heavy metals. In
addition we studied possible relationships between honey bee products and some
environmental markers.

2. Materials and Methods

2.1. SE'ITING AND CHARACTERISTIC OF HIVES

The environmental monitoring was performed in summertime (from July to Sep-

tember), using 12 colonies of Apis meUifera ligustica Spin bred in hives (Dadant-
Blatt model) at 10 combs. All the combs were replaced before setting them in
the sampling area. Hives were placed at about 50 m from an extranrban crossroad
(average of traffic density: 104 vehicles/day).
 HEAVY METALS CONTAMINATION 3

Four hives were equipped with a modified Gary trap (Marchetti et al., 1987),
in order to collect the dead foragers expelled by the colony. In addition, a 'pollen
catching' trap was fitted on four hives to collect the pollen from the body surface
of foragers before their entrance into the hive.
The combs in the hives were replaced weekly to verify a possible dynamics of
metal accumulation in honey bees and their products. The experiment was carded
out for nine weeks.

2.2. SAMPLING

We followed the survey design proposed by Italian Ministry of Agriculture and

Forests for researches aimed to evaluate the methods on biological materials and
standardized by Greatti et al. (1994).
- B e e - Foragers were caught at the entrance of the hive during the sampling
day. Dead bees expelled from the hives by the colony during the sampling
week were collected.
- B e e p r o d u c t s - Honey and royal jelly were collected weekly and kept in freezer
at-25 ~
- Environmental markers
- Pollen was collected in the pollen-catching trap.
- Propolis was gathered by scraping the internal surface of the hives.
- Flowers of T r i f o l i u m p r a t e n s e L. as the most representative specie of fodder
crop in the sampling area were collected at progressive distance from hives.
Sampling area was divided in four sub-areas, at 3, 10, 20 and 30 m radius
around the hives, where honey bee foraging was greater (Greatti e t a l . 1994).
Flowers in each sub-area were collected randomly.
- Rainwater was collected into pluviometers set at progressive distance (3-
30 m) from hives. All samples were kept in freezer at-25 ~ before analysis.

2.3. ANALYTICAL PROCEDURES

Collected foragers were immediately washed after sampling with distilled water
(milli Q) in order to quantify the water soluble Pb, Zn and Cd deposited on the
surface of their body. Washing solutions were filtered at 0.2 # m (nitro-cellulose
Whatman filter) before metal measurements. Washed foragers and dead bees were
oven dried at 105 ~ for 24 h and pre-treated for 12 h in concentrated HNO3
(1/10 w/v). Thereafter mineralizations were carried out by means of microwave
digestion system (C.E.M. corps MDS 81D) by successive 20 min steps of 50%-
70%-50% full power (700 MHz). After centrifugation at 8500 g for 10 min, solu-
tions were analyzed for Cd, Pb and Zn content by ICP-OES (Optima 3000 Perkin
Elmer). Analytical procedure was standardized using CRM 278 BCR reference
material. Honey bee products and the environmental markers were mineralized in
concentrated nitric acid (1/10 w/v) by microwave digestion in glass carbon vessels
4 L. LEITA ET AL.

set at 70% full power for 3 cycles at 20 min/each. Procedure was standardized using
CRM 062 BCR reference material and methodological suggestions described in
EUR Reports of EC Environment and quality of life.
Rainwater was filtered at 0.2 #m and directly analyzed for Pb, Cd and Zn content
by means of ICP-OES (Optima 3000 Perkin-Elmer)

3. Results and Discussion

In Table I are reported the amounts of Pb, Zn and Cd removed by distilled water
from the forager's surface, and the total content of metals measured by subsequent
mineralization of bees.
The amount of Pb removed by distilled water was ever lower than 1/~g ml- 1,
whereas the Pb accumulated in the foragers was larger from the second week of
sampling, and showed a tendency to increase throughout the experiment. Converse-
ly to expectation, the amount of water soluble Pb was lower than detectable limit
(1 #g g - i ) and seems to be strongly bound on the insect's surfaces or/and likely to
be accumulated in the bee's body.
A different response was found for Zn and Cd. The washing solutions contained
greater amounts of both metals in comparison to those accumulated. In particular,
the Cd accumulated in the foragers was surprisingly low (about one order of
magnitude), if compared with the corresponding Cd removed by washing from the
insect's surface. The amount of Zn in the washing solution was slightly greater
than that accumulated. As a consequence, it is reasonable to presume that Cd and
Zn deposited on the forager's surface probably on bee hairs which were able to be
a receptacle of particulate of atmospheric fallout and pollen foraging (Cesco et al.,
1994).
According with Accorti et aL (1990), dead bees collected in contaminated
environments, such as trafficked areas, contained large concentration of heavy
metals. We found a progressive increase of Pb, Cd and Zn concentration in dead
expelled bees throughout the experiment. The amount of heavy metals increased
after the first week of the experiment and did not differ markedly till the fifth week.
Afterwards Pb, Zn and Cd increased largely till the end of the experiment. Statistical
regression showed a well fitted linear relationship between metal accumulation and
the time of duration of exposure, as reported in Figure 1.
There is scarce information on relationship between the amount of Pb, Zn and
Cd in honey bee products and the degree and dynamics of environmental pollution.
We did not find any relationships between the heavy metals content in honey bee
products and time of sampling. Therefore Table II reports the mean (• SE) of the
total content of heavy metals in honey and royal jelly samples collected weekly.
Results obtained showed that Pb content in honey was largely greater than the limit
of 0.215 ppm suggested by Otto and Jekat (1977) for honey consumption, and the
amount of Cd was higher than that found by Celli et al. (1987) and Jones (1987).
Table I
Lead, Cadmium and Zinc (mean concentration 4- S.E.) removed by water washing from the surface of foragers and the amount of heavy
metals accumulated in their body measured after subsequent mineralization
rv
Weeks Lead Cadmium Zinc
water accumulated water accumulated water accumulated
<>
,.<
washing washing washing
#g/ml 4- S.E. #g/mg 4- S.E. #g/ml 4- S.E. /zg/mg 4- S.E. #g/ml 4- S.E. /~g/mg 4- S.E.
r"
<1 <1 8.4-1-2.2 1.9-1-0.5 79.14- 0.8 76.24- 7.1
<1 1.4 -4- 0.2 10.1 + 2.2 1.5 + 0.5 87.5 4- 14.2 66.4 -4- 12.8 o
z
<1 1.4 4- 0.9 10.1 4- 3.9 1.4 4- 0.1 88.8 4- 13.3 52.5 4- 13.8
<1 1.74-0.8 9.64-3.1 1.34-0.1 88.14-13.8 63.54-11.3 z
<1 1.9 4- 0.1 10.6 4- 3.2 1.2 4- 0.1 99.3 4- 28.4 54.4 4- 0.3
<I 2.24-0.8 9.64-1.9 1.34-0.1 87.24-10.6 54.54- 6.8 z
<1 1.84-1.7 11.54-1.7 1.24-0.2 83.04- 6.8 56.94- 6.4
<1 2.0 4- 1.4 10.1 4- 2.2 1.1 4- 0.1 87.4 4- 14.2 57.8 4- 8.7
<1 3.0 4- 1.6 10.1 4- 4.0 1.4 4- 0.2 65.8 4- 8.9 67.2 4- 1.3
6 L. LEITAET AL.

27 Pb
21
15
9 9 9 r=0.89 (P<0.01)
3 y = 0.241 + 2.622x

1100
cD 900
cD 700
"Q 500
300 9 r = 0 . 9 2 (P<O.O1)

100 y = 41.3 + 126.08x

i i i i i i i i |

7
r.-4
!

5
3
y ,= - 0:504,+ 0.7,86x

1 2 3 4 5 6 7 8 9

Weeks
Figure 1. Accumulation trend of Cd, Zn and Pb in dead bees collected throughout nine weeks of
experiment.

Table II
Content of Pb, Cd and Zn (mean 4- S.E.) in honey-bee products

Lead Cadmium Zinc

Honey (pg. g - i f.w.) 1.8 -t- 0.5 1.9 -I- 0.2 29.4 4- 3.6
Royal Jelly (pg. g - i f.w.) 13.1 4- 0.5 2.9 4- 0.6 86.6 4- 10.4

Royal jelly contained the largest amount of heavy metals, however there is no
information of its natural content of Zn.
In order to verify a possible dynamic and the status of the contamination of sam-
piing area, we analyzed some environmental marker, such as pollen in the catching
trap, propolis, rainwater and flowers of Trifolium pratense L.. As previously, we
found a great variability in the amounts of heavy metals collected weekly. Table III
 HEAVY METALS CONTAMINATION 7
Table III
Heavy metal content (mean 4- S.E.) in environmental markers of sampling
area
Lead Cadmium Zinc
Rainwater (#g. ml -m) 3.0 -4- 1.0 1.4 4- 0.2 101.3-4- 6.5
Flowers (#g. g-l d.w.) 10.74- 3.0 1.7 4- 0.3 75.3 + 15.0
Pollen (#g.g-i f.w.) 3.94-0.7 2.1 4-0.2 99.04- 9.2
Propolis (/~g. g-i f.w.) 13.7 4- 6.6 1.5 4- 0.3 325.2 4- 73.1

2.4
Y = 0.69 * X + 0.74
r --- 0.85 (P < 0.01) ~
(13
t--
2.2
0
--r
0 ;y -
2.0

1.8
r~

1.6

1.4 1.6 1.8 2.0 2.2

lxg Cd. dw of Trifoliumpratonse
Figure 2. Correlation between Cd contents of flowers of Trifoliumpratense L. and honey.

showed that Pb, Cd and Zn occurred in all materials. In particular, the content o f Pb
and Cd, pure contaminants, in samples underlined and characterized the environ-
mental pollution o f sampling area. In opposite o f Pb and Cd, Zn is a micronutrient
o f biota, and therefore is unevenly present. The Zn content in flowers o f Trifolium
pratense L. was in agreement with natural occurrence reported by Kabata-Pendias
and Pendias (1992), whereas other authors (e.g., Robson, 1993) reported natural
content markedly lower in plants. Differently, pollen from flowers occurring within
the bee's flight range, collected into the trap and, overall, propolis and rainwater
8 L. LEITAET AL.

contained great amounts o f Zn, which underlined, together with both toxic metals
Pb and Cd, the environmental contamination.
Because o f the particular flower morphology, Trifolium pratense L. specie is
largely foraged by bees for nectar collection (Cesco et al. 1994) and for honey
production. We did statistical analysis (ANOVA) in order to verify a possible rela-
tion between the honey bee products and environmental markers. Results obtained
showed significative relationship only between Cd content in honey and its amount
in flowers o f Trifolium pratense L. (Figure 2), whereas no relationships were found
for Zn and Pb. The lack o f statistical correlation for both metals may be caused by
the different chemical behaviour o f Zn, Pb and Cd in the biological and biogeo-
chemical cycles. In fact, conversely to most heavy metals which showed a tendency
to be scarcely mobile, Cd displayed more reactivity and mobility throughout the
environmental compartments (Verkleij and Schat, 1990).
Therefore, our results suggested that honey bee products and examined envi-
ronmental markers may be considered useful parameters to assess the presence o f
environmental contaminants. Moreover, the measurements o f heavy metals in the
dead bees may be considered a suitable tool also to verify a possible dynamics o f
accumulation of pollutants.

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 HEAVY METALSCONTAMINATION 9

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