Ogni antibiotico è efficace in relazione a un determinato gruppo di microrganismi comprare doxycycline senza ricettain caso di infezioni oculari vengono scelte gocce ed unguenti.

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Biol Trace Elem ResDOI 10.1007/s12011-013-9732-6 Biomonitoring with Honeybees of Heavy Metalsand Pesticides in Nature Reserves of the MarcheRegion (Italy) Sara Ruschioni & Paola Riolo & Roxana Luisa Minuz &Mariassunta Stefano & Maddalena Cannella &Claudio Porrini & Nunzio Isidoro Received: 29 April 2013 / Accepted: 6 June 2013 # Springer Science+Business Media New York 2013 Abstract The aim of this study was to carry out biomonitoring Keywords Honeybee . Biomonitoring . Heavy metal .
with honeybees (Apis mellifera L.) to assess the presence of Pesticide . Nature Reserves pesticides and heavy metals (cadmium, chromium, nickel, lead)in all of the ten nature reserves of the Marche Region (central–eastern Italy). The study was carried out during the spring and summer seasons when the honeybees were active, over 3 years(2008–2010). Twenty-two colonies of honeybees bred in hives Biomonitoring can be defined as the use of bio-organisms or were used. Samples of live and dead honeybees and of honey bio-materials to obtain information on certain characteristics were collected from 11 sampling stations from May to October of the biosphere. The relevant information in biomonitoring is in each year. No pesticide pollution was found. Significant commonly deduced from either changes in the behavior of the differences in heavy metal concentrations were found among monitored organism or from the concentration of specific years, months and sites, and in particular situations. The anal- substances in the tissues of the monitored organism The ysis reveals that high heavy-metal concentrations occurred interest in bioindicator-based techniques for the detection and exclusively in live honeybees. For the seasonal averages, the evaluation of environmental contaminants has been increasing most detected heavy metal was chromium, which exceeded the ]. With the correct selection of an organism, the general threshold more often than for the other elements, followed by advantage of the biomonitoring approach is related primarily cadmium and lead; nickel never exceeded the threshold. The to the permanent and common occurrence of the organism in data are discussed with an evaluation of the natural and an- the field, the ease of sampling, and the absence of any neces- thropic sources taken from the literature and from local situa- sary expensive technical equipment ]. Therefore, it is evi- tions that were likely to involve heavy metal pollution.
dent that the selection of a suitable organism as a bioindicatorrepresents a critical step in overall biomonitoring activities.
Electronic supplementary material The online version of this article The organism can be further selected on the basis of their contains supplementary material, accumulative and time-integrative behavior [].
which is available to authorized users.
In the literature, biomonitoring species for trace-element air S. Ruschioni P. Riolo (*) R. L. Minuz N. Isidoro pollution are often selected on the basis of criteria such as Dipartimento di Scienze Agrarie, Alimentari ed Ambientali, specificity ], accumulation ratios ], or the well-defined Università Politecnica delle Marche, via Brecce Bianche,60131 Ancona, Italy representation of a sampling site []. For the biomonitoring of atmospheric pollution, the honeybee (Apis mellifera L.) hasbeen the subject of various investigations, and can be consid- M. Stefano M. Cannella Centro Agrochimico Regionale, Azienda Servizi Settore "ideal bioindicator," as defined by Stöcker [A.
Agroalimentare delle Marche, Regione Marche, via Roncaglia, mellifera is an insect that is directly affected by the toxicolog- 20, 60035 Jesi, Italy ical conditions of its natural environment. It is a good biolog-ical indicator, as it is widespread and sensitive to environmen- tal changes. Indeed, honeybees are exposed to numerous Dipartimento di Scienze Agrarie, Università degli Studidi Bologna, via Fanin, 42, 40127 Bologna, Italy pollutants during their foraging activities, their body hair can Ruschioni et al.
easily retain atmospheric residues, and they can be contami- from natural sources and anthropogenic activities, even if, nated via food resources when gathering pollen and nectar especially for Cr, they originate more from widespread use from flowers, or through water ]. Therefore, since the late in various and specific industries , Cadmium (Cd) and 1970s, the honeybee has increasingly been used to monitor lead (Pb) are prominent examples of anthropogenic environ- pesticides and environmental pollution by heavy mental metal pollutants, and therefore these are considered to metals , in territorial and urban surveys.
be the principle toxic heavy metals Pesticides are scattered both in time and space, and The aim of the present study was to use honeybees as depending on the type of chemical compound, their stability, bioindicators for a regional survey of pesticide and heavy metal and their affinity for the target organism and surrounding environmental air pollution. A three-beekeeping-season study environment, they can be degraded by various environmen- was carried out in all of the ten nature reserves of the Marche tal factors over greater or lesser periods of time. Honeybees Region (central–eastern Italy), to determine the presence of are extremely sensitive to pesticides. Many honeybees that these pollutants in foraging honeybees and honey. Cd, Cr, Ni, come into direct contact with an insecticide will not have and Pb were chosen as the representative heavy metals, the enough strength to return to their hive and will die in the field levels of which in the atmosphere represent a reliable index of or during their return flight. Other honeybees with only marginal contact while visiting flowers of a treated speciesor gathering nectar and pollen from spontaneous speciescontaminated by pesticide "drift" will eventually die in the Materials and Methods hive. In this case, the honeybee acts as a direct indicator andprovides us with information on the form of the residues ]. The number of dead honeybees in front of a hive istherefore the most important variable to be considered for This biomonitoring study was performed over the years 2008, these contaminants [and this can vary according to a 2009 and 2010, in all of the ten nature reserves of the Marche number of factors: the toxicity (for honeybees) of the active Region (see Fig. ): Riserva Naturale di Abbadia di Fiastra (site ingredient used the presence and extension of bloom A: 43°12'02.77"N 13°24'24.34"E, 309 m above sea level among cultivated or spontaneous plants, the presence of (a.s.l.)), Parco del Conero (site B: 43°34'35.75" N honeybees on the site and at the time of a chemical treatment, 13°32'17.34"E, 67 m a.s.l.), Riserva Naturale Statale Gola the means used to distribute a pesticide, and the presence of del Furlo (site C: 43°38'33.22"N 12°45'13.99"E, 771 m wind, among other factors ].
a.s.l.), Parco Naturale della Gola della Rossa e di Frasassi (site In the Marche Region, as in other Italian regions, organ- D: 43°26'36.69"N 12°57'03.86"E, 339 m a.s.l.), Parco ophosphorus and pyrethroids are very widely used as in- Naturale Monte San Bartolo (site E: 43°56'28.68" N secticides for agriculture purposes ]. Fungicides are usu- 12°50'14.03"E, 96 m a.s.l.), Parco Naturale dei Monti Sibillini ally considered safe for honeybees; however, triazoles can be (Site F: 43°016'13.91"N 13°10'59.32"E, 778 m a.s.l.; site G: synergists for pyrethroids, and can thus induce negative side 42°59'57.96"N 13°06'52.14"E, 975 m a.s.l.), Riserva Naturale effects in bees ].
Ripa Bianca (site H: 43°32'04.26"N 13°17'28.55"E, 45 m The fundamental aspect that differentiates heavy metals a.s.l.), Parco del Sasso Simone Simoncello (site I: from other pollutants, like pesticides, is their introduction 43°45'55.33"N 12°20'03.21"E, 772 m a.s.l.), Riserva Naturale into the territory and their environmental fate. Heavy metals Regionale Sentina (site J: 42°53'57.91"N 13°54'15.97 E, 0 m are released in a continuous manner into the environment by a.s.l.), and Riserva Statale Montagna di Torricchio (site K: various natural and anthropic sources, and as they do not 42°58'00.07"N 13°02'15.22"E, 1,282 m a.s.l.). Sites C, D, F, decay and are characterized by latent toxicity, they are con- G, I, and K are mostly wilder areas, while sites A, B, and E are tinuously present in the environment and enter into the surrounded by agricultural environments, and sites H and J are biological cycles ]. They are predominately transferred surrounded by industrial and urban environments. Each sam- as molecules or particulate matter via the atmosphere, mostly pling station consisted of two healthy and homogenous hives on a large scale. The amounts of anthropogenically derived (Dadant-Blatt type, at ten combs), which were strategically heavy metals have increased continuously since the begin- deployed in the nature reserve areas (one sampling station for ning of the industrial revolution ]. Generally, they do not each area, except for Parco Naturale dei Monti Sibillini, which cause honeybee mortality, but they can be deposited on the had two sampling stations), and were constantly checked for body hairs and taken back to the hive with the pollen, or they sanitary purposes. The monitoring was performed each year might be absorbed together with the nectar of the flowers, or from May to October for each sampling site; for each season, through the honeydew produced by aphids.
dead honeybees were sampled about 20 times (once a week), Some compounds, such as chromium (Cr) and nickel (Ni), while live honeybees and honey were sampled five times are widely distributed in the environment, as they are released (once a month).


Biomonitoring with Honeybees in Nature Reserves (acrinathrin, alphamethrin, bifenthrin, cyfluthrin,cypermethrin, deltamethrin, esfenvalerate, fenvalerate,flucythrinate, fenpropathrin, fluvalinate, lambda-cyhalothrin,permethrin), and triazoles (bitertanol, bromuconazole,cyproconazole, diclobutrazol, esaconazole, fenbuconazole,flusilazole, flutriafol, myclobutanil, penconazole, prochloraz,propiconazole, tebuconazole, tetraconazole, triadimefon,triadimenol).
Biomonitoring of Heavy Metals Once a month, from May to October for each of the years,samples of honey were collected. Fresh, recently produced,and still unseasoned honey was collected from free cells. Inaddition, the collection of the honey samples was performed Fig. 1 Map showing Italy and the Marche Region. Black spots and reasonably far from the metallic wire that crossed and sup- letters indicate the position of sampling sites: a Riserva Naturale di ported the honeycombs to avoid any kind of metal contam- Abbadia di Fiastra, b Parco del Conero, c Riserva Naturale Statale Goladel Furlo, d Parco Naturale della Gola della Rossa e di Frasassi, e Parco ination. Each sample of honey (70 g) was collected in dupli- Naturale Monte San Bartolo, f, g Parco Naturale dei Monti Sibillini, h cate from each hive, after which it was stored at 4 °C.
Riserva Naturale Ripa Bianca, i Parco del Sasso Simone Simoncello, j Each 25-g honey sample was mixed with ca. 50 mL Riserva Naturale Regionale Sentina, k Riserva Statale Montagna di distilled water and heated in a water bath at 40 °C for 15 min, to improve and facilitate the handling of the mixture.
Biomonitoring of Pesticides Then, each sample was cooled and stored at −20 °C. Themineralization was performed for 3 h using a bi-position Each hive was equipped with a collection cage for dead heating mantle (Falck Instruments, Italy) equipped with re- honeybees, as an "underbasket" type [], which was posi- flux condensers (Sigma-Aldrich, Italy): each 5-mL sample tioned to collect the dead honeybees expelled by the colony.
was diluted with 10 mL 65 % aqueous solution of nitric acid Once a week, the colonies were checked and the number of (Merck, Italy) and 3 mL 30 % aqueous solution of hydrogen dead honeybees was recorded. When the mortality rate peroxide (Merck, Italy). Each sample was then cooled, trans- exceeded the critical threshold (250 honeybees per week ferred to a 50-mL volumetric flask, taken to 50 mL with bi- per sampling site) ], pesticide laboratory analyses were distilled water, and analyzed by inductively coupled plasma– performed on the dead honeybee samples.
atomic emission spectroscopy (ICP-AES) (Horiba, France) The determination of pesticides was carried out by gas- (modified from [Standard solutions consisted of 1.0 g/L chromatography (Carlo Erba, Italy) analysis using specific nitric acid and 5 % of each element (Cd, Cr, Ni, Pb), at the detection modes: electron capture detector, nitrogen phospho- highest purity available (Merck, Italy). Prior to use, all glass- rus detector, and gas chromatography–mass spectrometry. The ware were treated with a solution of 5 % nitric acid over- homogenized samples were mixed with 10 g diatomaceous night, to avoid any contamination, then rinsed with ultrapure earth. The mixture was transferred to a cartridge, and the water and dried. Dilute solutions were prepared from stan- extraction was carried out automatically by accelerated sol- dard solutions by dilution with 5 % nitric acid, as appropriate vent extraction (Dionex, CA, USA), with elution with for each element, and then mixed at five concentration levels, dichloromethane (Merck, Italy). The extract was then evapo- which were used for the construction of calibration curves.
rated, without drying, on a rotatory evaporator and taken up in Ultrapure water was used as the blank.
acetone (Merck, Italy). The following chemical classes ofpesticides were analyzed: organophosphorus (azinphos-ethyl, Live Foraging Honeybee Matrix azinphos-methyl, chlorpyrifos, chlorpyrifos-methyl,coumaphos, diazinon, dichlorvos, dimethoate, fenamiphos, Once a month, from May to October of each year, fenitrothion, fenthion, fonofos, forate, formothion, fosalone, about 100 honeybees were collected from each hive phosphamidon, heptenophos, malathion, methamidophos, using a modified hand vacuum (Philips, The Nether- methidathion, omethoate, parathion-ethyl, parathion-methyl, lands), and placed in sterile, plastic bags. The forager pirimiphos-methyl, pyrazophos, pyridaphenthion, quinalphos, honeybees were sacrificed in the laboratory by freezing tolclofos-methyl, trichlorfon, vamidothion), pyrethroid at ca. −20 °C, lyophilized at 40 °C, unified by grinding, Ruschioni et al.
and carefully mixed [modified from 36]. Samples of ca.
and site F (+0.01 mg/kg) and site I (+0.05 mg/kg) in 2009. The 1 g [] were mineralized by acid digestion and ana- seasonal averages for Cr (Table Online Resources) showed lyzed by ICP-AES, as described for honey matrix.
excess values in the live honeybee matrix for site B in 2009 (+0.01 mg/kg), site C in 2008 (+0.06 mg/kg) and 2010 (+0.03- mg/kg), site D in 2010 (+0.03 mg/kg), site F in 2008 (+ 0.01 mg/kg) and 2010 (+0.02 mg/kg), site I in 2008 (+0.48- Data from the pesticide biomonitoring were not included in mg/kg), and site K in 2008 (+0.017 mg/kg). The seasonal the statistical analysis due to the negative results. The envi- averages for Pb (Table , Online Resources) showed excess ronmental risk thresholds used in this study were defined by values for site A in 2008 (+0.27 mg/kg).
Porrini et al. (modified from [on the basis of previous In comparing the average site values for the heavy metals literature and their own experimental data. The mean thresh- in the live honeybee matrix within the years, there were old values were: for the honeybee matrix, Cd, 0.10 mg/kg; significant differences among the sites in 2008 for Cd Cr, 0.12 mg/kg; Ni, 0.30 mg/kg; and Pb, 0.70 mg/kg; and for (F=2.625, p<0.05) (Table Online Resources), in 2009 for the honey matrix, Cd, 0.01 mg/kg; Cr, 0.02 mg/kg; Ni, Ni (F=2.744, p<0.01) (Table Online Resources) and Pb 0.20 mg/kg; and Pb, 0.05 mg/kg.
(F=7.937, p<0.001) (Table Online Resources), and in 2010 The data were log-transformed to meet the assumptions of for Cd (F=3.726, p<0.01) (Table Online Resources). The normality, and analyzed by two-way ANOVA, followed by only significant differences among the sites in the honey Tukey tests (p<0.05), to reveal any differences among years, matrix were seen for Ni in 2009 (F=4.528, p<0.001) and months, and sites. The threshold overrun frequencies (percent) 2010 (F=3.921, p<0.001) (Table , Online Resources).
were calculated for each heavy metal in the live honeybee and By comparing the average monthly values of the heavy honey matrices.
metals within the years, significant differences among themonths were seen in the live honeybee matrix in 2008 for Cd(F=2.799, p<0.05) (Table , Online Resources) and Cr (F=4.678, p<0.01) (Table Online Resources), and in2009 for Cd (F=2.429, p<0.05) (Table Online Resources) Biomonitoring of Pesticides and Cr (F=4.169, p<0.01) (Table , Online Resources). Inthe honey matrix, this was only seen in 2010 for Ni During the beekeeping seasons of 2008, 2009 and 2010, the (F=5.150, p<0.01) (Table , Online Resources).
mortality rate of the foraging honeybees exceeded the critical By comparing the mean seasonal values of the heavy threshold only twice: for site E on June 28, 2008 (305 dead metals within each site, significant differences were ob- honeybees) and site J on May 15, 2009 (366 dead honey- served among the years (Table Online Resources). In the bees). However, laboratory analyses carried out in the col- live honeybee matrix, this was seen for site B for Cr lected samples of the dead honeybees did not show the (F=4.490, p<0.05) and Ni (F=9.719, p<0.01), for site E presence of any of the pesticides.
for Cr (F=6.509, p<0.01) and Pb (F=13.636, p<0.001), forsite F for Cd (F=5.134, p<0.05) and Ni (F=15.362, Biomonitoring of Heavy Metals p<0.001), for site G for Ni (F=29.319, p<0.001), and forsite H for Cr (F=3.905, p<0.5). For the honey matrix, this The heavy metals exceeded their respective thresholds in was seen only for Ni, for site C (F=9.207, p<0.01), for site D different periods, according to the sites. The monthly aver- (F=17.839, p<0.001), for site F (F=16.176, p<0.001), and ages for Cd in the live honeybee matrix exceeded the thresh- for site I (F=24.490, p<0.001).
old in 11.6 % (23/198) of cases (Fig. ). The monthlyaverages for Cr in the live honeybee matrix exceeded thethreshold in 19.7 % (39/198) of cases (Fig. while in the honey matrix, this occurred in 11.1 % (22/198) of cases(Fig. ). The monthly averages for Ni in the live honeybee The weekly screenings conducted to determine the mortality matrix exceeded the threshold in 5.1 % (10/198) of cases of the two hives in each of the biomonitoring sites showed (Fig. while in the honey matrix this occurred in 1.5 % threshold overruns for only two dates, although these analy- (3/198) of cases (Fig. ). The monthly averages of Pb in the ses did not reveal the presence of pesticides. The high mor- live honeybee matrix exceeded the threshold in 4.6 % tality was probably associated with the confirmed swarming.
(9/198) of cases (Fig. ).
Natural areas in the Marche Region have different types of The analysis of the seasonal averages for Cd (Table flora over different extents. As pesticides were not detected, Online Resources) showed values that exceeded the threshold this might indicate that the sites are completely surrounded in the live honeybee matrix for site C in 2008 (+0.05 mg/kg), by wild environments, although this assumes different values



Biomonitoring with Honeybees in Nature Reserves Fig. 2 Scatter plots showing the concentrations of Cd (a), Cr (b), Ni 2010, as indicated. The dashed lines show heavy metal threshold (c), and Pb (d) in the honeybee matrix, during May (M), June (J), July values: Cd, 0.10 mg/kg; Cr, 0.12 mg/kg; Ni, 0.30 mg/kg; Pb, (J), August (A), September (S), and October (O) of 2008, 2009 and 0.70 mg/kg. The different symbols represent the different sampling sites for areas in which the biomonitoring site is surrounded by showed that the Marche Region is one of the regions with anthropogenic and agricultural activities. Therefore, these the lowest distribution of pesticides per hectare ].
data show that during the 3 years of investigation, there The analysis of the seasonal averages showed higher was no contamination due to non-rational use of pesticides values of heavy metals exclusively in the live honeybees.
in any of these Nature Reserves of the Marche Region. This The most prevalent heavy metal was Cr, which exceeded the is in line with the statistical analyses conducted from 2001 to threshold more often than the other heavy metals, while the 2011 by the Italian National Institute of Statistics, which seasonal averages for Ni never exceeded the threshold. The Fig. 3 Scatter plots showing the concentrations of Cr (a) and Ni (b) in The dashed lines show heavy metal threshold values: Cr, 0.02 mg/kg; the honey matrix, during May (M), June (J), July (J), August (A), Ni, 0.20 mg/kg. Cd and Pb were not detected in the honey matrix. The September (S), and October (O) of 2008, 2009 and 2010, as indicated.
different symbols represent the different sampling sites Ruschioni et al.
overrun occurred mostly in the wilder mountain sites, as if During these 3 years of the survey, Cd exceeded the thresh- their altitude acted as a natural barrier for accumulation.
old only in the live honeybee matrix. Indeed, from the literature During the year in which the heavy metal thresholds were it appears that Cd is deposited at its highest levels in the more often exceeded, the weather conditions were very dry hemolymph of the bees []. Cd is naturally present in the soil (data not shown), which might have caused the failure of the and in sediments , but it mainly originates from domestic process of heavy metal leaching out of the flowers. However, and metal industry combustion processes, whereby it is it is important to note that in all of these cases, the threshold transported from the soil to plants, consequently contaminating was never exceeded in the corresponding honey matrix. This the nectar and the honey [. Cd is one of the most missing correspondence can be explained by the biology of dangerous heavy metals due to its high mobility and the small honeybees: after 3 weeks, the workers become foragers, and concentrations at which its effects on plants are seen The they gather pollen, nectar, honeydew, and water for the presence of this contaminant in the live honeybee matrix can colony until their death ], and as these honeybees are be explained according to Yaaqub et al. ], who showed that the only ones to get out of the hive, they are the ones 33 % to 72 % of the local Cd is supplied from the air, and involved in the accumulation of heavy metals from the according to Harrison and Williams [who explained that environment. Therefore, this might represent a point event airborne Cd is transferred predominately by large-scale atmo- of environmental pollution, which was enough to pollute the spheric transport. Moreover, the absence of Cd in the honey foragers only in a given period, but not enough to be accu- matrix might indicate low concentrations of this element, mulated in the honey.
which might not be sufficient to contaminate the nectar of the Accordingly, critical situations were observed for sites foraged plants.
where the thresholds were exceeded in particular months in Also in the case of Pb, the threshold was exceeded only in both matrices, so in the live honeybees and in the honey, this the live honeybee matrix. As suggested by Lambert et al. [ would confirm a continuous polluting event. These situa- as honeybees appear to be more sensitive to Pb contamination, tions occurred for only two sites: during the summer for Ni this could be linked to exposure to Pb during their flight, so the and in early autumn for Cr.
peaks of this contamination in the bees should reflect very The most common of these heavy metals in the Marche occasional episodes of atmospheric contamination. Perugini Region was definitely Cr, which showed the highest thresh- et al. reported similar results with a significant difference old overruns. Kotaś and Stasicka [and Seigneur and in Pb concentration in honeybees through comparing sites Constantinou ] demonstrated that 30 % to 40 % of the located in urban areas and sites located in nature reserves. In Cr in the atmosphere originates from natural sources, such as their case, the highest mean concentrations were detected in the weathering of rock constituents, wet precipitation, dry honeybees collected from hives for which the surroundings fallout from the atmosphere, and runoff from the terrestrial were characterized by intense air traffic and intense motor systems; the remaining 60 % to 70 % comes from anthropo- vehicle circulation. This hypothesis is not directly supported genic sources. Indeed, Cr is a significant worldwide problem by the present study because the nature reserves that showed ], with interest in it originating from widespread use of Cr the higher values of Pb were mostly surrounded by a wilder in various industries, such as for chrome colors and dyes, environment. However, this might also indicate drift, and the cement manufacture, and wood preservatives [].
accumulation of Pb from the polluted atmosphere of the Therefore, large quantities of Cr compounds are discharged nearest urbanized areas.
as liquid, solid, and gaseous pollutants into the environment In the literature, Ni is considered to be one of the many and will ultimately have significant adverse biological and trace metals that are widely distributed in the environment, as ecological effects. The amount of Cr at any particular time it is released from both natural sources and anthropogenic and location will thus depend mostly on the intensity of the activity ]. Ni has many industrial and commercial uses, industrial processes in the proximity of the sources, the and it has been increasing in worldwide importance with the amount of Cr released, and the meteorological factors ].
application of new technologies [Although Ni is In the present study, it can be noted that the Cr threshold was omnipresent and is vital for the function of many organisms, exceeded during the month of October for almost all of the its high concentrations in some areas from both anthropo- sites. This situation appears to be caused by the regional genic release and naturally varying levels might be toxic to industrial activities and the weather conditions: the higher living organisms [Despite this, during these 3 years of frequency of excess over the thresholds might have been survey, Ni exceeded the threshold only a few times in the live driven by the combination of anthropogenic activities and honeybees and honey. This can be explained as it finds its the lack of rainfall recorded in the period before the sam- way into the ambient air as a result of the combustion of coal, pling. In support of this thesis, the Cr threshold was not diesel oil and fuel oil, the incineration of waste and sewage, exceeded during the year in which the rainfall during the and from miscellaneous sources , and these are late summer was higher (data not shown).
activities that are slightly evident in the Marche Region.
Biomonitoring with Honeybees in Nature Reserves These data for the heavy metal content of the honey also "Use of Apis mellifera in biomonitoring of Nature Reserves." The com-ments and suggestions made by three anonymous reviewers helped us to support the previous data from Leita et al. [Fakhim- improve this manuscript.
Zadeh and Lodenius [], and Porrini et al. [], inwhich there was again relatively low contamination ofhoney. This is probably due to "filtering" by the hon- eybees, as honey has a considerably lower heavy metalcontent than live honeybees.
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World J Gastroenterol 2012 March 21; 18(11): 1141-1153 ISSN 1007-9327 (print) ISSN 2219-2840 (online) © 2012 Baishideng. All rights reserved. EDITORIAL Genetically modified mouse models for the study of nonalcoholic fatty liver disease Perumal Nagarajan, M Jerald Mahesh Kumar, Ramasamy Venkatesan, Subeer S Majundar, Ramesh C Juyal