(Rattus rattus L.) and Algerian mice

15 Pages·2005·3.56 MB·English

EnvironmentalPollution139(2006)561e575
www.elsevier.com/locate/envpol
Tissues and hair residues and histopathology in wild rats
(Rattus rattus L.) and Algerian mice (Mus spretus Lataste)
from an abandoned mine area (Southeast Portugal)
R. Pereiraa,b,*, M.L. Pereira a, R. Ribeiro c, F. Gonc¸alves a
aDepartamentodeBiologiadaUniversidadedeAveiro,CampusUniversita´riodeSantiago,3810-193Aveiro,Portugal
bInstitutoPiaget,CampusAcade´micodeViseu,EstradadoAltodoGaio,Lordosa,3515-776Viseu,Portugal
cInstitutodoAmbienteeVida,DepartamentodeZoologiadaUniversidadedeCoimbra,
LargoMarqueˆsdePombal,3004-517Coimbra,Portugal
Received20February2004;accepted23April2005
The bioaccumulation of As and Cd and signs of renal histopathological injury proved the value of
Algerian mice as a bioindicator species in the risk assessment of contaminated sites.
Abstract
DatagatheredinthisstudysuggestedtheexposureofratsandAlgerianmice,livinginanabandonedminingarea,toamixtureof
heavymetals.Althoughsimilarhistopathologicalfeatureswererecordedintheliverandspleenofbothspecies,theAlgerianmouse
has proved to be the strongest bioaccumulator species. Hair was considered to be a good biological material to monitor
environmentalcontaminationofCrinrats.Significantpositiveassociationswerefoundbetweenthelevelsofthiselementinhair/
kidney (rZ0.826, nZ9, p!0.01) and hair/liver (rZ0.697, nZ9, pZ0.037). Although no association was found between the
levelsofAsrecordedinthehairandintheorgans,thelevelsofthiselementrecordedinthehair,ofbothspecies,weresignificantly
higher in animals captured in the mining area, which met the data from the organs analysed. Nevertheless, more studies will be
neededto reduce uncertaintyaboutcauseeeffectrelationships.
(cid:1)2005Elsevier Ltd. Allrights reserved.
Keywords:Rodents;Heavymetals;Exposureassessment;Liver;Spleen;Kidney;Hair;Histopathologicalchanges
1. Introduction decades, after mineral exploitation has ceased (Helms,
1993; Pereira et al., 1995, 1999a; Lopes et al., 1999).
Although metals are naturally occurring elements, Terrestrial vertebrates have been reported to be
their concentration in environmental compartments is exposed to heavy metals in contaminated areas and to
significantly increased by anthropogenic activities, such bioaccumulate them in different tissues (Hyva¨rinen and
as mining. Metallic and non-metallic ore extraction Nygre´n,1993;Burgeretal.,1994,2000;Gochfeldetal.,
activities are responsible for serious impacts on the 1996;Erryetal.,1999;Pereiraetal.,1999b;Ka˚la˚setal.,
environment,whichmostofthetimespersistforseveral 2000; Mertens et al., 2001). Therefore, animals have
long served to evaluate inorganic compound exposures,
especially for Cd, Hg and Pb (Talmage and Walton,
* Correspondingauthor.DepartamentodeBiologiadaUniversidade
1991). Data collected from animals, which are exposed
de Aveiro, Campus Universita´rio de Santiago, 3810-193 Aveiro,
to contaminants in their natural habitat, are very
Portugal.Tel.:C351234370200x22712;fax:C351234370777.
E-mailaddress:[email protected](R.Pereira). important for human and environmental health risk
0269-7491/$-seefrontmatter(cid:1)2005ElsevierLtd.Allrightsreserved.
doi:10.1016/j.envpol.2005.04.038
562 R.Pereiraetal./EnvironmentalPollution139(2006)561e575
assessments. Such data provide information about performed in Portugal and Spain (Ruı´z-Laguna et al.,
environmental and food chain contamination with 2001; Nunes et al., 2001a, 2001b; Lopes et al., 2002).
chemicals or chemical mixtures, potential wildlife and The present study isintegrated intherisk assessment
human exposures, and about hazard effects on animals process planned for the abandoned St. Domingos mine
themselves (NRC, 1991). (Pereiraetal.,2004).Thespecificobjectivesofthiswork
Chronic exposures to heavy metals have been were:(i)toassesstheexposureofratsandAlgerianmice
associated with various effects on blood biochemistry tometalsandarsenicpresentinthevicinityofthemine;
(WHO, 1998; Silva et al., 1999; Liu et al., 2000), (ii) to identify adverse effects, namely histopathological
enzymes and transport proteins activity (Foulkes, 1996; and weight changes, in the liver, kidney and spleen of
Vogiatzis and Loumbourdis, 1998; Wright et al., 1998; rats and mice, and to compare them with exposure
Liu et al., 2000; Lopes et al., 2002), and on cells levels; (iii) to make some insights about the bioavail-
structure and function (Markovich and James, 1999). ability of metals, to wild mammals, on the mining
Most of these effects are yielded by hepatotoxic and area; (iv) to discuss the suitability of histopathological
nefrotoxic mechanisms causing severe histopathological features as an endpoint in wildlife and human health
alterations (Silva et al., 1999; Jeong et al., 2000; Liu riskassessmentinmetal-contaminatedscenarios,and(v)
et al., 2000). Therefore, tissue lesions are an important to evaluate the suitability of hair as a monitoring tool
assessment endpoint for the effects induced by chronic for assessing wild mammals exposure. For the matter,
exposure to heavy metals in natural conditions, a fact the constraints imposed by the European Union
that is corroborated by Talmage and Walton (1991), legislation in force (EC, 1986), regarding the protection
Stehr et al. (1997) and Silva et al. (1999). Even when of animals used for experimental and other scientific
cellular responses are not toxic manifestations, such as purposes, were considered in this study.
degenerationornecrosis,butadaptiveones,theymaybe
importantindicatorsofexposurethatshouldbeassessed
(Haschek and Rousseaux, 1998). 2. Material and methods
By obvious reasons, field conditions are always
difficult to mimic in lab experiments, especially when 2.1. Study area
wild organisms are exposed to complex mixtures of
contaminants where synergistic and/or antagonistic TheSt.Domingosmineisacupricpyritemine,located
effectsaretobeexpected.Additionally,availablemodels in Southeast Alentejo (Portugal), where tons of mining
for exposure and bioaccumulation prediction can in- tails where left behind, exposed to atmospheric con-
troduce considerable uncertainty into screening-level ditions(Fig.1).Theexploitationfinishedin1965without
ecological risk assessment (Torres and Johnson, 2001). any attempt to take reclamation measures in order to
Thus, in order to overcome these constraints, wild rats prevent the aerial transportation of the finest particles
(Rattus rattus L.) and Algerian mice (Mus spretus and the secondary pyrite oxidation, with subsequent
Lataste) were the species chosen in this study to assess production of acidic conditions, favourable for heavy
an integrated exposure to a complex mixture of heavy metals mobilization (Helms, 1993; Gerke et al., 1998).
metals, and subsequent toxic effects, in the vicinity of Acid mine drainage (AMD) has been responsible for
aderelict pyritemine.Thesespeciessatisfied thecriteria seriousthreatstoecosystemssincehighconcentrationsof
to be considered good indicators, namely: (i) they have toxic metals were found in the aquatic compartment of
alargegeographicdistributionandcanbefoundinboth the St. Domingos mine area (Pereira et al., 1995, 2000;
contaminated and non-contaminated areas; (ii) they Lopes et al., 1999). The impacts of AMD on aquatic
may cohabit with humans and eat their food, which organisms and communities have already been assessed
makesthempotentialindicatorsofhumanexposure;(iii) with laboratory and in situ bioassays, and field studies
theyareacomponentofsometerrestrialecosystemsand (Canteiro,1994;Lopesetal.,1999;Pereiraetal.,1999a,
occupyamiddlepositioninmanyfoodchains;(iv)they 2000; Castro et al., 2003). Few researches have been
contact with soil during their entire life cycle, being performed in the terrestrial compartment of the mine
exposed to heavy metals mainly by ingestion of area. Total metal concentrations, pH, organic matter,
contaminated food or soil and through dermal absorp- and conductivity, in surface soils, were assessed at
tion;(v)theyhavesmallhomeranges,typicallylessthan differentdistancesfromthemine(Pereiraetal.,inpress)
90m(MacDonaldandBarret,1993),whichmakesthem (Table1).HigherconcentrationsofAs,Cu,Fe,Pb,and
appropriate site-specific indicators of contamination; Zn were recorded in themining areawhen compared to
and finally, (vi) their populations are usually large thenorthpartofthemine(Fig.1).As,Cd,Crwereabove
enough to support harvesting without a major adverse toxic levels in both areas, according to the soil quality
effect at the population level (NRC, 1991). Regarding criteriarecommendbyMEEandDEPA(1995).Cuand
the Algerian mouse, it has been widely used as a Pb overcame these criteria only in the mining area.
bioindicator species in some environmental studies However, the low pH values recorded by Pereira et al.
R.Pereiraetal./EnvironmentalPollution139(2006)561e575 563
Corte do Pinto
Tapada
Grande
N (TG)
Tapada Pequena
Aveiro
n
a
ce Mina de
O n S. DomingosMine Pit
antic Spai
Atl Lisbon Sulphur mill
(M4)
Guadiana River Acid mine drainage Montes Altos
Mina de S.
Domingos
Treatment
ponds
Atlantic Ocean Mosteirão
stream Telheiros
Chança River
Santana
de Cambas
Chança Reservoir
Malagon River
Pomarão Chança
Dam
Guadiana River
Residential areas
1Km
Fig.1.MapofthestudyarealocatedinSoutheastPortugal.
(1999b, in press) suggested a potentially greater bio- recordedhigherlevelsofCd(max.value2.08mg/kgwet
availability of metals in the vicinity of mine. The weight)andFe(max.value647mg/kgwetweight)inthe
assessmentofsoilenzymeactivitiesintheSt.Domingos liverofratscapturedneartheoldsulphurmill(M4)than
mine area, to evaluate the effects of soil contaminants inthosecapturedinthenorthpartoftheTapadaGrande
on its microbial community and subsequently on soil lake (TG) (Fig. 1). In opposition, Zn concentrations
functions,alsosuggestedagreatbioavailabilityofmetals (max. value 331mg/kg wet weight) were higher in livers
in the mining area (Pereira et al., in press). A previous ofratscapturedatTG.Theauthorsdidnotanalyseother
study on metal bioaccumulation (Pereira et al., 1999b) metalsrecordedinthesoilandwatercompartments.
564 R.Pereiraetal./EnvironmentalPollution139(2006)561e575
Table1
TotalAsandmetalconcentrations(mg/kg),pH,conductivity(Cond.,mS/cm)andorganicmatter(OM,%)atTGandM4sitesintheSt.Domingos
minearea(Pereiraetal.,inpress)
Fe Cu Cr Zn Pb Ni Mn As Cd pH Cond. OM
TG 47,000 32 231 87 40 44 1608 20 3 6.4G0.2 86.9G67.0 7.68G3.5
M4 49,000 281 114 108 1124 11 156 306 1 4.7G0.7 74.7G39.8 5.8G1.8
pH,conductivity(Cond.)andorganicmatter(OM)eAverageGSD.
2.2. Sampling procedure 2.3. Sample preparation for light and
electron microscopy
SmallmammalsweresampledatTG,3kmapartfrom
theminepit,andM4,neartheoldsulphurmill(Fig.1).The Small sections of liver, spleen and kidney were
choiceofthesesiteswassupportedbyphysical,chemical collected and fixed for 24h in Bouin’s fluid. Dehydra-
and soil microbial parameters, which gave some insights tion in graded concentrations of ethanol and sample
aboutthedegreeofsoilcontaminationandtoxiceffectson inclusion in paraffin wax was sequentially performed.
soilfunctions(Pereiraetal.,inpress).FromApriltoJuly Sections(5e7mmthick)wereobtainedandstainedwith
2000,ratsandmicewerecapturedinthree5-dayperiods, hematoxylineeosin for light microscopic examination.
usinglivetrapsbaitedwithamixtureofcannedtunafish Observations were made and photographs taken using
andchildren’spap.Thetrappedanimalsweretakenalive a microscope model Olympus BH2-RFC equipped with
to the laboratory, where they were anaesthetised and an automatic photomicrographic system Model PM-
sacrificedaccordingtolegalprocedures.Trapswereplaced 10ADS.Theremainingportionsoftheorganswerestored
on the field some days prior to the capture allowing inpolypropylenetubesat(cid:2)20 (cid:1)Cuntilchemicalanalysis.
animalstofamiliarisewiththenewobject(Fig.2). For electron microscopy, small fragments of liver
After being weighted, the sex of each animal was werefixedwith2.5%glutaraldehydein0.1Mcacodilate
determined and standard mammal body measurements buffer, and then washed in the same buffer. Specimens
were recorded. Liver, kidneys and spleen were dissected were postfixed in 2% buffered osmonium tetroxide,
and weighed to the nearest 0.1mg. Eye lenses were dehydrated through graded alcohols, and embedded in
removed and preserved in a borate buffered 4% Epon 812 (158 EMS(cid:2)). Thin sections were made with
formaldehyde solution. As soon as possible, eye lenses adiamondknifeanddouble-stainedwithuranylacetate
were dried at 60 (cid:1)C, until constant weight. The weight (Merck(cid:2)) and lead citrate (17800 EMS(cid:2)). Observations
of eye lenses is considered a good criterion to assess were carried out using a Hitachi transmission electron
mammals’ age because its development is continuous microscope, operating at 100kV.
during the animals’ entire life by accumulation of
insoluble proteins (Bourlie`re and Spi, 1975; Wheeler 2.4. Chemical analysis
andKing,1980).Hairsampleswerealsocollected,from
the inguinal and lumbar regions and stored in poly- Tissue samples were oven-dried at 105 (cid:1)C until
ethylene bags, closed with a zipper. After collection, constant weight and dry weight was determined. Wet
sampleswereimmediatelytransportedtothelaboratory ashing was performed in a sand bath at approximately
and stored at (cid:2)20 (cid:1)C, prior to analysis. 60 (cid:1)C, in closed 50-ml polypropylene tubes using
the HNO (extrapure, Merck(cid:2))/H O (proanalysis,
3 2 2
Merck(cid:2)) procedure. Hydrogen peroxide was added to
the samples in 1-ml aliquots until they were clear. The
samples were diluted to a final volume with Milli-Q(cid:2)
water obtained from an ultrapure water System (Ultra
Clear SG(cid:2)). Wet ashing was considered an appropriate
procedure for the preparation of biological samples for
chemical analysis, since it is performed at a relatively
lowtemperaturereducingthepossibilityofanalytesloss,
especially the most volatile ones (Miller-Ihli, 1989;
Hoening and Kesabiec, 1996).
Before chemical analysis, hair samples were cut into
smallpieces(2e3mm)andwashedfivetimes,according
to the method proposed by IAEA (1977 in Subrama-
nian, 1996), following the sequence: acetoneeMilli-Q(cid:2)
watereMilli-Q(cid:2) watereMilli-Q(cid:2) watereacetone. Hair
Fig.2.Livetrapsinthefield. samples were immersed in 25ml of acetone or water,
R.Pereiraetal./EnvironmentalPollution139(2006)561e575 565
eachtime,during20minwithmagneticstirring.Washed Table2
sampleswereoven-driedat60 (cid:1)C,during24h,andthen Bodyandorgansweightandcorrespondingratiosfromratsandmice
capturedatM4andTGsites
weighed. The washing step is controversial (Bozsai,
1992; Subramanian, 1996), but it is crucial for the M4 TG
analysis of hair samples in the removal of exogenous AverageGSD n AverageGSD n
dirt. Rattusrattus
A GBC Model 932 Plus Atomic Absorption Spec- Bodyweight(bw) 87.469G47.56 5 98.959G64.87 4
trometer (AAS) was used for flame atomic absorption Kidneyswt(average) 0.361G0.19 5 0.380G0.23 4
Kidneyswt/bw 0.005G0.002 5 0.004G0.0 4
analysis of Fe, Mg and Zn. For Graphite Furnace ana-
Liverwt 5.154G3.59 5 6.092G1.99 4
lysis of As, Cd, Cr, Cu, Mn, Ni, and Pb, this apparatus
Liverwt/bw 0.057G0.02 5 0.062G0.001 4
was equipped with a GBC-GF3000 Graphite Furnace, Spleenwt 0.101G0.07a 5 0.237G0.09a 4
deuterium background correction and a furnace auto- Spleenwt/bw 0.002G0.001 5 0.003G0.001 4
samplerPAL3000.Standardswerepreparedusingstock
Musspretus
solutions Specpur(cid:2) 1000mg/ml (Alfa Aesar(cid:2), Johnson Bodyweight(bw) 21.236G4.32 8 18.571G7.32 7
Matthey Company). Dilutions were made using HNO Kidneyswt(average) 0.156G0.04 8 0.181G0.12 7
3
at 2%. For each element, three standards were used to Kidneywt/bw 0.007G0.002 8 0.013G0.02 7
Liverwt 1.006G0.30 8 0.889G0.34 7
covertheanalyticalworkingrangeoftheinstrument.The
Liverwt/bw 0.047G0.01 8 0.049G0.01 7
standardadditionmethodwasperformedinsomespare
Spleenwt 0.023G0.01 8 0.021G0.01 7
hairandbovineliverandkidneysamples.Recoveriesof Spleenwt/bw 0.001G0.0 8 0.001G0.001 7
know amounts of trace elements added to the samples
bw,bodyweight(g);wt,wetweight(g).
variedbetween82and108%. a Statisticallysignificantdifferencesbetweensites.
Ni(NO ) /Mg(NO ) and diammonium hydrogen
3 2 3 2
phosphate(1%)wereusedasmatrixmodifierstothermally enceswerefoundbetweenrelativeweights(organweight/
stabilisearsenicandlead,respectively(Krynitsky,1987). body weight) of eye lenses, liver, kidney, and spleen for
ratsandmicefrombothsites.
2.5. Statistical analysis The heavy metal and As concentrations were
recorded in the liver, kidney and spleen of both species
For the comparison of trace element concentrat- (Tables 3 and 4). The liver and spleen were the target
ions between different groups of individuals, one-way organs for essential elements such as Fe, Mg and Zn.
ANOVAswereperformed.Inordertomeettheassump- The average level of Mg was significantly higher in the
tionsofthisstatisticalprocedure,dataweretransformed liverofratsfromM4(FZ201.349,d.f.Z8,p!0.001)
using the equation: x#Zlog(xC1) (Zar, 1996). Ratios thaninthosefromTG,whiletheoppositewasobserved
were transformed by the equation, x#Zarcsen(x). for Fe and Zn (Fe: FZ10.229, d.f.Z8, pZ0.015; Zn:
Pearson’s correlation coefficients (r) were calculated to FZ37.510, d.f.Z8, p!0.001). Although no signifi-
investigate associations between heavy metals and As cantdifferenceswererecordedbetweensites,thehighest
concentrationsrecordedinthedifferenttissuesandhair. average concentration of Fe was recorded in the spleen
Foreachelement,associationsamongtheconcentrations ofratsfromM4(Table3).Theaverageconcentrationof
found in the different tissues were also analysed by the Mg in the kidneys was also significantly higher in rats
same statistical procedure. Statistical analyses were from M4 (FZ5.932, d.f.Z8, pZ0.045). Considering
performed with transformed data after normality and non-essential elements, the spleen of rats from M4 was
thehomogeneityofvarianceshavebeenconfirmed.Raw also the target organ for the bioaccumulation of As
data are presented in Tables 2e5, in order to facilitate (FZ9.528, d.f.Z8, pZ0.018) (Table 3).
comparisonwithdatareportedbyotherauthors. IntheliverofAlgerianmice,theaveragelevelsofMg
(FZ9.501, d.f.Z14, pZ0.009) and Fe (FZ7.690,
3. Results d.f.Z14, pZ0.016) were significantly higher at M4
thanatTG(Table4).Similartorats,thehighestaverage
3.1. Tissue residues analysis concentration of Fe was recorded in the spleen of mice
from M4, although no significant differences were
All the wild rats were males, except one, and all the recorded (Table 4). The average concentration of Mg
mice were females, which reduced the variability associ- in the kidneys was also significantly higher in the mice
ated with sex. No significant differences were found fromM4(FZ8.381,d.f.Z14,pZ0.013).Thekidneys
(pO0.05) in the body parameters recorded (Table 2), of mice from M4 presented significantly higher average
between animals captured at the TG and M4 sites, in concentrations of As (FZ9.530, d.f.Z13, pZ0.019)
either species. An exception was observed in rats from and Cd (FZ5.633, d.f.Z14, pZ0.034) (Table 4).
M4, which had significantly lighter spleens (FZ6.522, Significantly higher concentrations of As were found
d.f.Z8, pZ0.038). Additionally, no significant differ- in hair of both species from M4 than from TG
566 R.Pereiraetal./EnvironmentalPollution139(2006)561e575
Table3
ConcentrationsofmetalsandAsonratsfromtheTGandM4sites
Liver Kidney Spleen
AverageGSEM n AverageGSEM n AverageGSEM n
Cr
)
TG 0.513G0.221 4 0.345G0.051 4 1.231G0.332 4
)
M4 0.117G0.029 5 0.072G0.012 5 0.185 1
Cu
TG 12.147G0.956 4 13.998G3.896 4 11.095G2.261 3
M4 15.073G4.320 5 22.104G3.296 5 14.007G2.679 3
Fe
)
TG 316.811G31.112 4 292.538G90.770 4 747.448G251.428 4
)
M4 189.543G25.510 5 164.506G24.856 5 1863.135G845.696 4
Mg
) )
TG 0.016G0.003 4 0.029G0.011 5 0.05G0.022 5
) )
M4 0.382G0.02 5 0.214G0.066 4 0.353G0.111 4
Ni
TG 0.060G0.021 4 0.060G0.021 4 0.841G0.301 4
M4 0.266G0.142 5 0.065G0.020 5 0.577G0.330 5
Zn
) )
TG 0.141G0.014 4 0.107G0.03 4 0.06G0.015 4
) )
M4 0.048G0.008 5 0.076G0.008 5 0.120G0.012 5
As
)
TG 0.003G0.003 4 1.041G0.342 4 0.678G0.309 4
)
M4 12.733G9.915 5 2.198G0.602 5 9.993G2.651 5
Cd
TG 0.381G0.025 4 0.409G0.076 4 0.506G0.163 4
M4 0.320G0.140 5 1.275G0.489 5 0.747G0.089 3
Pba
TG e 0.643 1 2.706 1
M4 5.953 1 3.344 1 16.592e82.745 2
Onlyconcentrationsabovethedetectionlimitwerepresented.Fe,MgandZnconcentrationsexpressedasmg/g,dryweight.As,Cu,Cd,Cr,Niand
Pbconcentrationsexpressedasmg/g,dryweight.
)
Statisticallysignificantdifference(p!0.05).
a Onlyconcentrationrangesarepresented.
(rats: FZ59.432, d.f.Z8, p!0.001; mice: FZ26.188, The highest average concentrations were always re-
d.f.Z13, p!0.001). The concentrations of Ni (FZ corded in Algerian mice.
22.627, d.f.Z8, pZ0.002), Cr (FZ35.592, d.f.Z8, Significantpositiveassociationswererecordedbetween
p!0.001)and Cu (FZ22.452,d.f.Z8,pZ0.002)were non-essentialandessentialelementsintheliverofrats(As/
significantlyhigherinthehairofratsfromTG.Although Zn: rZ0.692, pZ0.0387) and mice (Cd/Fe: rZ0.651,
nosignificantdifferenceswerefound,thehighestaverage pZ0.009;As/Mg:rZ0.543,pZ0.045).Althoughstron-
concentration of Cd was recorded in animals from M4. ger, the same kind of associations was recorded in the
This observation meets the data obtained for liver and spleenofrats(As/Mg:rZ0.841,pZ0.008)andmice(Cd/
kidneysamples (Tables 3e5). Thesedatasuggestedthat Mg:rZ0.710,pZ0.022).Interactionsbetweenessential
haircanonlybeusedasanindicatorofmetallevelsinthe elements in the liver, kidney and hair were diverse and
kidney (rZ0.826; nZ9; p!0.01) and liver (rZ0.697; difficulttopredict(Table6).
nZ9; pZ0.037) of rats for Cr. However, data for the
concentrations of As in the hair of both species were 3.2. Histopathological analysis
significantly higher in animals from M4, which was in
agreement with data from the analysed organs. Lead The liver of rats from M4 demonstrated a strong
concentrationswereabovethedetectionlimitintoofew hepatocyte vacuolation. The natural arrangement of
samplestomakeappropriatecomparisonsand,therefore, these cells in cords was also visibly disturbed, in
onlyrangesarepresentedinTables3and4. opposition totheliveroftherats from TG(Fig. 3a and
Interspecies comparisons revealed significant differ- b). There were no signs of morphological injury in the
encesbetweentheliverconcentrationsofFe(FZ8.027, hepatic portal vein of animalsfrom eithersite (Fig. 3b).
pZ0.015), Cu (FZ5.254, pZ0.043) and Zn (FZ LiverspecimensfromallratscapturedatM4hadsimilar
12.568, pZ0.005). The same was observed for the histological changes, except for the liver of one rat that
hair concentrations of Zn (FZ12.508, pZ0.005), Cu also showed signs of haemorrhage. The ultrastructural
(FZ8.221, pZ0.015) and Ni (FZ5.819, pZ0.034). observation of rats’ hepatocytes confirmed the presence
R.Pereiraetal./EnvironmentalPollution139(2006)561e575 567
Table4
ConcentrationsofmetalsandAsonmicefromTGandM4sites
Liver Kidney Spleen
AverageGSEM n AverageGSEM n AverageGSEM n
Cr
TG 0.361G0.068 7 1.441G0.343 7 14.921G6.127 7
M4 0.651G0.238 8 0.985G0.361 8 8.357G2.961 5
Cu
)
TG 38.623G7.796 7 26.059G6.145 7 6.577G3.100 6
)
M4 29.034G3.969 8 47.781G13.104 8 21.831G4.670 6
Fe
)
TG 333.456G53.518 7 219.024G52.043 7 e e
)
M4 681.185G126.299 8 280.537G38.004 8 1919.311G2061.786 4
Mg
) ) )
TG 0.250G0.071 7 0.306G0.061 7 0.318G0.140 7
) ) )
M4 0.548G0.066 8 0.517G0.122 8 0.767G0.124 5
Ni
TG 0.186G0.073 7 0.359G0.092 7 0.758 1
M4 0.561G0.237 8 2.175G0.823 8 12.526G4.423 7
Zn
)
TG 0.108G0.022 7 0.093G0.007 7 0.137G0.096 7
)
M4 0.094G0.009 8 0.112G0.027 8 1.340G1.167 3
As
)
TG 0.148G0.026 6 0.861G0.267 6 1.734G0.608 6
)
M4 1.792G0.946 8 3.887G0.817 8 e e
Cd
)
TG 0.293G0.087 7 0.379G0.119 7 0.270G0.042 7
)
M4 1.014G0.337 8 1.122G0.273 8 1.191G0.639 8
Pba
TG 2.562 1 6.316e6.738 2 e e
M4 0.869 1 19.555 1 e e
Onlyconcentrationsabovethedetectionlimitwerepresented.Fe,MgandZnconcentrationsexpressedasmg/g,dryweight.As,Cu,Cd,Cr,Niand
Pbconcentrationsexpressedasmg/g,dryweight.
)
Statisticallysignificantdifference(p!0.05).
a Onlyconcentrationrangesarepresented.
oflargevacuolesinthecytoplasm(Fig.4).Althoughless authors(e.g.,Halbrooketal.,1993;ShoreandDouben,
pronounced, hepatocyte vacuolation and an abnormal 1994; Nunes et al., 2001b), with wild and laboratory
arrangementofepitheliumcellswerealsoobservedinthe mammals, which usually observe reductions in the
AlgerianmicefromM4,inoppositiontothosefromTG weight of body and organs of animals exposed to en-
(Fig.5aandb). vironmental contaminants. Still, as it was pointed out
The kidneys of mice from M4 showed glomerulla by Nunes et al. (2001b), reductions recorded in body
swelling(Fig.6aandb).Nolesionswereobservedinthe measurementsandtraitsofwildmammalsshouldnotbe
kidneys of rats from either site (data not shown). directly attributed to contaminants, because resources
The observation of histological sections of spleen may be available differently between contaminated and
showed cell depletion near the fibrocollagenous capsule uncontaminated areas, leading to a differential growth
in rats from both sites (Fig. 7a and b). However, in the rate of local populations. Spleens’ atrophy may explain
spleen of rats from the vicinity of the mine the the decrease in weight recorded for this organ in rats
distinction between red and white pulp was not clear from M4, when compared to those from TG.
andcapsulethicknessincreased.RegardingtheAlgerian The selection of a reference site is often the most
mice, the most pronounced histopathological features difficulttaskinfieldstudiesbecauseitshouldpresentthe
observed in animals from M4 were a thick fibrocollag- same physical and chemical properties, when compared
enous capsule and the lack of cells in the periphery of to the contaminated sites, and simultaneously demon-
the organ (Fig. 8a and b). strate low toxicity of metals and other contaminants
(Indeherbergetal.,1998).IntheSt.Domingosminerisk
assessment process this problem also occurred, because
4. Discussion this area is located in the Iberian Pyrite Belt, a metal-
logenic province that crosses the Southeast Alentejo,
Except for the spleen of rats, the results obtained for towards Spain (Webb, 1958). Therefore, high concen-
body parameters contradict those obtained by other trations of heavy metals are to be expected even at
568 R.Pereiraetal./EnvironmentalPollution139(2006)561e575
Table5 Table6
ConcentrationsofmetalsandAsinthehairofratsandmicefromTG Pearsoncorrelationcoefficientsfortherelationshipsbetweenelements
andM4 in the organs of the Algerian mice and rats from the St. Domingos
minearea
Rattusrattus Musspretus
Rattusrattus Musspretus
AverageGSEM n AverageGSEM n
Liver Liver
Cr
) Fe/Mg (cid:2)0.764* Fe/Mg 0.523*
TG 1.309G0.197 4 1.673G0.618 7
) Fe/Zn (cid:2)0.757* Cr/Ni 0.737**
M4 0.232G0.066 5 0.579G0.165 8
Mg/Zn 0.924*** As/Mg 0.543*
Cu
) As/Zn 0.692* Cd/Fe 0.651**
TG 29.272G10.254 4 10.475G3.081 7
)
M4 3.856G0.756 5 21.385G4.731 8 Kidney Kidney
Fe Cr/Mg (cid:2)0.693* Cu/Ni 0.748***
TG 324.858G155.768 4 109.269G51.304 7 Cu/Zn 0.600*
M4 88.362G43.991 5 97.388G28.117 8
Spleen Spleen
Mg
As/Mg 0.841** Cd/Mg 0.710*
TG 0.071G0.008 4 0.481G0.103 7
M4 0.296G0.125 5 0.489G0.079 8 Hair Hair
Ni Fe/Cu 0.930*** Cd/Ni 0.685**
)
TG 0.178G0.024 4 0.283G0.073 7 Cr/Ni 0.954***
)
M4 0.069G0.008 5 0.701G0.204 8 Cr/As (cid:2)0.945***
Zn Ni/As (cid:2)0.876**
TG 0.160G0.042 4 0.257G0.032 7
*p%0.05;**p%0.01;***p%0.001.
M4 0.095G0.028 5 0.257G0.042 8
As
) )
TG 0.336G0.203 4 0.228G0.051 7
) )
M4 2.456G0.184 5 3.497G0.546 8 exposures (WHO, 1981; Hunder et al., 1999; Liu et al.,
Cd
2000). The toxicity of As in mammals was found to be
TG 0.228G0.027 4 0.227G0.052 7
related with levels above 3mg/g in the liver and kidney
M4 0.401G0.214 5 0.432G0.083 8
Pba (Gupta,1998)andanimaldatasuggestthatAsexposure
TG 0.031e4.299 2 e e may have chronic effects on the kidneys (WHO, 1981).
M4 19.245e61.290 2 0.460e0.946 2 Liuetal.(2000)recordedthatglomerularswellingisone
Onlyconcentrationsabovethedetectionlimitwerepresented.Fe,Mg of the degenerative changes that usually occur in mice
andZnconcentrationsexpressedasmg/g,dryweight.As,Cu,Cd,Cr, chronically exposed to As. These changes were also
NiandPbconcentrationsexpressedasmg/g,dryweight.
) observed in mice from M4. Although no positive
Statisticallysignificantdifference(p!0.05).
a Onlyconcentrationrangesarepresented. correlation between the levels of Cu and As was found
inthekidneyofmice,thepresenceofhighlevelsofboth
elements was recorded in animals captured at M4.
agreatdistancefromtheminebut,thoseconcentrations Differentauthors(e.g.,Hunderetal.,1999;Ademuyiwa
maynotreflectheavymetalstoxicitybecauseitdepends and Elsenhans, 2000) have reported this finding in the
on their availability, which in turn is determined by kidney of rats, which is probably due to a functional
several different physical and chemical factors (Esnaola relationship regarding the renal retention of these
and Milla´n, 1998; Rieuwerts et al., 1998). Although elements.Thelackofapositivecorrelationinourstudy
differentmethodshavebeenproposedfortheevaluation may result from interactions with other elements.
of metals availability in soils (e.g. Sterckeman et al., Both kidney and liver have been mentioned as the
1996; Esnaola and Milla´n, 1998), in situations of preferentialorgansforCdbioaccumulation(Leitaetal.,
environmental contamination, biomonitoring is usually 1991; Talmage and Walton, 1991; Alonso et al., 2002)
a powerful tool since it can provide valuable informa- and, once more, we have confirmed this fact for the
tionabouthazards,theirbioavailabilityandsubsequent Algerian mice. The kidney is the organ that first attains
impact on biota (Talmage and Walton, 1991). In the its critical concentration of Cd in chronic exposures,
St.DomingosmineareasignificantlyhigherlevelsofAs and histopathological changes are some of the early
andCdwererecordedinthekidneyofmicefromtheM4 symptoms (S´wiergosz-Kowalewska, 2001). Glomerular
site when compared to those captured at TG, which dysfunction was also reported as one of the effects on
suggested the great bioavailability of these elements in thekidneyyieldedby long-term exposure toCd(WHO,
theminingarea.Thesame wasobservedforratsbutno 1992). Regarding Pb, although its levels were recorded
significant differences were found among animals from in very few samples, suggesting that small mammals
both sites. Arsenical compounds can be present in the were not exposed to this element, it should not be
organism and the kidney is the major organ for As ignored that 90% of the total body burden of Pb is
elimination from the body, which contributes in a great found in bone (Talmage and Walton, 1991). Therefore,
extent for the susceptibility of this organ to As the biological samples analysed were not the most
R.Pereiraetal./EnvironmentalPollution139(2006)561e575 569
CV
H CV
H
S
a b
Fig.3.(a)LiverfromaratcapturedatTGwithoutsignsofmorphologicalinjuryatthecentralvein(CV)andinthesinusoidalchannels(S)(100!).
Epitheliashowednormalfeatures;(b)liverfromaratcapturedatM4displayingstronghepatocyte(H)vacuolation(100!).
appropriate to assess chronic low level exposures. that the bioaccumulation of this metal is frequently
Additionally, Shore and Douben (1994) have already higher in reference sites. These observations may be
observed the small capacity of wild rats to bioaccumu- explained by competition mechanisms between essential
late Pb in contaminated environments. and non-essential elements regarding metal enzyme
Chromiumwasmainlyrecordedintheorgans(except complexes. The opposite was observed in the liver of
for the liver) of rats and mice from TG. This met the Algerian mice from M4 which bioaccumulated the
data obtained for soil samples, since, the concentration highest levels of Cr. The strong positive association
ofthismetalinTGwasabout2-foldwhatwasrecorded foundbetweenCrandNi(rZ0.737,pZ0.0017)(Table6)
inM4(Pereiraetal.,inpress)anditwasabovethetoxic could have been responsible for such an aspect.
level defined for the soil compartment (MEE and Therefore, analogous mechanisms of bioaccumulation
DEPA, 1995). Similar to the observations in our study, for both elements, in the liver of Algerian mice, could
Talmage and Walton (1991), in their review of several explain the high levels of Cr recorded in this organ in
studies performed with wild mammals, also observed opposition to what was observed in kidney and spleen.
N
Fig.4.UltrastructuraldetailofahepatocyteofaratcapturedatM4(77,000!).Arrowsshowstrongvacuolationofcytoplasm;nucleus(N).
570 R.Pereiraetal./EnvironmentalPollution139(2006)561e575
H
H
a b
Fig.5.LiverfromAlgerianmicecapturedinSt.Domingosminearea.(a)TG(400!);(b)M4(400!).Arrowsshowhepatocytes(H).
The liver of rats and mice was the main target organ non-contaminated area. The low levels of essential
for some essential elements and which agrees with the elements recorded in these wild mammals can be
role of this organ in homeostatic mechanisms. The liver explainedbytheexposuretoamixtureofcontaminants
is the major organ primarily exposed to toxic and and the subsequent interactions between them. In spite
essential substances that enter the organism through ofthesignificantassociationsfoundfortheliveramong
inhalation or ingestion. Its main blood supply comes different essential trace elements (Table 6), these data
fromtheintestines(McCuskeyandSipes,1997;Haschek require confirmation. Several significant positive associ-
and Rousseaux, 1998). The liver extracts metals from ations between essential elements and As and Cd were
plasma, metabolises, stores and redistributes them in also recorded. When compared with the levels recorded
variousformseitherintothebileorbackintotheblood intheotherorgans,theFecontentsinthespleenofboth
stream (Bla´zovics et al., 2002). The highest levels of species were the highest, which results from the main
Cu, Zn and Fe recorded in the liver of the Algerian functions of spleen, namely the destruction of aged or
mice, from the St. Domingos mine area (TG and M4 damagedredbloodcellsandthestorageofFeasferritin
sites), were similar to those recorded by Lopes et al. or hemosiderin for future recycling (Ross et al., 1995;
(2002) in mice from the same species captured in a JunqueiraandCarneiro,1999).Thehistologicalchanges
BC
BC
G
G
a b
Fig.6.KidneyfromAlgerianmicecapturedinSt.Domingosminearea.(a)TG(400!);(b)M4.Arrowsshowglomerulus(G)andBowmancapsule(BC).