the use of honeybees and honey as environmental ...key words: honeybees, honey, environmental...

Draft

THE USE OF HONEYBEES AND HONEY AS ENVIRONMENTAL

BIOINDICATORS FOR METALS AND RADIONUCLIDES: A

REVIEW

Journal: Environmental Reviews

Manuscript ID er-2017-0029.R3

Manuscript Type: Review

Date Submitted by the Author: 21-Jun-2017

Complete List of Authors: Herrero Latorre, Carlos; Universidade de Santiago de Compostela, Departamento de Química Analítica, Nutrición y Bromatología Barciela García, Julia; Universidade de Santiago de Comnpostela, Química Analítica, Nutrición y Bromatología García Martín, Sagrario; Universidade de Santiago de Compostela, Química Analítica, Nutrición y Bromatología Peña Crecente, Rosa M; Universidade de Santiago de Compostela, Departamento de Química Analítica, Nutrición y Bromatología

Keyword: honeybees, honey, environmental bioindicator, metals, radionuclides

https://mc06.manuscriptcentral.com/er-pubs

Environmental Reviews

Draft

1

THE USE OF HONEYBEES AND HONEY AS ENVIRONMENTAL

BIOINDICATORS FOR METALS AND RADIONUCLIDES: A REVIEW

C. Herrero-Latorre, J. Barciela-García, S. García-Martín, R.M. Peña-Crecente

Universidade de Santiago de Compostela, Dpto. Química Analítica, Nutrición y

Bromatología, Facultad de Ciencias, Alfonso X el Sabio s/n, 27002 Lugo, Spain

Corresponding author:

Carlos Herrero-Latorre

Universidade de Santiago de Compostela, Dpto. Química Analítica, Nutrición y

Bromatología, Facultad de Ciencias, Alfonso X el Sabio s/n, 27002 Lugo, Spain

Telephone number: 34 982 824 064

Fax number: 34 982 824 001

e-mail address: [email protected]

Word count: 13962

Page 1 of 46



Draft

2

ABSTRACT

Honeybees interact strongly with vegetables, air, soil and water in the vicinity of

the hive and, as a consequence, pollutants from these sources are translated to the

honeybees themselves and to the hive products. Therefore, over the last decades of the

past century, honeybees and honey have been proposed as possible bioindicators for the

study of the environmental status of the area surrounding the hive.

This work is a critical review on the use of the hive as a passive sampling device

and on the use of honeybees and honey as environmental bioindicator substrates for

metals and radionuclides. The design of sampling networks, sampling procedures,

sample pretreatments, analytical techniques, data analysis and other influencing factors

in this area are also reviewed on the basis of more than 80 references.

Key words: Honeybees, honey, environmental bioindicator, metals, radionuclides.

INDEX

1. Introduction

2. Honeybees and honey as pollution bioindicators

2.1. Sample collection: the hive as a passive sampling device

2.2. Sample pretreatment and analytical determination techniques

2.3. The use of honeybees and honey for assessing pollution

3. Considerations on the use of honeybees and honey as environmental biomarkers

4. Conclusion

5. References

Page 2 of 46



Draft

3

1. INTRODUCTION

Honeybees (Apis mellifera L.) forage in a wide range of vegetables and water

sources in different places and therefore they strongly interact with the environment

around the hive. Consequently, honeybees, honey and other products of the hive reflect

the pollutants that are present in (or on) the forage plants, the atmosphere, the water and

the soil of the area in which the hive is located. This fact led to the proposal that

honeybees themselves, honey and other related products could be potential bioindicators

to monitor pollution in the vicinity of the hive (Devillers and Pham-Delègue 2002). The

use of honeybees and their products –as well as other insects such as ants (Grześ 2010)–

for environmental monitoring purposes is not a new idea. Svoboda (1961) published the

first report on the adverse effects of arsenic on honeybees caused by industrial pollution

in certain areas of Czechoslovakia. The same author (Svoboda, 1962) proposed a

system for monitoring 90Sr from nuclear experiments through the radioactivity produced

by this radioisotope in honey. Therefore, hive-products have been used to monitor

different types of contaminants, particularly metals and radionuclides. Heavy metals and

radionuclides (from the Chernobyl catastrophe) are ubiquitous contaminants in most

environmental scenarios, and both pollutant types have important influence on living

organisms. Moreover, the dynamics of these contaminants in the biosphere are the same

or very similar in most of cases because they have similar chemical properties.

Metals are found naturally in the earth and they become concentrated by

anthropogenic activities. The main sources of metals include mining and industrial

wastes, vehicle emissions, urban emissions, wastewater and agricultural activities,

amongst others. Over the last decades of the past century, different studies based on

honeybees, honey or other hive products have been carried out to evaluate and monitor

pollution due to heavy metals in industrial and urban areas. With regard to

Page 3 of 46



Draft

4

radionuclides, two main sources are responsible for artificial radionuclide production

and dispersion throughout the biosphere: atomic weapons tests and accidents in nuclear

plants. Other minor emissions of radioactive material can be produced in nuclear

processing and reprocessing plants, atomic and medical laboratories and other nuclear

facilities. The first studies on the levels of radionuclides in honeybees and honey were

carried out in the United States in New York (Gilbert and Lisk 1978) and in Los

Alamos National Laboratory, New Mexico (Fresquez et al. 1997a, 1997b). The accident

at Chernobyl (Ukraine) in 1986 released a great quantity and variety of radionuclides

into the atmosphere during a period of at least nine days. These nuclides were

subsequently transported by the winds and reached large areas of Europe. Therefore,

from these data the effect of radionuclides (such as 137Cs, 134Cs, 131I, 60Co, and others) in

European countries (Porrini et al. 2002a) was also studied by evaluating the two

substrates considered here, namely honeybees and honey. In general, two main

approaches have been applied to obtain useful information on the environmental status

of the hive surroundings. Firstly, the use of information provided by the honeybees

themselves and, secondly, the use of information obtained by chemical analysis of other

bee products, particularly honey. The use of both strategies constitutes a very interesting

and economical way to obtain useful samples for testing the environmental status of the

area in question. This review examines the published literature on the use of honeybees

and honey as bioindicators to assess environmental metal and radionuclide pollution and

the important effects that these two contaminants have on living organisms. For both

groups of contaminants the design of the monitoring network, the monitoring strategies,

the sampling procedures and sample pretreatments, the different analytical techniques

employed for their determination and data analysis are all discussed. In addition, the

question as to whether honeybees or honey is the best environmental marker remains a

Page 4 of 46



Draft

5

matter of debate and, as a consequence, the use of both substrates is critically evaluated.

2. HONEYBEES AND HONEY AS POLLUTION BIOINDICATORS

Honeybees can fly up to 4–5 km from the hive in all directions, but the majority

of flights (due to the energetic consumption of bees) are in the range up to 2 km.

Therefore, the area that is realistically sampled by honeybees is represented by a

circular area of approximately 12 km2 surrounding the hive (Hoopingarner and Waller

1993), although other authors consider larger (Seeley 1995) or smaller (Crane 1984)

areas. On the basis of the extensive area foraged by bees, and taking into account that

the production of 1 kg of honey requires more than 100,000 foraging flights, it is clear

that both honeybees and honey could be appropriate random sample substrates that may

be highly representative of the average levels of bioavailable pollutants in the foraging

area environment. Other diverse bee products (such as propolis, wax and pollen) have

been employed as sampling materials to monitor environmental pollution (Celli and

Maccagnani 2003; Kalbande et al. 2008), but honeybees and honey are the most

commonly employed matrices among all the hive products.

When honeybees collect nectar, honeydew, pollen and other plant exudates, they

come into contact with plants, soil, air and water. In contaminated areas, both metals

and radionuclides from different sources can be globally distributed throughout the

biosphere (and the stratosphere in the case of radionuclides from weapons tests), and

therefore they enter the food chain by vegetal and animal uptake. If the surroundings of

the hive are polluted by heavy metals or artificial radionuclides, contaminants

distributed in these media are ultimately incorporated into the honeybees and the hive

products (Figure 1). This contamination results in an alteration (generally elevation) of

the levels of these undesired pollutants (Pohl et al. 2009). Thus, different analytical

Page 5 of 46



Draft

6

aspects should be taken into account during sample collection, sample pretreatment and

the determination procedures employed when honeybees and honey are used as

environmental biomarkers.

2.1. Sample collection: the hive as a passive sampling device

The economic cost of monitoring with honeybees or honey is markedly lower

than for other traditional sampling procedures. Hives are low cost devices for spatial

and temporal surveys and their management does not require specialist personnel. In

addition, beehives do not require a power supply and they can therefore be placed in

isolated locations in which infrastructure to monitor the environment is not available

(Van der Steen 2016). Moreover, honeybees and honey have other advantages as

monitoring substrates. Apis mellifera L. is a species of particular interest for this goal

for the following reasons: (i) its management is well known by humans since ancient

times; (ii) honeybees have a high rate of reproduction; (iii) the physiology, behavior and

ecology of honeybees are well known; and (iv) they are ubiquitous organisms with

modest food requirements (Porrini et al. 2002b; Badiou-Bénéteau et al. 2013). On the

other hand, the use of honey has other different advantages: (i) the sample produced by

stationary apiaries is descriptive of long temporal patterns of the foraging periods; (ii)

the samples are readily available and easy to obtain and manage; (iii) honey is a water

soluble matrix and it is chemically simple, and (iv) in general the use of honey reduces

the sample pretreatment required prior to analysis in comparison with honeybees. For

these reasons, both of the aforementioned substrates have been used as appropriate

environmental bioindicators.

It is clear that sampling is the critical step when honeybees or hive-products are

used to obtain environmental chemical information on the distribution of pollutants in

the considered area. The experimental design, the number of colonies or apiaries to be

Page 6 of 46



Draft

7

considered, where and when a colony should be sampled, the substrate to be employed

to extract environmental information, the corresponding size of the sample according to

the substrate used, amongst other factors, all have an influence and they need to be

carefully considered. Despite the information outlined above, in a large proportion of

published papers (see Tables 1–3), the sampling design commonly employed is simple

and it involves locating a certain number of hives in the polluted area to be sampled.

Another set of honeybee colonies was also placed in unpolluted zones for comparison

purposes (as a control). The number of sampling colonies or apiaries, the number of

honeybees or honey samples analyzed and their size are highly variable. It is also

remarkable that in a high percentage of these studies, information concerning the

criteria followed for choosing these relevant parameters is not provided. Moreover, and

with the exception of extensive monitoring works, a significant number of studies used

a limited number of samples obtained from a reduced number of sampling points over a

short period of time. This fact, as stated by various authors themselves, limits the

conclusions that can be drawn about the environmental health of the sampled area.

In an interesting recent study Van der Steen (2016) proposed that the whole

honeybee colony should be considered as a passive bio-sampler for pollutants and that

this approach works on three levels (see Figure 2). The first level concerns the use of

hives/apiaries in the area to be studied for sampling the environment of the surroundings

(field sampling), the second involves the sub-sampling of these hives to obtain

representative amounts of honeybees or other hive-products such as honey

(colonies/apiary sub-sampling), and the third consists of analytical determinations on

honeybees or honey and data processing to provide useful environmental information

(chemical determinations and data analysis). From this point of view it is evident that it

is possible (or necessary) to use statistical tools in all three blocks: (i) in order to design

Page 7 of 46



Draft

8

the spatial and temporal extent of the monitoring network in the field, including the

number of sampling points to be considered, (ii) in order to decide how, where, when

and in what quantity the considered hives should be appropriately subsampled to obtain

representative subsamples of honeybees or honey, and finally (iii) in order to evaluate

the chemical data obtained in the previous block. It is not the objective of this work to

present the different statistical methods available for such tasks. For this goal, interested

readers can consult the excellent review paper by Pirk et al. (2013), in which certain

guidelines on the statistical designs for honeybee-based research are presented.

Moreover, the specific chapter on statistical guidelines in the first volume of BeeBook

on standard methods for Apis mellifera research edited by V. Dietemann et al. (2016)

(also including supplementary examples) is also a good source for a deeper

understanding of this aspect. Two examples of the application of this strategy of

considering the whole hive as a bio-sampler for monitoring metal concentrations are

summarized in Figure 3. In both cases, hives were subsampled for adult honeybees as

the measurement substrate. It can be seen that a framework consisting of seven steps

was defined in order to use appropriately the honeybee colony as a passive sampling

device. Once the target analytes had been selected (a set of 18 metals), the colonies

could be placed according to the ubiety and distribution of the pollutants in the random

foraging area. The number of colonies and the choice of sampling with individual hives

or with pooled samples from each apiary must be taken based on the objective of the

study. In the first work (Van der Steen et al. 2012), performed with the goal of studying

the potential of honeybees to detect and monitor spatial and temporal metal

concentrations, three sampling locations in the Netherlands (urban, rural and industrial)

were employed with three honeybee colonies per location (a total of 9 colonies) during a

period of three months. Individual colonies were measured to ascertain whether there

Page 8 of 46



Draft

9

were differences between them. However, in the second paper (Van der Steen et al.

2016), which concerned a surveillance study for metals in honeybees in the Netherlands

carried out in 2008, a very high number of 150 apiaries in 9 regions of the country were

necessary in order to achieve an adequate sampling procedure. The sample size was

calculated on the basis of binomial probability theory and taking into account two

factors: the percentage of foraging bees carrying the target analyte to the hive and the

probability of detection of the target analyte (Pirk et al. 2013). Once the colonies had

acted as a passive sampling device during the appropriate time, the next step was to

subsample the hive to obtain a representative honeybee subsample. The subsampling of

the hive can be carried out in both sacrificial and non-sacrificial ways. In the sacrificial

approach, the bees (or other hive-products) are killed (or destroyed) for analysis,

whereas in the non-sacrificial strategy the bees are not killed and the target matter is

picked up from the body surface of the bees by means of appropriate devices. The

number of honeybees that constitutes an adequate subsample was also calculated on the

basis of the binomial factors mentioned above. With this approach, it is necessary to

acquire several hundred bees for low concentration analytes, while for more abundant

targets several teens are enough (Pirk et al. 2013). In both cases at hand, representative

sacrificial subsamples composed of 100–150 worker honeybees were taken from the

outer frame of each hive. Finally, the metal determinations were performed by using

inductively coupled plasma optical emission spectrometry (ICP-OES) after acid

mineralization of the honeybee subsamples. Thus, following this (or a similar)

framework strategy, representative samples can be attained with a good chance of

detecting the target analyte.

The subsampling process for collecting honeybees or honey merits a few further

comments. In the case of sacrificial honeybee subsampling, diverse types of structures,

Page 9 of 46



Draft

10

traps and devices for the collection of bees have been employed, such as Gary´s cages,

underbasket traps, dead bee traps and electronic bee counters, amongst others (see

Porrini et al., 2002b for a detailed description). For non-sacrificial honeybee collection

tubes internally lined with a plastic sheet (Halbwirth et al. 2014) or beehold tubes

covered by a thin transparent PVC foil holding a sticky polyethylene (PEG) layer were

used. The plastic sheet or PEG layer physically adheres the particles attached to the hair

and feet of the honeybees entering the hive (Van der Steen 2016). In the case of honey,

the acquisition of the sample is simpler because it can be obtained by the extraction

process commonly used by beekeepers, and the only question is how to obtain sufficient

subsamples to achieve good representativeness of the final pooled sample. It is

important to note that appropriate materials and procedures should be employed in order

to avoid contamination of the sample, both for the collection of honeybees and the

extraction of honey (especially in centrifugation and storage vessels). In addition, for

this purpose some authors recommend the use of apiaries particularly constructed using

fir wood and with all other elements free from any metal parts (including the hive door

and honeycomb spacer) (Conti and Botrè 2001). If the analysis is not performed

immediately, the appropriate storage of the sample is another important issue. For

suitable conservation of the sample, honeybees must be frozen (or lyophilized), dried

and pulverized, and honey must be stored in glass containers at 4 ºC in the dark.

2.2. Sample pretreatment and analytical determination techniques

Once the hive has been used for field sampling and the appropriate substrate

subsample has been obtained from it, honeybees or honey must be pretreated before

chemical measurements are made. The sample pretreatment clearly depends on two

main factors: the type of contaminant to be determined (and therefore the analytical

technique employed) and the substrate employed. In the following subchapter, the

Page 10 of 46



Draft

11

different sample pretreatments of honeybees or honey for the determination of metals or

radionuclides are considered and discussed.

2.2.1. Metals and Heavy Metals. Metal determinations in biological samples can be

made with appropriate figures of merit by using diverse atomic spectrometry

techniques. Therefore, the pretreatment of honeybee or honey samples involves

mineralization of the substrate in order to eliminate the organic matrix and to obtain an

acidic solution containing the metal ions. In addition, the mineralization step has other

advantages. Firstly, the similarity between the matrices of the sample and standard

solutions of metals allows direct calibration in instrumental determinations, thus

avoiding the use of the standard addition method. Secondly, the problems caused by

carbonaceous residues of honey in burners, nebulizers and graphite furnaces of the

atomic spectrometers used for metal determination are prevented (Pohl 2009). As can be

seen from the results in Table 1, in most of the published papers wet-acid digestion is

the method of choice for the honeybee substrate. A few grams of dried (or lyophilized)

and homogenized honeybee sample were treated with HNO3(c) or different mixtures of

H2O2 and HNO3 (Leita et al. 1996; Fakhimzadeh and Lodenius 2000a, 2000b; Perugini

et al. 2011; Sadegh et al. 2012; Satta et al. 2012; Silici et al. 2013; Gutiérrez et al. 2015)

at high temperature and/or with microwave-assisted systems. Other acids such as H2SO4

(Lambert et al. 2012) and HCl (Van der Steen et al. 2012) have also been used. After

this mineralization step, the determination of a set of metals using different atomic

spectrometric techniques was performed on the solution obtained after appropriate

dilution. The digestion step is avoided in cases where the measurements are aimed at

studying the metals deposited on the honeybee (e.g., for the evaluation of air pollution).

In this case, collected foragers were directly washed with dilute HNO3 in order to

extract soluble metals from the surface of their bodies (Leita et al. 1996), and the

Page 11 of 46



Draft

12

resulting acidic solution was subsequently subjected to chemical analysis.

When honey was employed as the sampling substrate, both dry or acid wet

decomposition were used in order to produce a measurable solution. By employing

these two mineralization procedures, the honey organic matrix was removed and the

metals contained in the sample were appropriately transferred to the acid solution as

ions. It can be seen from the references cited in Table 2 that two main approaches have

been applied for honey mineralization: (i) dry ash up to 450–750 ºC (Üren et al. 1998;

Samimi et al. 2001; Bratu and Giorgescu 2005; Wieczorek et al. 2006; Rodríguez-

García et al. 2006; Yarsan et al. 2007; Lambert et al. 2012) and (ii) wet acid digestions

with HNO3 or other acids, frequently mixed with H2O2, using open vessels or sealed

PTFE bombs in microwave-assisted systems (Devilliers et al. 2002a; Stankowska et al.

2008; Yücel and Sultanoğlu 2012; Berinde and Michnea 2013). Several authors have

carried out comparative studies between the two mineralization procedures and it was

concluded that acid digestion is preferable (Fredes and Montenegro 2006; Raeymaekers

2006). These results were confirmed by analyzing certified reference materials and it

was demonstrated that acid microwave-assisted digestion is the best procedure for

mineralization as it gives the highest recoveries in the measured samples (Tuzen et al.

2007). However, in spite of this evidence, dry ash continues to be a widely used

approach, but in these cases the possible losses of volatile metals that could occur in

open‐vessels should be prevented (Korn et al. 2008).

The determination of metals at low concentrations in the acidic solutions

resulting from the sample pretreatment of honeybees and honey has been performed

using different analytical techniques involving potentiometric stripping analysis (Muñoz

and Palmero 2006), anodic stripping voltammetry (Sanna et al. 2000), X-ray

fluorescence spectroscopy (Golob et al. 2005), ion chromatography (Buldini et al. 2001)

Page 12 of 46



Draft

13

and two-dimensional thin-layer chromatography (Horvat et al. 2002), amongst others.

However, in the majority of monitoring studies, the analytical measurement of the

different metals has been carried out using analytical atomic spectrometric-based

methods (See Tables 1 and 2 and the review articles by Pohl (2009) and Solayman et al.

(2016)). The main advantages of atomic spectrometric techniques are the excellent

selectivity and precision, high or very high sensitivity, and the elevated sample

throughput. In addition, most atomic techniques are easy to use and they are not

expensive. The prevalence of atomic techniques over other approaches is evidenced by

the fact that only one monitoring study based on non-atomic spectrometric techniques

has been published to date (Fermo et al. 2013). In this case, ion chromatography was

used for the determination of diverse ions in honey samples for environmental purposes.

Flame atomic absorption spectrometry (FAAS) and flame atomic emission

spectrometry (FAES) have been employed for the low-cost and rapid determination of

metals present in honey in high concentrations, i.e., in the range of µg mL–1 (such as Ca,

Cu, K, Fe, Li, Mg, Mn, Na and Zn) (Üren et al. 1998; Przybylowski and Wilezyńska

2001; Bratu and Giorgescu 2005; Wieczorek et al. 2006). Electrothermal atomic

absorption spectrometry (ETAAS) (Conti and Botrè 2001; Yarsan et al. 2007; Perugini

et al. 2011; Lambert et al. 2012; Sadegh et al. 2012; Satta et al. 2012; Gutiérrez et al.

2015), given its higher sensitivity, has been employed for the measurement of trace and

heavy metals present in concentrations in the range of ng mL1 (such as Al, Cd, Co, Cr,

Pd, etc.). In certain cases, a combination of FAAS and ETAAS or ICP has been used on

the basis of the concentration levels of the metals to be determined (Fakhimzadeh and

Lodenius 2000a, 2000b; Fredes and Montenegro 2006; Raeymaekers 2006; Rodríguez-

García et al. 2006; Stankowska et al. 2008; Berinde and Michnea 2013; Silici et al.

2013). Moreover, other atomic techniques, such as hydride generation-atomic

Page 13 of 46



Draft

14

absorption spectrometry (HG-AAS) and cold vapor atomic fluorescence spectrometry

(CVAFS) (Raeymaekers 2006), have been employed in environmental studies with

appropriate results obtained for the determination of Hg, As and Se. However,

inductively coupled plasma spectrometry (ICP), due to the great benefit of its

multielemental character, seems to be the most appropriate analytical technique to be

considered in further developments. The two ICP variants were successfully used for

multielemental determinations in a single measurement, i.e., inductively coupled plasma

optical emission spectrometry (ICP-OES) (Leita et al. 1996; Devillers et al. 2002a;

Raeymaekers 2006; Yücel and Sultanoğlu 2012; Van der Steen et al. 2012, 2015) and

inductively coupled plasma atomic emission spectrometry-mass spectrometry (ICP-MS)

(Bogdanov et al. 2007). ICP-MS has two other advantages over other ICP approaches:

(i) When this technique was applied, a noticeable improvement in sensitivity was

achieved in comparison to other atomic techniques (the detection limits attained by ICP-

MS are very impressive, in the range of ng L–1 or lower). Thus, ICP-MS is the technique

of choice when a very high sensitivity is needed. And (ii), due to the special

characteristics of the mass spectrometry detector, an increasing trend towards the use

ICP-MS as a tool in isotopic analysis is evident (Vanhaecke and Degryse 2012).

Perhaps new complementary tools in monitoring metal studies could be developed

based on using stable isotope ratios (of H, C, N, S, Si and others) measured by MS (or

derived techniques) as additional information for classifying an area as contaminated or

not, and for further ensuring the unpolluted status of control areas.

2.2.2. Radionuclides. Sample pretreatment is not necessary in the case of radionuclides

due to the nature and the high selectivity of the analytical technique employed for their

determination: gamma-ray spectrometry (GRS). Detailed information about the

principles and practice of GRS can be found in the text by Gilmore (2008). In brief, a

Page 14 of 46



Draft

15

radionuclide is an unstable atom due to an excess of nuclear energy. This excess energy

from the nucleus is diminished (to become a stable isotope) by producing new particles

(alpha or beta particles) and gamma radiation. The different radionuclides emit gamma-

rays with diverse intensities and energies (ranging from 15 keV to 10 MeV) and, as a

consequence, the measurement of the intensity of the ionizing radiation emitted is a

reliable way to determine the presence (qualitative) and the concentration (quantitative)

of the radionuclide(s) in question. Moreover, since the gamma spectrum is characteristic

of each gamma-emitting radionuclide, GRS constitutes an extremely selective and

sensitive analytical technique for the quantitative measurement of different

radionuclides in environmental samples.

The great advantages of this technique are: (i) GRS analysis can be performed

directly on several tens of grams of untreated sample (honey or honeybees) directly

placed in the measurement beaker of the gamma-ray spectrometer without any sample

pretreatment; (ii) the good analytical figures of merit achieved in terms of accuracy,

precision, sensitivity and specificity. For these reasons, radionuclide measurements are

much simpler than in the case of metals. Another advantage of the determination of the

radionuclides by means of GRS is its general applicability. In contrast to metals

analysis, the determination of radionuclides was performed in all cases without any

sample pretreatment and using the same GRS analytical technique (see Table 3). These

characteristics facilitate the comparison of results from different scenarios and

countries.

2.3. The use of honeybees and honey for assessing pollution

In this subchapter, a literature survey of different works in which honeybees or

honeys were employed in environmental studies as biomarkers is presented on the basis

of the type of contaminant evaluated.

Page 15 of 46



Draft

16

2.3.1. Metals and heavy metals. Honeybees and honey have been employed in an effort

to biomonitor environmental pollution due to metals. The question of which substrate is

the most appropriate is yet to be answered definitively and this has been under

discussion in the literature published from 1970s to the present day. In 1975, the

pioneering paper by Tong et al. (1975) demonstrated that honey collected in New York

State in the vicinity of a highway contained elevated levels of metals emitted by traffic.

On the other hand, Jones (1987) suggested that the analysis of honeybees could be a

better environmental indicator than honey, especially for atmospheric deposition. Also,

Bromenshenk et al. (1985) employed honeybees as effective monitors for environmental

contaminants (As, Cd and F) in Puget Sound, WA, USA. Following these approaches,

in the past few decades different studies have been carried out using both honeybees and

honey. The studies in which honeybees have been used as the optimal matrix to detect

environmental pollution are summarized in Table 1. Fakhimzadeh and Lodenius (2000a,

2000b) evaluated honeybees, honey and pollen as possible heavy metal indicators in

Finland, and they concluded that honey and pollen are not good environmental markers

(significant differences were not found in metal pollution in these two matrices), while

bees themselves could be better candidates for this goal. Porrini et al. (2002b) and Celli

and Maccagnani (2003) confirmed this result in studies carried out in Italy. A similar

conclusion was also reached by Bogdanov (2006), who stated that honey and other bee

products (such as pollen, propolis, etc.) are inadequate for use as bioindicators for Cd

and Pb, due to the natural data variability with these metals. In addition, a range of other

studies were carried out using honeybees for monitoring purposes in Czechoslovakia

(Veleminsky et al. 1990), Italy (Leita et al. 1996; Conti and Botrè 2001; Porrini et al.

2002b; Perugini et al. 2011; Satta et al. 2012), France (Lambert et al. 2012), Iran

(Sadegh et al. 2012), The Netherlands (Van der Steen et al. 2012, 2015, 2016), Spain

Page 16 of 46



Draft

17

(Gutiérrez et al. 2015) and Turkey (Silici et al. 2013) (details in Table 1). In all of these

cases, the contamination levels measured in the collected honeybees are correlated with

the environmental conditions of the area studied.

A range of papers concerning the use of honey as an environmental marker have

been published since the 1980s in different countries, especially in Italy, where this field

is very active. Balestra et al. (1992) has worked since 1989 on the determination of

heavy metals (Cd, Cr, Ni, and Pb) in honey, pollen and larvae using automatic

monitoring devices near Modena. Other authors, such as Conti and Botrè (2001),

highlighted the usefulness of this substrate as biomarker on the basis of a monitoring

program for heavy metals performed in Rome and its province. Since the 1990s more

than 15 monitoring studies have been published using honey was as environmental

marker in a wide variety of locations of Europe and around the world: Switzerland

(Bogdanov et al. 2007), Turkey (Üren et al. 1998; Tuzen et al. 2007; Yarsan et al. 2007;

Yücel and Sultanoğlu 2012), Poland (Przybylowski and Wilezyńska 2001; Wieczorek et

al. 2006), France (Devilliers et al. 2002a; Lambert et al. 2012), Romania (Bratu and

Georgescu 2005; Zugravu et al. 2009; Berinde and Michnea 2013), Spain (Rodríguez-

García et al. 2006), Macedonia (Stankowska et al. 2008), Germany (Raeymaekers

2006), Chile (Fredes and Montenegro 2006), Italy and the Balkan countries (Fermo et

al. 2013), and Egypt (Rashed et al. 2009). Details of these studies are summarized in

Table 2. Nevertheless, other authors questioned the use of honey due to the lack of

evidence for metal bioaccumulation (Pohl 2009). In addition, the great variability in the

low concentrations of heavy metals detected in different types of honey (according to

geographical origin, floral type, season, climatic conditions, etc.) impedes the

establishment of consensus background levels and indicates that this substrate could be

insensitive for the detection of differences in anthropogenic sources (Tuzen et al. 2007).

Page 17 of 46



Draft

18

However, as it can be seen from the literature, the two approaches using honeybees and

honey have been used. In a study performed in Italy by Leita et al. (1996) determined

three metals (Cd, Pb, Zn) in honey, in mineralized honeybees (absorbed metals) and on

the body surface of honeybees (deposited metals). Based on the results obtained from

this study, these authors concluded that honey is very useful for detecting the presence

of pollutants, while measurements on honeybees (both in mineralized and washed

samples) also serve to follow the dynamics of metal bioaccumulation. From this point

of view, it seems that the two approaches could provide complementary ways to

evaluate environmental pollution due to metals.

It should be noted that other monitoring studies involving the use of other hive-

products alone have not been proposed, except in the case of Kalbande et al. (2008),

who used pollen to biomonitor heavy metals in an urban environment.

2.3.2. Radionuclides. Both honey and honeybees are also employed as substrates to

study the pollution due to radionuclides. The first approach to control radioactive

isotopes was carried out in 1979 in the USA. The surroundings of the Los Alamos

National Laboratory were monitored (from 1979 to 1996) using dead bees as a research

substrate. The results indicate that honeybees could be used advantageously in

comparison to honey: tritium levels in the environment were adequately related to the

content in honeybees but were poorly correlated with the content of this radionuclide in

honey (Fresquez et al. 1997a, 1997b). In addition, studies on radionuclide levels in three

vegetables (white sweet clover, salt cedar and rabbit brush) did not show any significant

differences between these substrates (Haarmann 1998a). Furthermore, bioaccumulation

of natural (22Na) and artificial (60Co) isotopes was detected in honeybees but not in

vegetables (Haarmann 1998b). Tonelli et al. (1990) stated that pollen is the best

bioindicator for radionuclides because this substrate presented higher radionuclide

Page 18 of 46



Draft

19

activity under the same conditions when compared to bees and honey. However, in spite

of this consideration, the majority of European studies on the presence of different

radionuclides carried out in Germany (Bunzl et al. 1988; Assmann-Werthmüller et al.

1991), France (Devillers et al. 2002b), Italy (Panatto et al. 2007; Schiuma et al. 2015;

Meli et al. 2016), Croatia (Barišic et al. 1992, 1994, 1999, 2002; Kezić et al. 1997) and

Poland (Borowska et al. 2013) in the period 1988–2014 were developed by analyzing

honey samples as the substrate (See Table 3 for details). This fact can be explained for

two reasons: (i) due to the sensitivity of GRS, the gamma-activity in honey is sufficient

for a correct radioisotope measurement with monitoring purposes, and (ii) the

acquisition of the honey sample is much easier than for other hive products such as

honeybees or pollen.

3. CONSIDERATIONS ON THE USE OF HONEYBEES AND HONEY AS

ENVIRONMENTAL BIOMARKERS

It is evident that hive products are interesting substrates for monitoring the

environmental status of the area surrounding the colony. The purpose of this work is not

to discuss the levels of the different metals and radionuclides in diverse places

throughout the world because the immense variability in the scenarios affected by the

different boundary conditions (pollution sources, soil, geographical location, matrix

substrate, etc.) would make this task extraordinarily long and of little value. However,

several points with general impact on the strategies for sampling the environment by

using honeybees or honey should be discussed.

(i) Honeybees or honey as the sampling substrate? Honeybees and honey are the main

substrates used to obtain pollution information among all hive-products, and only

limited applications have been published based on other hive products (wax, pollen and

Page 19 of 46



Draft

20

propolis). The use of honeybees themselves or honey as the better environmental

marker remains a matter of debate. However, based on our literature survey, several

points should be considered using bees and honey as environmental indicators. In order

to estimate the performance of these two substrates for monitoring metals and

radionuclides, an evaluation as radar plots is presented in Figure 4 on the basis of five

parameters, SA: Sample availability, EST: Ease of sample treatment, ATA: Analytical

technique availability, EAT: Ease of analytical technique, IP: Information provided. It

can be seen that both honeybees and honey are very useful for monitoring radionuclides.

Sample pretreatment is not required and the widespread use of GRS as an analytical

measurement technique led to consistent and comparable results between different areas

under surveillance in distinct countries worldwide. For monitoring of metals, from a

literature survey it was concluded that appropriate results were also achieved with both

matrices when measured by commonly available and easy analytical techniques such as

FAAS, ETAAS or ICP. However, the comparison of results must be filtered on the basis

of different digestion or ashing procedures employed for the sample preparation, the

diverse analytical atomic spectroscopic techniques used for measurement and the

external influences on the metal content of the substrate, such as soil type and botanical

origin of the sample in the case of honey. In general, honeybees are better substrates for

metal pollution when compared with honey. Experimental results indicate that

honeybees are a more sensitive biomarker matrix for metals than honey (Lambert et al.

2012; Satta et al. 2012) and that they are also able to detect early anthropogenic changes

in environmental conditions (Sadegh et al. 2012). Several comparative studies stated

that honey is not appropriate as an indicator for metal pollution in comparison with bees

themselves. Fakhimzadeh and Lodenius (2000a, 2000b) reported cases in Finland in

which highly polluted honeybees produced unpolluted honey, leading these authors to

Page 20 of 46



Draft

21

conclude that honey may not be a good bio-indicator. Conti and Botre (2001), in a study

in Italy, and Silici et al. (2013) in Turkey, concluded that honeybees themselves better

reflect the status of environmental health than honey. The conclusions drawn from these

various studies suggest that honeybees should be selected as the preferred sampling

matrix for heavy metal monitoring.

The use of bees either dead or alive also merits consideration. In general, the

number of dead bees over a given period of time has been established as a means to

determine the threshold for pesticide impact. If this threshold is surpassed, then analysis

of dead bodies is performed for identifying and quantifying the compound responsible

for the poisoning. Dead bees can also be used for pesticide and metal accumulation

studies (Leita et al. 1996), but live bees (and foragers in particular) are preferred for

monitoring metals (Satta et al. 2012). A bioindication study (Ruschioni et al. 2013)

revealed that high levels of heavy metals are better detected in live than in dead bees. In

addition, sampling with live bees presents other advantages over dead bees such as: (i)

the sample size (number of bees to be sampled) can be selected without death

restrictions; (ii) the age cohort of bees can be selected by sampling in different locations

inside the hive (Van der Steen 2016); (iii) other hive-products (such as pollen) can be

simultaneously sampled when using the appropriate traps; and (iv) the honeybee trail

outside the hive can be tracked and even directed using modern micro-sensors and

sound and chemical signals (Bromenshenk et al. 2015).

(ii) External influencing factors. Factors not directly related to contamination sources

can influence the concentration of metals and radionuclides in the substrates analyzed.

Therefore, these factors must be taken into account when the experimental design is

carried out in order to minimize their effect. Since elements are transferred from the soil

to the plants through the root system, and they pass to the nectar and then finally to the

Page 21 of 46



Draft

22

honeybees and honey (Bogdanov et al. 2007), the soil composition of the foraging area

could affect the levels of metals and radionuclides. Previous studies demonstrated that

honey samples with high natural amounts of minerals offer a higher resistance to reflect

the effects of anthropogenic contamination sources (Üren et al. 1998). However, to date,

a direct relationship between metal content in soil and in honey or honeybees has not

been established because metal uptake can be strongly influenced by soil type and

conditions, pH, rhizosphere, metal bioavailability, etc. However, information about the

soil composition in sampled and control areas could help to elucidate the influence of

this aspect.

Another critical factor when honey is used as a biomarker is the botanical origin

(Yarsan et al. 2007). The uptake of metals by plants is notably affected by the

nutritional requirements of the vegetable: e.g., plants with low nutrient requirements

produce nectar with low metal concentrations (Wieczorek et al. 2006); aromatic plants

concentrate pollutants more than herbaceous plants and honeys produced from the

nectar of deciduous trees have lower metal contents than honeys from evergreen ones

(Devillers et al. 2002a). These facts are also supported by different studies in which the

metal composition of honey was employed to identify the botanical origin of the

product (see the reviews of Pohl (2009); Gonzalvez et al. (2009); Drivelos and

Georgiou (2012); Solayman et al. (2016)). In relation to the radionuclides, several

authors demonstrated that the concentration of these pollutants (such as 239Pu, 240Pu,

137Cs, 90Sr and 40K) in honey could also be dependent on the floral type (Bunzl and

Kracke 1981). In addition, Molzahn and Assmann-Werthmüller (1993) reported

differences in the cesium transfer factor from soil to plant for different vegetables (this

explain the relatively high Cs contamination of heather honeys compared with other

floral honeys). Since the botanical origin influences the metal and radionuclide contents,

Page 22 of 46



Draft

23

when using honey as a bioindicator, information provided by palynological studies

should be included in the analysis. Ideally, honeys employed for this purpose should be

of the same floral origin. Evidently, since in practice this cannot be achieved in most of

cases, the conclusions obtained on the different areas should take into account if honeys

from different botanical origins were considered.

Other external influences on the metal content of the hive-related products, such

as the use of metal components and metal-based wood preservatives in commercial

beehives, must be avoided (Van der Steen 2012).

(iii) Selection of analytes. In general, the selection of the analytes to be measured was

carried out by taking into account the characteristics of the possible pollution sources.

However, the choice of a set of common analytes could be an interesting objective. The

adoption of a list of consensual analytes is an appropriate way to achieve comparable

inter-study results with a lower number of assays. In the case of metals, (with the

exception of particular cases in which the contamination source can be directly related

to one or several specific elements), special attention should be paid to Cd, Cu, Pb, and

Zn (used in more than 75% of the studies surveyed), followed by Cr, Fe, Mn and Ni

(used in 50–75% of the works). In contrast, the analysis of other elements with minor

information on environmental status could be avoided. For the case of radionuclides,

137Cs (used in more than 75% of cases) and 134Cs were considered as the most

interesting markers for contamination produced by nuclear weapons tests, nuclear

accidents and nuclear facilities (Devillers et al. 2002b). Therefore, most efforts should

be focused on these two radionuclides as general radioactivity biomarkers. In addition,

40K seems to be the best candidate for evaluating natural radioactivity.

(iv) The extent and design of the monitoring network. In a recent paper on the statistical

guidelines for research based on honeybees (Pirk et al. 2013), the authors stated that

Page 23 of 46



Draft

24

without a priori conception of the experimental design based on statistical knowledge,

the resulting conclusions obtained can be poor and frequently biased. Therefore, the

design of the sampling network is a fundamental aspect in these studies. The number of

sampling stations and the number of samples analyzed per station should be carefully

planned on the basis of the sampled area and the possible pollution sources. In order to

guarantee representative information, the sampling experimental design might be

assisted by using statistical distribution tools based on the probability of detection of the

considered pollutant in the colonies or apiaries. In addition, when the colonies/apiaries

are subsampled for honeybees and honey, the appropriate sample size can also be

calculated on the basis of statistical considerations in order to obtain, in each case, a

subsample that is representative of the passive sampling device: i.e., the whole hive.

Examples of different networks used on the basis of the determined analytes can be

consulted in Tables 1-3. The general guidelines for the establishment of the network

appropriate size and the number of subsampled colonies is outlined in section 2.1 as

well as in Pirk et al. (2013), Dietemann et al. (2016), and Van der Steen (2016).

In most cases, pollution levels of the sampled area are evaluated by comparing

to areas considered to be unpolluted zones. As a consequence, the selection of locations

designed for control stations warrants special attention. It is assumed that

unambiguously unpolluted areas in natural wildlife reserves or isolated sites have been

used as the best options. However, these areas are not, per definition, unpolluted zones

because atmospheric deposition of metals can take place over large distances from the

emission source (Steinnes, 2013, Van der Steen et al. 2015, 2016). More detailed

studies are required to determine background levels in control areas so that comparisons

can be made among diverse locations used for such tasks in different countries. This

could present an interesting study for establishing common criteria for the selection of

Page 24 of 46



Draft

25

unpolluted control zones.

(v) Sample pretreatment and analytical determination techniques. For metals, it is clear

that the lack of standardized determination protocols constitutes a serious drawback.

The high diversity of sample pretreatment procedures and analytical techniques applied

for honeybees and honey in diverse geographical locations and from different origin

makes it difficult to compare the measurements carried out by different authors in

distinct locations. Although several studies dealing with the effect of different sample

pretreatment procedures on the final results have been published (Fredes and

Montenegro 2006; Tuzen et al. 2007), the need to define a common protocol for

pretreatment and analysis of these substrates has become evident for enabling

comparisons between results, optimizing data collection and minimizing the time and

effort required. As a guideline for establishing future standardized protocols for metals,

microwave-assisted wet acid digestion followed by measurement by ICP-OES or ICP-

MS seems to be the best approach. Following this strategy, the combination of the

appropriate sample pretreatment with a determination technique characterized by its

multielemental character and very low detection limits is considered as the optimum

option.

(vi) Data analysis. In general, the consideration of diverse zones as polluted is made by

comparison with a control zone considered to be unpolluted. Therefore, statistical tools

should be applied in order to test the differences between these areas for each metal or

radionuclide considered; e.g., for parametric data, an ANOVA-test or a paired T-test can

be used to determine whether there are any statistically significant differences between

the means for each metal for the independent studied areas; for-non parametric data two

types of non-parametric data test, the Kolmogorov–Smirnov two-sample test or the

Mann–Whitney U-test, were also used (Porrini et al. 2002b). To test the existence of

Page 25 of 46



Draft

26

significant differences in the whole set of metals (or radionuclides), multivariate

analysis of variance (MANOVA) could be applied (Warne 2014).

Moreover, because some of the analytical techniques employed (such as ICP)

are multielemental ones, the number of metals determined as explanatory variables for

each sample (as well as the number of samples analyzed) can be increased. Therefore,

multivariate chemometric procedures such as principal component analysis or

hierarchical cluster analysis can also be used as display techniques to visualize the data

sets in a reduced dimension and to extract useful information on the relationship

between variables and samples (see as examples the works of Devillers et al. (2002a);

Fredes and Montenegro (2006); Raeymaeker (2006); Rodríguez-García et al. (2006);

Bogdanov et al. (2007); Stankowska et al. (2008); Yücel et al. (2012); Fermo et al.

(2013)). Additionally, classification models based on pattern recognition multivariate

procedures (such as discriminant analysis, soft independent modeling of class analogy,

neural networks and others) could be used for classifying a zone as polluted or

unpolluted on the basis of the levels of metals and radionuclides measured.

5. CONCLUSION

It has been demonstrated that honeybees and honey constitute excellent

substrates for monitoring the environment surrounding the hive. However, to ensure the

success of this approach and to obtain comparable results among a variety of scenarios,

the different steps of the analytical process need to be carried out carefully. Future items

to be considered for an improvement of this approach include the following: (i)

Selection of the appropriate substrate (honeybees or honey); (ii) use of sampling and

sub-sampling strategies appropriately designed, application of standardized sample

pretreatment procedures (wet-acid digestion) and common analytical techniques (ICP-

Page 26 of 46



Draft

27

MS or related one) for metals and GRS for radionuclides; (iii) avoidance of external

undesired influences; (iv) providing botanical origin information when using honey as

the substrate, and (v) extracting useful information from the data with the aid of

multivariate and chemometric procedures.

Page 27 of 46



Draft

28

6. REFERENCES

Assmann-Werthmüller, U., Werthmüller, K., and Molzahn, D. 1991. Cesium contamination of heather

honey. J. Radioanal. Nucl. Ch. 149:123-129.

Badiou-Bénéteau A., Benneveau A., Géret F., Delatte H., Becker N., Brunet J.L., and Reynaud B.,

Belzunces L.P. 2013. Honeybee biomarkers as promising tools to monitor environmental quality.

Environ. Int. 60:31-41.

Balestra V., Celli G., and Porrini C. 1992. Bees, honey, larvae and pollen in biomonitoring atmospheric

pollution. Aerobiologia 8:122-126.

Barišic D., Lulić S., Kezić N., and Vertačnik A. 1992. 137Cs in flowers, pollen and honey from the

Republic of Croatia four years after the Chernobyl accident. Apidologie 23:71-78.

Barišic, D., Lazarić, K. D, Lulić, S., Vertačnik, A., Dražić, M., and Kezić, N. 1994. 40K, 134Cs, and 137Cs

in pollen, honey and soil surface layer in Croatia. Apidologie 25:585-595.

Barišic D., Vertačnik A., Bromenshenk J,J., Kezić N., Lulić, S., Hus, M., Kraljević, P., and Šeletković, Z.

1999. Radionuclides and selected elements in soil and honey from Gorski Kotar, Croatia.

Apidologie 30:277-287.

Barišić, D., Bromenshenk, J.J., Kezić, N., and Vertačnik, A. 2002. The role of honey bees in

environmental monitoring in Croatia. In Honey Bees: Estimating the Environmental Impact of

Chemicals. Edited by Devillers, J., and Pham-Delègue, M.H. Taylor and Francis, London, U.K.,

pp 160-185.

Berinde, Z.M., and Michnea, A.M. 2013. A comparative study on the evolution of environmental and

honey pollution with heavy metals. J. Sci. Arts 2:173-180.

Bogdanov, S. 2006. Contaminants of bee products. Apidologie 37:1-18.

Bogdanov, S., Haldimann, M., Luginbuhl, W., and Gallmann, P. 2007. Minerals in honey:

Environmental, geographical and botanical aspects. J. Apicult. Res. 46:269-275.

Borowska, M.H., Kapala, J., Puścion-Jakubik, A., Horembala, J., and Markiewicz-Źukowska, R. 2013.

Radioactivity of honeys from Poland after the Fukushima accident. B. Environ. Contam. Tox.

91:489-492.

Bratu, I., and Georgescu, C. 2005. Chemical contamination of bee honey-identifying sensor of the

environment pollution. J. Cent. Eur. Agric. 1:467-470.

Bromenshenk, J.J., Carlson, S.R., Simpson, J.C., and Thomas, J.M. 1985. Pollution monitoring of Puget

Page 28 of 46



Draft

29

Sound with honey bees. Science 227:632-634.

Bromenshenk, J.J., Henderson, C.B., Seccomb, R.A., Welch, P.M., Debnam S.E., Firth, D.R. 2015. Bees

as biosensors: chemosensory ability, honey bee monitoring systems, and emergent sensor

technologies derived from the pollinator syndrome. Biosensors 5:678-711.

Buldini, P.L., Cavalli, S., Mevolic, A., and Sharmad, J.L. 2001. Ion chromatographic and voltammetric

determination of heavy and transition metals in honey. Food Chem. 73:487–495.

Bunzl, K., and Kracke, W. 1981. 239/240Pu, 137Cs, 90Sr, and 40K in different types of honey. Health Phys.

41:554-558.

Bunzl, K., and Kracke, W. 1988. Transfer of Chernobyl-derived 134Cs, 137Cs, 131I and 103Ru from flowers

to honey and pollen. J. Environ. Radioactiv. 6:261-269.

Celli, G., and Maccagnani, B. 2003. Honey bees as indicators of environmental pollution. Bull.

Insectology 56:137-139.

Conti, M.E., and Botrè, F. 2001. Honeybees and their products as potential bioindicators of heavy metal

contamination. Environ. Monit. Assess. 69:267-282.

Crane, E. 1984. Bees, honey and pollen as indicators of metals in the environment. Bee world 55:47-49.

Devillers, J., and Pham-Delègue, M.H. 2002. Honey Bees: Estimating the Environmental Impact of

Chemicals. Taylor and Francis, London, U.K.

Devillers, J., Doré, J.C., Marenco, M., Poirier-Duchêne, F., Galand, N., and Viel, C. 2002a.

Chemometrical analysis of 18 metallic and nonmetallic elements found in honeys sold in France. J.

Agric. Food Chem.5:5998–6007.

Devillers, J., Ghouma-Tomasella, N., and Doré, J.C. 2002b. Cesium-134 and Cesium-137 in French

honeys collected after the Chernobyl accident. In Honey Bees:Estimating the Environmental

Impact of Chemicals. Edited by Devillers, J., and Pham-Delègue, M.H.. Taylor and Francis,

London, U.K pp. 151-159.

Dietemann, V., Ellis, J.D., and Neumann, P. (eds.). (2016). COLOSS BeeBook Volume I: Standard

methods for Apis mellifera research. Institute of Bee Health. University of Bern. Bern,

Switzerland. Available from http://www.ibrabee.org.uk/bee-science?layout=edit&id=3740

[accessed 5 April 2017].

Drivelos, S.A., and Georgiou, C.A. 2012. Multi-element and multi-isotope-ratio analysis to determine the

geographical origin of foods in the European Union. TrAC-Trend. Anal. Chem. 40:38-51.

Page 29 of 46



Draft

30

Fakhimzadeh, K., and Lodenius, M. 2000a. Honey, pollen and bees as indicator of metal pollution. Acta

U. Carol. Environ. 14:13-20.

Fakhimzadeh, K., and Lodenius, M. 2000b. Heavy metals in Finnish honey, pollen and honey bees.

Apiacta 32:85-95.

Fermo, P., Beretta, G., Facinio, R.M., Gelmini, F., and Piazzalunga, A. 2013. Ionic profile of honey as

potential indicator of botanical origin and global environmental pollution. Environ. Pollut.

178:173-181.

Fredes, C., and Montenegro, G. 2006. Heavy metals on other trace elements contents in Chilean honey.

Cienc. Investig. Agrar. 33:50-58.

Fresquez, P.R., Amstrong, D.R., and Pratt, L.H. 1997a. Tritium concentrations in bees and honey at Los

Alamos National Laboratory:1979-1996, LA-13202-MS. Los Alamos National Laboratory UC-

902 and UC-908, NM, USA.

Fresquez, P.R., Amstrong, D.R., and Pratt, L.H. 1997b. Radionuclides in bees and honey within and

around Los Alamos National Laboratory. J. Environ. Sci. Heal. A 32:1309-1323.

Gilbert, M.D., and Lisk, D.J. 1978. Honey as an environmental indicator of radionuclide contamination.

B. Environ. Contam. Tox. 19:32-34.

Gilmore, G.R. 2008. Practical Gamma-ray spectrometry. John Wiley and Sons, Chichester, UK.

Golob, T., Doberśek, U., Kump, P., and Nečemer, M. 2005. Determination of trace metals and minor

elements in Slovenian honeys by total X-ray fluorescence spectroscopy. Food Chem. 91:593-600.

Gonzalvez, A., Armenta, S., and de la Guardia, M. 2009. Trace-element composition and stable-isotope

ratio for discrimination of foods with Protected Designation of Origin. TrAC-Trend. Anal. Chem.

28:1295-1311.

Grześ, I.M. 2010. Ants and heavy metal pollution. A review. Eur. J. Soil Biol. 46:350-355.

Gutiérrez, M., Molero, R., Gaju, M., Van der Steen, J., Porrini, C., and Ruiz, J.A. 2015. Assessment of

heavy metal pollution in Cordoba (Spain) by biomonitoring foraging honeybee. Environ. Monit.

Asses. 187: 651-665.

Haarmann, T. 1998a. Honey bees as indicators of radionuclide contamination: comparative studies of

contaminant levels in forager and nurse bees and in the flowers of three plant species. Arch.

Environ. Con. Tox. 35:287-294.

Haarmann, T. 1988b. Honey bees (Hymenoptera Apidae) as indicators of radionuclide contamination:

Page 30 of 46



Draft

31

investigating contaminant redistribution using concentrations in water, flowers and honey bees. J.

Econ. Entomol. 91:1072-1077.

Halbwirth, H., Gosch, C., Stich Persen, U., Marco-Noales, E., Lòpez, M.M., Moosbeckhofer, R.,

Gottsberger, R., Gusina, V., and Petrutis, K. 2014. Endbericht Forschungsprojekt Nr. 100867

(Phytfire): ERA-NET.

Hoopingarner, R.A., and Waller, G.D. 1993. Crop pollination. In The Hive and the Honey Bee. Dadant &

Sons, Hamilton. Edited by Graham JM. IL, USA pp. 1043–1082.

Horvat, A., Šoljić, Z., and Debelić, M. 2002. Qualitative identification of metal ions in honey by two-

dimensional thin-layer chromatography. JPC-J. Planar Chromat. 15:367-370.

Jones, K.C. 1987. Honey as an indicator of heavy metal contamination. Water Air Soil Poll. 33:178-189.

Kalbande, D.M., Dhadse, S.N., Chaudhari, P.R., and Wate, S.R. 2008. Biomonitoring heavy metals by

pollen in urban environment. Environ. Monit. Assess. 138:233-238.

Kezić, N., Hus, M., Seletković, Z., Kraljević, P., Pechhacker, H., Barišic, D., Lulić, S., and Vertačnik, A.

1997. Honey-dew honey as a long term indicator of 137Cs pollution. International Atomic Energy

Agency, Vienna, Austria.

Korn, A., Santos da Boa Morte, E., Muniz Batista dos Santos, D.C., Teixeira Castro, J., Pereira Barbosa,

J.T., Paixão Teixeira, A., Pires Fernandes, A., Welz, B.,, Carvalho dos Santos W.P., Batista

Guimarães Nunes dos Santos, E., and Korn, M. 2008. Sample preparation for the determination of

metals in food samples using spectroanalytical methods—A review. Appl. Spectrosc. Rev. 43:67-

92.

Lambert, O., Piroux, M., Puyo, S., Thorin, C., Larhantec, M., Delbac, F., and Pouliquen, H. 2012. Bees,

honey and pollen as sentinels for lead environmental contamination. Environ. Pollut. 170:254-259.

Leita, L., Muhbachova, G., Cesco, S., Baqrbattini, R., and Mondini, C. 1996. Investigation of the use of

honey bees and honey bee products to assess heavy metals contamination. Environ. Monit. Assess.

43:1-9.

Meli, M.A., Desideri, D., Roselli, C., Feduzi, L., and Benedetti, C. 2016. Radioactivity in honey of the

central Italy. Food Chem. 201: 349-355.

Molzahn, D., and Assmann-Werthmüller, U. 1993. Caesium radioactivity in several selected species of

honey. Sci. Total Environ. 130-131:95-108.

Muñoz, E., and Palmero, S. 2006. Determination of heavy metals in honey by potentiometric stripping

Page 31 of 46



Draft

32

analysis using a continuous flow methodology. Food Chem. 94:478-483.

Panatto, D., Gasparini, R., Lai, P., Rovatti, P., and Gallelli, G. 2007. Long-term decline of 137Cs

concentration in honey in the second decade after Chernobyl accident. Sci. Total Environ.

382:147-152.

Perugini, M., Manera, M., Grotta, L., Abete, M.C., Tarasco, R., and Amonera, M. 2011. Heavy metal

(Hg, Cr, Cd and Pb) contamination in urban areas and wildlife reserves: honeybees as

bioindicators. Biol. Trace Elem. Res. 140:170-176.

Pirk, C.W., Miranda, J.R., Kramer, M., Murray, T.E., Nazzi, F., Shutler, D., Van de Steen, J.M., and Van

Dooremalen, C. 2013. Statistical guidelines for Apis mellifera research. J. Apic. Res. 52:1-24.

Pohl, P. 2009. Determination of metal content in honey by atomic absorption and emission

spectrometries. TrAC-Trend. Anal. Chem. 29:117-128.

Pohl, P., Sergiel, I., and Stecka, H. 2009. Determination and fractionation of metals in honey. CRC Cr.

Rev. Anal. Chem. 39:276-288.

Porrini, C., Ghini, S., Girotti, S., Sabatini, A.G., Gattavecchia, E., and Celli, G. 2002b. Use of honey bees

as bioindicators of environmental pollution in Italy. In Honey Bees: Estimating the Environmental

Impact of Chemicals. Edited by Devillers, J., and Pham-Delègue, M.H. Taylor and Francis,

London, U.K. pp. 186-247.

Porrini, C., Sabatini, A.G., Girotti, S., Ghini, S., Medrzycki, P., Grillenzoni, F., Bortolotti, L.,

Gattavecchia, E., and Celli, G. 2002a. Honey bees and bee products as monitors of the

environmental contamination. Apiacta 38:63-70.

Przybylowski, P., and Wilezyńska, A. 2001. Honey as an environmental marker. Food Chem. 74:2889-

291.

Raeymaekers, R. 2006. A prospective biomonitoring campaign with honey bees in a district of Upper-

Bavaria (Germany). Environ. Monitor. Assess. 116:233-243.

Rashed, M.N., El-Haty, M.T.A., and Mohamed, S.M. 2009. Bee honey as environmental indicator for

pollution with heavy metals. Toxicol. Environ. Chem. 91:389-403.

Rodríguez García, J.C., Iglesias Rodríguez, R., Peña Crecente, R.M, Barciela García, J., García Martín,

S., and Herrero Latorre, C. 2006. Preliminary chemometric study on the use of honey as an

environmental marker in Galicia (NW Spain). J. Agric. Food Chem. 54:7206-7212.

Ruschioni, S., Riolo, P., Minuz, R.L., Stefano, M., Cannella, M., Porrini, C., Isidoro, N. 2013.

Page 32 of 46



Draft

33

Biomonitoring with honeybees of heavy metals and pesticides in nature reserves of the Marche

Region (Italy). Biol. Trace Elem. Res. 154:226-33.

Sadegh, A., Mozafari, A.A., Bahmani, R., and Shokri, K. 2012. Use of honeybees as bio-indicators of

environmental pollution in the Kourdistan province of Iran. J. Apicult. Sci. 56:83-88.

Samimi, A., Maymand, O.M., and Mehrtabatabaei, M. 2001. Determination of Cadmium and Arsenic by

bee honey based on the study of Ja´far abad area from Saved City from Iran. Water Geosci. 199-

202.

Sanna, G., Pilo, M.I., Piu, P.C., Tapparo, A., and Seeber, R. 2000. Determination of heavy metals in

honey by anodic stripping voltammetry at microelectrodes. Anal. Chim. Acta 415:165–173

Satta, A., Verdinelli, M., Ruiu, L., Buffa, F., Salis, S., Sassu, A., and Floris, I. 2012. Combination of

beehive matrices analysis and ant biodiversity to study heavy metal pollution impact in a post-

mining area (Sardinia, Italy). Environ. Sci. Pollut. R. 19:3977-3988.

Schiuma, F., Viti, E., Grippo, A.M., Ghini, S., and Porrini, C., Pace, S. 2015. Biomonitotaggio dei

radionuclii tramite Api nel territorio comunale di Rotondella-MT (2012). Associazioni Scientifiche

per le Informazioni Territoriali e Ambientali. XIX Conferenza Nazionale ASITA, Lecco, Italia (30

september-1 october 2015), pp 939-947.

Seeley, T.D. 1995. The wisdom of the hive. The social physiology of honey bee colonies. Harvard

University Press, Cambridge, MA, USA pp. 47.

Silici, S., Oluozlu, O.L., Tuzen, M., and Soylak, M. 2013. Honeybee and honey as monitors for heavy

metal contamination near the thermal power plant in Mugla, Turkey. Toxicol. Ind. Health 1-10.

Solayman, M., Islam, M.A., Sudip, P., Yousuf, A., Khalil, M.I., Alam, N., and Gan, S.H. 2016.

Physicochemical properties, minerals, trace elements, and heavy metals in honey of different

origins: a comprehensive review. Compr. Rev. Food Sci. F. 15:219-233.

Stankowska, E., Stafilov, T., and Šajn, R. 2008. Monitoring of trace elements in honey from the Republic

of Macedonia by atomic absorption spectrometry. Environ. Monit. Assess. 142:117-126.

Steinnes, E. 2013. Heavy metal contamination of the terrestrial environment from long-range atmospheric

transport: Evidence from 35 years of research in Norway. E3S Web of Conferences 1, 35001.

Svoboda, J. 1961. Industrial poisoning of bees by arsenic. Ved. Pr. Vyzk. Ustavu Vcelarskeho CSAZV

2:55–60.

Svoboda, J. 1962. Teneur en strontium 90 dans les abeilles et dans leurs produits. Bull. Apicole 5:101–

Page 33 of 46



Draft

34

103.

Tonelli, D., Gattavecchia, E., Ghini, S., Porrini, C., Celli, C., and Mercuri, A.M. 1990. Honey bees and

their products as indicators of environmental radioactive pollution. J. Radioanal. Nucl. Ch.

141:427-436.

Tong, S.S.C., Morse, R.A., Bache, C.A., and Lisk, D.J. 1975. Elemental analysis of honey as an indicator

of pollution. Arch. Environ. Health 30:329-332.

Tuzen, M., Silici, S., Mendil, D., and Soylak, M. 2007. Trace element levels in honeys from different

regions of Turkey. Food Chem. 103:325-330.

Üren, A., Şerifoğlu, A., and Sarikahya, Y. 1998. Distribution of elements in honey and effect of a

thermoelectric power plant on the elements content. Food Chem. 61:185-190.

Vanhaecke, F., and Degryse, P. 2012. Isotopic analysis. Fundamentals and applications using ICP-MS.

Wiley-VCH Verlag & Co., Weinheim, Germany.

Van der Steen, J.J.M. 2016. The colony of the honeybee (Apis mellifera L) as a bio-sampler for pollutants

and plant pathogens. PhD Thesis. Wageningen University. Netherlands. Available at:

http://edepot.wur.nl/375348.

Van der Steen, J.J.M., de Kraker, J., and Grotenhuis, T. 2012. Spatial and temporal variation of metal

concentrations in adult honeybees (Apis mellifera L.). Environ. Monit. Assess. 184: 4119-4126.

Van der Steen, J.J.M., de Kraker, J., and Grotenhuis, T. 2015. Assessment of the potential of honeybees

apis mellifera L.) in bimonitoring of air pollution by Cadmium, Lead and Vanadium. J. Environ.

Protec. 6: 96-102.

Van der Steen, J.J.M., Cornelissen, B., Blacquière, T., Pijnenburg, J.E.M.L., and Sverijnen, M. 2016.

Think regionally, act locally: metals in honeybee workers in the Netherlands (surveillance study

2008). Environ. Monit. Asses. 188: 463.

Veleminsky, M., Láznička, P., and Stary, P. 1990. Honeybees (Apis mellifera L.) as environmental

monitors of heavy metals in Czechoslovakia. Acta Entomol. Bohemos. 87:37-44.

Warne, R.T. (2014). A primer on multivariate analysis of variance (MANOVA) for behavioral scientists.

Practical Assessment Research Evaluation 19: 1-10.

Wieczorek, J., Wieczorek, K., and Mozolewski, W. 2006. Can be honey serve as an environmental

marker? Pol. J. Environ. Stud. 15:203-207.

Yarsan, E., Karaca, F., Ibrahim, I.G., Dikmen, B., Koksal, A., and Das, Y.K. 2007. Contents of some

Page 34 of 46



Draft

35

metals in honeys from different regions in Turkey. B. Environ. Contam. Tox. 79:255-258.

Yücel, Y., and Sultanoğlu, P. 2012. Determination of industrial pollution effects on citrus honey with

chemometric approach. Food Chem. 135:170-178.

Zugravu, C.A., Parvu, M., Patrascu, D., and Stoian, A. 2009. Correlations between lead and cadmium

pollution of honey and environmental heavy metal presence in two Romanian counties. Bull.

UASVM Agric. 66:230-233

.

Page 35 of 46



Draft

Figure 1.- Diffusion of polluting substances in the environment and translation to honeybees and honeybee products. (adapted from Solayman et al. 2016, with permission of John Wiley & Sons).

400x112mm (96 x 96 DPI)

Page 36 of 46



Draft

Figure 2.- Scheme in three blocks for using beehives as a passive sampling device.

225x229mm (96 x 96 DPI)

Page 37 of 46



Draft

Figure 3.- Seven-step framework for the use of the hive as a sampling device for the evaluation of spatial and temporal metal concentrations in adult honeybees.

164x148mm (96 x 96 DPI)

Page 38 of 46



Draft

Figure 4. Radar plot for the characteristics of the monitoring methods using honeybees and honey as environmental markers for metals, radionuclides and pesticides. (SA: Sample availability; EST: Ease of

sample treatment; ATA: Analytical technique availability; EAT: Ease of analytical technique; IP: Information provided).

168x175mm (96 x 96 DPI)

Page 39 of 46



Draft

Table 1.- Monitoring studies using honeybees as bioindicators to examine metal and heavy metal pollution.

Elements

analyzed Sample pretreatment

Determination

Technique

Sampling

Notes Ref Country/

Sampling (S) or

published (P) year

Location Sites and samples

As, Cd, F Dry ash at 600ºC AAS USA July-Sept. 1982 (S)

Puget Sound, Washington

72 sampling sites covering

7500 km2 − Honeybees are effective as a large-scale biomarkers

Bromenshenk

et al. 1985

Cd, Cu, Fe, Mn, Pb,

Zn

0.5 g of dried bee sample were

digested with HNO3 and H2O2 and

diluted to a final volume of 25 mL

FAAS and

ETAAS

Finland

1994 (S)

12 sites

around

Finland

62 honeybee samples from industrial, urban and control

sites

− Pollen and honey were also

studied.

− Honeybees are a better bioindicator for metals than

honey.

Fakhimzadeh and Lodenius

2000a

Cd, Cu, Pb, Zn 0.5 g of dried bee sample were

digested with HNO3 and H2O2

diluted to a final volume of 25 mL

FAAS and

ETAAS

Finland

1994 (S)

12 sites

around

Finland

62 honeybee samples from

polluted and unpolluted sites − Pollen and honey were also

studied.

− Honeybees are a better

bioindicator for metals than

honey.

Fakhimzadeh

and Lodenius

2000b

Cd, Pb, Zn Wet mineralization with HNO3

(1/10 w/v) in a microwave-assisted

digestion system

For metals deposited in the surface of the bee body, collected foragers

were washed with Milli-Q water

ICP-OES Italy

1995 (S)

----- 12 colonies placed near

extraurban crossroad with

high traffic density

− Honey, pollen and propolis,

were also studied.

− Honey products are useful to

detect presence of pollutants.

− Dead bees are useful to verify

the dynamics of the

accumulation of pollutants.

Leita et al.

1996

Cd, Cr. Pb Wet mineralization with HNO3 (in a

microwave-assisted digestion

system

ETAAS Italy

1998 (S)

Rome and

surroundings

5 sampling sites (1 in Rome,

4 in the surroundings)

30 honey samples (6 for each

sampling site)

− Pollen, propolis, wax and

honey were also studied.

− Honeybees and other hive-products are better

bioindicators for metals than

honey.

Conti and

Botrè 2001

Cr, Cd, Hg, Pb 0.7 of lyophilized sample were

subjected to wet mineralization

using a mixture HNO3:H2O2 7:1.5

ETAAS and

TDA-AAS

Italy

2001 (S.)

Abruzzi and

Latium

regions

4 sampling sites located in

wildlife reserves and 4

sampling sites in urban areas

− Honeybees are able to detect

heavy metal concentrations in

reserves considered “clean”

areas.

Perugini et al.

2011

Cd, Pb, V A sample of 25 worker bees were

frozen, dried and mineralized using

HNO3:HCl 1:3 and diluted to a final

volume of 50 mL

ICP-OES Netherlands

Jul- Sept 2005 (S.)

Maastricht,

Buggenum,

Hoek van

Holland

12 honeybee samples: 4 samples for sampling site (3)

each 14 day intervals

− No relationship between metal

concentration in honeybees

and in air were detected. Bees

cannot be a reliable alternative

to air analysis

Van der Steen et al. 2015

Al, As, Cd, Co, Cr,

Cu, Li, Mn, Mo,

A sample of 25 worker bees were

frozen, dried and mineralized using

ICP-OES Netherlands

2006 (S.)

Maastricht,

Buggenum,

18 duplicated honeybee

samples from three locations

in the Netherlands

− Results suggest that honeybees can serve to detect temporal

Van der Steen

et al. 2012

Page 40 of 46



Draft

Ni, Pb, Sb, Se, Sn,

Sr, Ti, V, Zn

HNO3:HCl 1:3 and diluted to a final

volume of

50 mL

Hoek van

Holland

and spatial patterns of metal

concentrations.

Cd, Cr, Pb 0.4 g of bee sample were treated

with HNO3 in a microwave-assisted

mineralization system (300-600 W)

followed by dissolution in water to

a final volume of 25 mL

ETAAS Italy

2007-2008 (S)

Post mining

area in

SW Sardinia


two mining sites (24) and a control site (12)


studied.

− Results indicate that honeybees

are the most sensitive matrix for contamination.

Satta et al.

2012

Cr, Cd, Ni, Pb Bee sample (0.5 g) was mineralized

using HNO3:H2O2 1:1 in a

microwave-assisted mineralization system

ETAAS Spain

2007-2010 (S)

Córdoba 192 honeybee samples from

five sampling sites

(urban, industrial and

control)

− Biomonitoring approach

proved detection of metals in locations that were missed by

standard measurement stations

Gutiérrez et

al. 2015

Al, As, Ba, Cd, Co,

Cr, Cu, Li, Mn,

Mo, Ni, Sb, Se, Sn,

St, Ti, V, Zn

A sample of 25 worker bees were

dried and mineralized in aqua regia

at 170ºC and diluted to a final

volume of 50 mL

ICP-OES Netherlands

2008 (S.)

150 locations

throughout the

Netherlands

150 apiaries were sampled − Results demonstrated spatial

differences between the sampled regions and local

variations per metal.

− The land uses influence the metal content of honeybees

Van der Steen

et al. 2016

Pb 0.2 g of ground dry honeybees were

treated with H2SO4 and dry ash (up

to 750 ºC) followed by dissolution

in HNO3 to a final volume of 10 mL

ETAAS France

2008-2009 (S)

Pays de la

Loire

Honeybee samples from 4

types of area: Hedgerow (47), Cultivated (38), Urban

(39), Island (15)


studied.

− Results indicate that honeybees are the most sensitive matrix

for detecting Pb contamination.

Lambert et al.

2012

Al, Cd, Cr, Cu, Fe,

Mn, Ni, Pb, Zn

1.0 mg of bee sample was

mineralized using HNO3:H2O2 3:1

in a microwave-assisted digestion

system up to 550 W

FAAS and

ETAAS

Turkey

2010 (S)

Mugla Region 11 honeybee samples in the

vicinity of a thermal power plant

− 6 honeydew samples were

comparatively analyzed.

− Honeybees are a better

bioindicator for metals than

honey.

Silici et al.

2013

As, Ba, Ca, Fe, Hg, K, Li, Mn, Na,

1.0 mg of dried and homogenized bee sample was mineralized using

HNO3 (c.) in a microwave-assisted

digestion system

ETAAS Iran

2011 (S)

Maravian, Kamyaran,

Bijat,

Ghorveh


four counties of the province

of Kurdistan

− Honeybees are able to detect early anthropogenic changes in

environmental conditions.

Sedegh et al.

2012

Page 41 of 46



Draft

Table 2.- Monitoring studies using honey as a bioindicator to examine metal and heavy metal pollution.

Elements

determined Sample pretreatment

Determination

Technique

Sampling

Notes Ref Country/

Sampling or published

year

Location Sites and samples

Ag, Cd, Cu, Pb 5-15 g of honey were treated with equal (w/w) quantities of 1% (v/v) solution of

HNO3

ETAAS United Kingdom 1950-1951 (S)

25 different location in

UK

50 honey samples − Pollen and soil were also studied.

− Honey is insensitive to spatial differences in contamination

− Honeybees could be better environmental indicator

Jones 1987

Cd, Cr, Ni, Pb ----- Automatic monitoring

devices

Italy 1989 (S)

Modena 5 monitoring stations (with 2 hives each).

Monthly samples during a year

− Pollen and larvae were also studied.

Balestra et al. 1992

Ca, Cd, Cu, Fe, K, Mg, Mn, Zn

Cd, Pb: Dry ash (450 ºC) followed by formation of their APDC complexes and

extraction in MIBK Remaining metals: Dry ash (600ºC)

FAAS Turkey 1993-1994 (S)

Yatagan and other sites in

Turkey

74 liquid honeys − Honeys with low mineral content are useful to detect contamination sources.

− Honeys with high mineral content possess resistance to the effects of pollution.

Üren et al. 2006

Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn

----- ICP-SFMS Switzerland 1998-2001 (S)

95 places covering

Swiss production

areas

95 honey samples with known floral origin divided

in four groups: City, Village, Rural and Mountain

− Chemometric methods were used for data study.

− Results suggest that botanical origin has a greater influence than environmental aspects in mineral composition of honey.

Bogdanov et al. 2007

Cd, Pb, Zn ----- FAAS Poland 2001 (P)

Pomeranian Region

15 honey samples − Results suggest that honey is useful for assessing the presence of metal pollution.

Przybylowski and

Wilezyńska 2001

As, Cd, Cu, Fe, Pb, Zn

Dry ash FAAS and ICP-OES

Iran 2001 (P)

Ja`far Abad area

Saveh City

----- − Results suggest high levels of Cd and As due to pollution.

Samimi et al. 2001

Al, Ag, Ca, Cd, Cr, Co, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, P, Pb, S, Zn

Wet digestion using HNO3 (66% v/v) ICP-OES France 2002 (P)

63 French honeys plus 23 honeys

from other 11 countries

86 honey samples sold in France with known botanical

and geographical origins


− Results suggest that the heavy metal content in honey is highly dependent on botanical origin.

Devilliers et al. 2002a

Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Sr, Zn

- Wet digestion using HNO3 (75%) by dissolution to final volume of 10 mL

- Dry ash (600 ºC) followed by dissolution in HCl (1:2) to a final

ICP-OES Chile 2001-2003 (P)

IV and X Administrative

Regions

47 honey samples with known floral origin


− Acid digestion method is preferable.

− Honeys stored in aluminum

Fredes and Montenegro

2006

Page 42 of 46



Draft

volume of 10 mL - For Hg: wet digestion in PTFE flask

assisted by microwave energy

containers exhibit higher metal contamination levels.

Al, Co, Cr, Cu, Fe, Mn, Ni, Zn

Dry ash (500 ºC) followed by dissolution in 2N HNO3 to a final

volume of 30 mL

ETAAS Turkey 2002-2003 (P)

Central Anatolia, East

Anatolia, South Anatolia Aegean Region, Mediterranean

Region and Black Sea

Region

44 natural liquid honey samples from 6 different

regions of Turkey

− Results show that mineral content is highly dependent on floral type.

− The presence of certain elements in honey can be influenced by geographical conditions and the use of metal containers.

Yarsan et al. 2007

As, Cd, Cu, Pb, Zn Dry ash (700 ºC) followed by dissolution in HNO3 (1:6) to a final

volume of 25 mL

FAAS Romania 2005 (P)

Sibiu County, Transylvania

5 polluted sites − Results suggest that honey could be used to detect metal contamination.

Bratu and Georgescu

2005

Cu, Cd, Pb, Zn Wet digestion using HNO3 FAAS and ETAAS

Romania 2005-2012 (S)

Baia Mare 8 honey samples (from 2005-2012) in the vicinity of an

industrial area

− Contamination levels (Cd, Pb) in honey are correlated with their concentrations in air.

Berinde and Michnea

2013

Al, Cd, Cu, Cr, Fe, Mn, Ni, Pb, Se, Zn

- Dry ash (450 ºC) followed by dissolution in HNO3 (25% v/v) to a final volume of 10 mL

- Wet digestion using 2:1 HNO3:H2O2 followed by dissolution to a final volume of 10 mL

- Wet digestion using 3:1 HNO3:H2O2

in a microwave assisted digestion system followed by dissolution to a final volume of 5 mL

FAAS and ETAAS

Turkey 2005 (S)

West Anatolia, Central

Anatolia, East and South Anatolia,

Mediterranean Region and Black Sea

Region

25 multifloral honey samples from 6 different regions of

Turkey

− Microwave-digestion method (high recoveries in certified reference material) is preferred over wet digestion and dry ash.

− Results confirmed that trace element concentration in honeys is correlated with the degree of trace element concentration in the environment.

Tuzen et al. 2007

Ca, Cd, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Zn

Dry ash (550 ºC) followed by dissolution in 1M HCl to a final volume

of 10 mL

FAAS and ETAAS

Spain 2006 (P)

Galicia 40 samples from industrial, urban and rural sites

− Chemometric methods were used for site differentiation.

− Honey is useful to differentiate polluted (industrial and urban) from unpolluted rural areas.

Rodríguez-García et al.

2006

Al, B, Ba, Bi, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Si, Ti, V, Zn

- Wet digestion using HNO3 (65%) - Dry ash (450 ºC) - For Hg: wet digestion in PTFE flask

assisted by microwave energy

ICP-OES ETAAS (Cd, Pb)

CVAAS (Hg)

Germany 2006 (P)

Altötting District, Bavaria

60 honey samples covering all Altötting District


− Honey is a good substrate for detecting anthropogenic and geogenic anomalies.

Raeymaeker 2006

Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Zn

Wet digestion using 1:1 HNO3:H2O2 in a microwave assisted digestion system

followed by dissolution to a final volume of 50 mL

FAAS and ETAAS

Macedonia 2008 (P)

12 regions of the country

123 honey samples − Chemometric methods were used for data study.

− Levels of different metals were associated with geochemical and anthropogenic influences.

Stankowska et al. 2008

Cd, Pb ----- AAS Romania 2008 (S)

Dambovita and Arges

108 honey samples from 2 counties in the vicinity of

Bucharest divided into polluted (54) and unpolluted

(54) areas

− Honey is a good environmental marker for heavy metal pollution.

− Cd and Pb levels are related with the pollution degree.

Zugravu et al. 2009

Page 43 of 46



Draft

Pb Dry ash (up to 750 ºC) followed by dissolution in HNO3 (c.) to a final

volume of 5 mL

ETAAS France 2008-2009 (S)

Pays de la Loire

Honey samples from 4 types of area: Hedgerow (47),

Cultivated (38), Urban (39), Island (15)

− Pollen and honeybees were also studied.

− Results indicate that honey is the least contaminated matrix.

Lambert et al. 2012

Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn

Wet digestion of 2 g of honey using 3:1 HNO3:HCl mixture in covered and heated Teflon beaker, followed by dissolution with HCl 1M to a final

volume of 50 mL

FAAS Egypt 2009 (P)

Edfu, Esna, Kom Omb,

Aneeba

48 honey samples divided into polluted (Edfu, Kom

Ombo) and unpolluted areas

− Soil and flower samples were also analyzed.

− Results suggest the use of honey as a bioindicator, but the floral origin also influences the mineral content of honey.

Rashed et al. 2009

Na, Ca, Mg, NH4+,

Cl-, Br-, SO42, NO3

-

PO43-

Honey samples were dissolved in Milli-Q water (0.1g mL-1), vortexed for 5 min and filtered through 0.45 µm membrane

and directly analyzed by ion chromatography

Ion Chromatography

(IC)

Different Italian Regions and six Western Balkan

countries 2009-2011 (S)

Italy (14), Slovenia (2), Croatia (2), Serbia (10), Kosovo(7),

Macedonia (2), Albania (8)

45 honey samples with known floral origin from

seven countries


− Sulfate and phosphate were considered as potential bio-indicators of anthropogenic contamination.

Fermo et al. 2013

Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, Se, Sn, Sr, Zn

Wet digestion with a 9:1 HNO3:H2O2

mixture using in a microwave assisted digestion system followed by

dissolution to a final volume of 5 mL

ICP-OES Turkey 2012 (P)

Hatay Region 15 citrus honey samples from 15 industrialized and non-

industrialized areas of Hatay


− Results show that trace element concentrations in citrus honey are clearly correlated with the level of trace element pollution in the environment.

Yücel et al. 2012

Page 44 of 46



Draft

1

Table 3.- Monitoring studies using honeybees and honey as bioindicators to examine pollution due to radionuclides.

Radionuclides

determined Determination

Technique

Sampling

Notes Ref Country/

Sampling or

published year

Location Sites and type of sample

3H

Gamma-Ray

Spectrometry

USA

Honeybees:

1982-1993 (S)

Honey:

1979-1996 (S)

Los Alamos,

New Mexico

13 sampling points in and

around Los Alamos National

Laboratory plus 5 sampling

stations in background areas

Honey and honeybee samples

were obtained at these points

over an 18 year period.

− Honeybees and honey were used to monitor the presence and distribution of

tritium at Los Alamos National Laboratory.

− The amounts of 3H in bees and honey on Los Alamos lands were poorly

correlated.

− 5 mL of moisture obtained from sample distillation were mixed with 15 mL of a

scintillation solution, and counted with a scintillation counter during 50 min. for

3H.

Haarmann

1998a, 1998b

134Cs, 137Cs, 131I, 103Ru

Gamma-Ray

Spectrometry Germany

1986 (S)

Munich 10 honey samples − Pollen was also analyzed.

− Levels of 137Cs, 134Cs were higher in pollen than in honey.

Bunzl and

Kracke 1988

134Cs, 137Cs, 131I, 103Ru

Gamma-Ray

Spectrometry

Italy

1988 (S)

Six regions of

Italy

120 honey samples

20 samples of pollen

9 samples of bees

− Results suggest that pollen is the best bioindicator for radionuclides, also bees

could be used as markers, and honey was the worst indicator.

Tonelli et al.

1990

134Cs, 137Cs Gamma-Ray

Spectrometry

Germany

1988 (S)

Lüneburger

Heide

26 heather honey samples from

1986 plus 10 honey samples

from 1985 harvest

− Also Calluna vulgaris plants and soil were analyzed.

− High Cesium activity in German honeys is related with Chernobyl pollution.

− High transfer of Cesium from soil to the Calluna vulgaris plants was

detected.

Assmann-

Werthmüller

et al. 1991

134Cs, 137Cs, Gamma-Ray

Spectrometry

France

1986-89 (S)

16 French

departments

46 honey samples − Honey samples are polluted by 134Cs, 137Cs emitted from the Chernobyl

accident.

− A decreasing gradient in radionuclide levels from East to West France was

detected.

− In 14 samples analyzed in 2000, levels of 134Cs had been reduced to traces,

and 137Cs was significantly reduced.

Devilliers et

al. 2002b

137Cs Gamma-Ray

Spectrometry

Croatia

1990 (S)

16 different

locations

25 honey samples − Pollen and flowers were also analyzed.

− Cs activity in honey is 4.5 times less than in pollen.

Barišic et al.

1992

134Cs, 137Cs, 40K, Gamma-Ray

Spectrometry

Croatia

1990-1992 (S)

Zagreb,

Pokupsko,

Daruva,

Grubišno,

Cetekovac

----- − Pollen and soil were also analyzed.

− 40K activity is one order of magnitude greater in pollen than in honey. Significant differences in 40K activity were detected on the basis of honey

type.

Barišic et al.

1994

137Cs, 40K Gamma-Ray

Spectrometry

Croatia

1993-1995 (S)

Gorski Kotar 35 honeydew samples − Results suggest that honeydew is the best bioindicator for radionuclides in

relation to pollen.

Kezić et al.

1997

137Cs, 40K, Gamma-Ray

Spectrometry

X-Ray

Croatia

1994-1998 (S)

Gorski Kotar 20 honeydew honeys and 21 mixed honeys

(12 sampling stations)

− Other metals were measured.

− Honeydew honeys were considered as an optimal indicator for 137Cs, even for

a long time after contamination.

Barišic et al. 1999

Page 45 of 46



Draft

2

Spectrometry

3H, 7Be, 57Co, 54Mn, 22Na, 181W

Gamma-Ray

Spectrometry

USA

1996 (S)

Los Alamos,

New Mexico

3 monthly dead bee samples (forager and nurse bees) from 3

different colonies

− Flowers of white sweet clover, salt cedar and rabbit brush were also analyzed. No significant differences were detected in the amounts of radionuclides in

these three plants.

− No differences were detected in contaminant levels in forager and nurse bees.

Haarmann 1998a

3H, 56Co, 60Co, 54Mn, 22Na, 181W

Gamma-Ray Spectrometry

USA 1995-1996 (S)

Los Alamos, New Mexico

15 dead bee samples from two

colonies (8+7) in 1995

6 dead bee samples from two

colonies (2+2) in 1996

− Water and floral samples were also analyzed.

− Results showed significant bioaccumulation of 60Co, and 22Na in honeybees

but not in flowers.

Haarmann

1998b

137Cs Gamma-Ray

Spectrometry

Italy

2001-2004 (S)

Six Different

locations of

Liguria

336 honey samples of the six Ligurian locations

− Honey contamination is related with the soil geomorphology.

− Differences in radioactive contamination were detected on the basis of floral

type of honey.

− Chestnut honey seems to be the best marker.

Panatto et al. 2007

137Cs, 40K Gamma-Ray

Spectrometry

Poland

2011-2012 (S)

Podlaise region 106 honeys from 5 different

floral origins − Cs activity was the same as in 2010, one year before the Fukushima accident.

− Cs activity was 5 times lower than in 1998, 12 years after the Chernobyl

accident.

− The diminution of the K and Cs activity stopped in 2011-2012, and the

authors related this fact with a possible influence of the Fukushima accident.

Borowska et

al. 2013

228Ac, 7Be, 214Bi, 40K, 212Pb, 214Pb, 235U, 226Ra, 208Tl

Gamma-Ray

Spectrometry

Italy

2012 (S)

Rotondella 3 sampling stations

Weekly samples of dead bees

obtained in “underbasket” trap

− Dead bees were considered useful bioindicators for radionuclides.

− Only 40K and 7Be were detected in abnormal quantities in relation to

background levels.

Schiuma et al.

2013

137Cs, 40K, 226Ra, 228Ra

210Po, 232Th, 228Th, 235U, 238U

Gamma-Ray

Spectrometry

Alpha-Ray

Spectrometry

Italy

2013 (S)

Four different

sampling areas

in Marche

Region

27 honey samples with

different botanical origins − Radionuclides present in honey samples depended on the area foraged, the

mineral composition of soil, the floral type.

− Other influencing factors are the preferential absorbability of the radionuclide

by the vegetal, use of fertilizers, irrigation water and climatic conditions.

Meli et al.

2016

Page 46 of 46



the use of honeybees and honey as environmental ...key words: honeybees, honey, environmental...

Documents