Bioaccumulation of As, Cd, Cu, Fe and Pb in wild grasses affected by the Aznalcóllar mine spill (SW Spain

The Science of the Total Environment 290(2002) 105–120

0048-9697/02/$ - see front matter� 2002 Elsevier Science B.V. All rights reserved.PII: S0048-9697Ž01.01070-1

Bioaccumulation of As, Cd, Cu, Fe and Pb in wild grassesaffected by the Aznalcollar mine spill(SW Spain)´

P. Madejon*, J.M. Murillo, T. Maranon, F. Cabrera, R. Lopez´ ˜ ´ ´

Instituto de Recursos Naturales y Agrobiologıa de Sevilla, CSIC, P.O. Box 1052, 41080 Seville, Spain´

Received 19 July 2001; accepted 5 October 2001

Abstract

The collapse of the tailing dam in the Aznalcollar pyrite mine(SW Spain) occurred in April 1998 and affectedápproximately 4300 ha along the Agrio and Guadiamar valleys. An urgent soil cleaning up and remediation programmewas started just after the accident. Eighteen months later, mineral nutrients and trace elements concentration in soiland two wild grasses —Cynodon dactylon andSorghum halepense — have been studied. Three types of conditionsare distinguished:(a) unaffected soils(control); (b) cleaned up and remediated soils(remediated); and (c) sludge-covered soils left in a fenced plot(non-remediated). As, Cd, Cu and Pb in grasses reached toxic levels for the foodweb in the non-remediated plot, while on remediated soils only Cd reached a toxic level in grass tissues. However,Pb and, to a lesser extent As and Fe, reached also toxic levels in unwashed plants(as they would be ingested byanimals) in remediated soils. Both native grasses seem tolerant of trace elements pollution and suitable for stabilisationof spill-affected soils.� 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cynodon dactylon; Sorghum halepense; Heavy metal; Guadiamar river

1. Introduction

Spills of mining wastes are a relative frequentsource of trace element pollution. The failure of atailing pond dam in the pyrite mine of Aznalcollar´(SW Spain), in April 1998, released a toxic spillof approximately 5 000 000 m that affected a total3

of 4286 ha along the Agrio and Guadiamar rivervalleys. A strip approximately 300–400-m-wideand 40-km-length, on both sides of the river

*Corresponding author. Tel.:q34-95-462-4711; fax:q34-95-462-4002.

E-mail address: [email protected](P. Madejon).´

channel was covered with a 2–30-cm-thick layerof a toxic black sludge(Grimalt et al., 1999).

After the mine spill, an emergency soil clean-up procedure quickly started. The toxic sludgecovering the ground was mechanically removedand disposed of in a mine open-pit. This cleaningup operation, lasting approximately 6 months,removed the sludge and a major portion of thecontaminated soil surface; however, the affectedzone continues to have a consistent pollution oftrace metals with a fairly irregular distribution(Moreno et al., 2001). Soil remediation was carriedout adding organic matter and calcium-rich amend-

106 P. Madejon et al. / The Science of the Total Environment 290 (2002) 105–120´

ments. On the other hand, a large-scale restorationplan included the purchase of the land(formerlydevoted to crops and pastures) and the design ofa public nature reserve, acting as a ‘green corridor’between the lowlands(Donana National Park) and˜

the mountains(Sierra Morena Natural Park) (Juntade Andalucıa, 1999). The successful management´

of this ‘green corridor’ would depend on theimmobilisation of trace elements still present inthe affected soils.

The restoration of a dense vegetation cover isthe most useful and widespread method to physi-cally stabilise the mine wastes and to reduce metalpollution effects (Bargagli, 1998). Differentautochthonous plant species, well adapted to thelocal conditions and relatively tolerant to highconcentration of metals in soil, should be used forthe early stages of revegetation of the ‘greencorridor’. Wild grasses can be a suitable materialfor this purpose(see a review of metal tolerantherbaceous species in McLaughlin et al., 2000).One candidate is the Bermuda grass(Cynodondactylon), a widespread creeping grass, which maytolerate pollution of trace elements(e.g. up to30 000 mg kg of As in soil) and thus is usefuly1

for stabilising spill-affected soils(Smith et al.,1998). In the process of vegetation restoration, acontinuous monitoring of trace element uptake andallocation in the selected grasses(or other plants)must be carried out in order to regulate and avoid(as much as possible) a consistent transfer of thesetrace elements along terrestrial food chains.

Here, we study the accumulation of trace ele-ments in the aboveground organs of wild grassesgrowing in the spill-affected soils of the Guadia-mar floodplain. We have selected two grass species— Bermuda grass(Cynodon dactylon) and wildsorghum(Sorghum halepense) — growing natu-rally in the trace elements polluted soils. Theconcentration of trace elements found in thesegrasses are compared with the toxicity levels forplants and animals, given in the literature; in caseof potential danger for animal feeding, we shouldrecommend the managers of the ‘green corridor’to take the appropriate measures.

2. Material and methods

2.1. Study area

Six sampling sites were selected along the Gua-diamar floodplain(Fig. 1). Two of these(sites 1and 2) were sited in locations not affected by themine-spill (control sites). Site 1 (near Gerenabridge, at 681193199N, 378329299W), was locatedupstream from the Aznalcollar mine and thus notáffected by the spill. Site 2(Soberbina farm, at681292899N, 3782792899W) was located approxi-mately 4.5 km from the mine, but on an upperriver terrace and was thus neither affected by thespill. Three of the sites were located on the areaaffected by the spill, where cleaning up of sludgeand remediation of polluted soil took place. Site 3was at Doblas bridge(12 km from the mine, at681395999N, 3782491899W), site 4 at Aznalcazar´bridge (25 km from the mine, at 681691299N,3781891699W) and site 5 at Vado del Quema(31km from the mine, at 681692799N, 3781591699W).Additionally, a fenced plot where the sludge hadnot been removed(for research purposes) was alsosampled(site 6, at 681692099N, 3781591799W) nearVado del Quema.

2.2. Plant and soil sampling

Two wild grass species, relatively abundant inthe affected area, were selected for the study:Cynodon dactylon (L.) Pers. var.affinis (Caro andSanchez) Romero Zarco(Bermuda grass, collectedín sites 1 to 6) andSorghum halepense (L.) Pers,(wild sorghum, collected in sites 2, 4 and 6).Nomenclature follows Valdes et al.(1987); for´simplicity they will be referred asCynodon andSorghum hereafter.

In each sampling site above ground organs ofthe grasses were clipped in three different patcheswith a distance among them of at least 30 m.Sampling was carried out during late October 1999(18 months after the spill). Plant samples(mixtureof stems and leaves, but excluding reproductivestructures) were washed(for 10 s approximately)with a solution of phosphate-free detergent, thenwith a 0.1 N HCl solution and finally with distilledwater. Plant material was then dried at 708 C,

107P. Madejon et al. / The Science of the Total Environment 290 (2002) 105–120´

Fig. 1. Area of the spill-affected zone and situation of the sampling sites in the Guadiamar valley(adapted from IGME, 1998).


Table 1Analysis of BCR reference samples(mean values"95% CI, mg kg dry matter)y1

Element CRM 279(sea lettuce) CRM 281(ryegrass)

Certified Experimental Certified Experimental

As 3.09"0.20 2.69"0.11 0.057"0.004 0.118"0.014Cd 0.274"0.022 0.202"0.007 0.120"0.003 0.117"0.005Cu 13.14"0.37 11.63"0.73 9.65"0.38 9.76"0.09Mn (2030"31.5) 1758"64.8 81.6"2.6 76.7"0.4Pb 13.48"0.36 12.47"1.09 2.38"0.11 2.29"0.07Zn 51.3"1.2 52.18"3.29 31.5"1.4 32.7"0.2

Value in parentheses is indicative; experimental values are calculated fromns6 (sea lettuce) andns5 (ryegrass).

ground and passed through a 500-mm stainless-steel sieve. A plant subsample was directly groundwithout previous decontamination by washing;with this treatment we study the actual concentra-tion of trace elements in the wild grasses(withadhered soil on plant surfaces) and their possibletoxic impact through their consumption as forage.

The washing techniques applied seemed effec-tive for sample decontamination. Ti concentrationvalues were rather low in washed tops of bothspecies(in the range of 5 to 10 mg kg , data noty1

shown), indicating a reasonably low level of exter-nal contamination. A measurement of Ti contentis a sensitive methodology to detect this type ofcontamination: given that Ti in plants(0.15 to 80mg kg ) is far lower than in soils(3300 mgy1

kg , mean content for world-wide soils, Kabata-y1

Pendias and Pendias, 1992), when levels of Tiexceed 100 mg kg in plants, they are usuallyy1

attributed to soil contamination(Cherney et al.,1983).

Soil samples, at 0–25 cm depth, were taken ineach patch where sampled grasses were rooted. Inthe case of site 6, only one sample of soil coveredwith the sludge was taken. Soil samples wereoven-dried at 408 C and crushed to pass through a2-mm sieve, and then ground to-60 mm for Sand trace element determinations.

2.3. Chemical analysis

Plant material was analysed for N by Kjeldahldigestion. Mineral nutrients(P, K, S, Ca and Mg),heavy metals and trace elements(As, Cd, Cu, Fe,Mn, Ni, Pb, Tl and Zn) were extracted by wet

oxidation with concentrated HNO under pressure3

in a microwave digester. Analysis of mineral nutri-ents, Fe and Mn in the extracts thus obtained wasperformed by ICP-OES(inductively coupled plas-ma spectrophotometry). Analysis of trace elementswas performed by ICP-MS(inductively coupledplasma-mass spectroscopy). Unwashed plant sam-ples were only analysed by ICP-OES.

The accuracy and precision of the analyticalmethod was assessed by carrying out analyses oftwo BCR (Community Bureau of Reference) ref-erence samples: CRM 279(Sea lettuce) and CRM281 (Ryegrass) (Griepink and Muntau, 1987,1988). The values obtained by ICP-MS and com-parative certified values for the same referencematerial are shown in Table 1.

Soil samples(-2 mm) were analysed for pHpotentiometrically in a 1:2.5 soil–water suspen-sion. Total carbonate content was determined bythe manometric method(Demolon and Leroux,1952). Size particle distribution was measured bythe hydrometer method(Gee and Bauder, 1986).Sulfur and trace elements(As, Cu, Pb and Zn)were determined by ICP-OES, after digesting thesamples (-60 mm) with a mixture of conc.HNO and HCl (‘aqua regia’); these values are3

referred to as ‘total’ concentration. Available con-centration of trace elements was determined byICP-OES after extracting the samples(-60 mm)with a 0.05-M EDTA solution. Total and availableconcentrations of trace elements are given on adry weight basis.

To evaluate the soil pollution severity, we usedthe pollution load index(PLI as defined by Tom-linson et al., 1980). This index is based on the


Table 2Soil characterisation(0–25 cm)

Site Soil pH CaCO3 Mean texturetype (%)

1 C 7.8 -1 Loamy sand2 C 7.6 23.5 Clay loam3 R 8.0 25.1 Clay loam4 R 7.7 5.8 Sandy loam5 R 8.1 6.7 Clay loam6 S 6.2 -1 Silty clay loam

Soil type: Cscontrol soil; Rsremediated soil; and Sssludge-covered soil.

value of the concentration factor(CF) of eachmetal in the soil. TheCF is the ratio obtained bydividing the concentration of each metal in thesoil by the base line or background values(theconcentrations of As, Cu, Pb and Zn for ‘normal’soil according to Bowen, 1979 have been used).For each sampling site,PLI is calculated as thenth root of the product of the obtainednCF. Valuesof PLI close to one indicates heavy metal loadsnear the background level, while values above oneindicate soil pollution(Cabrera et al., 1999).

2.4. Statistical analysis

The concentration of each trace element, in bothwashed and unwashed plant samples, was com-pared separately for the two grass species. Threedifferent habitats were considered according to thesoil pollution level:(a) control soils(as would bethe original conditions before the mine accident);(b) soils covered with sludge(as was the generalsituation after the mine spill); and (c) remediatedsoils (as is the general situation in the affectedarea after cleaning up the sludge and other soilremediation operations).

Analyses of variance(ANOVA), consideringone factor(soil pollution level), were performedfor the concentration of each trace element in thegrass. A multiple comparison of means was deter-mined by the ‘post-hoc’ Tukey test for unequalsample size. A significance level ofP-0.05 wasused throughout the study. When the normality testfailed (Kolmogorov–Smirnov test), the variableswere logarithmically transformed and normalitywas then passed in all the cases.

Correlation analysis was performed between theconcentration of trace elements inCynodon plantsand their availability(after EDTA extraction) inthe soil(excluding site 6). This preliminary infor-mation will indicate the suitability of this frequentplant as bioindicator of trace elements in the soil(Bargagli, 1998).

The SPSS(1999) for Windows program wasused for the statistical analyses mentioned above.

3. Results and discussion

3.1. Soil pollution

There is a large variety of soil types in theGuadiamar valley(Cabrera et al., 1999) and con-

sequently, the effects of the mine spill also varied.The main characteristics of the sampled soils areshown in Table 2. Soil of site 1(upstream fromthe mine) is sandy and without calcium carbonate.Soils of site 2(control, in upper terrace) and ofsites 3, 4 and 5(remediated) have a pH)7 andshow moderate to high calcium carbonate content.Soil of site 6(covered with sludge) has a pH-7and a negligible amount of carbonate.

The pollution load index(PLI) calculated forthe two control sites was lower than one, asexpected; whereas in the soil covered with sludgewas up to 22(Table 3). The remediated soilsranged from 5 to 12; that is, there was an importantreduction of soil pollution(approx. half ofPLI)caused by the clean-up operations(includingremoval of sludge and adding amendments); how-ever, remediated soils still remained contaminatedby heavy metals and trace elements in the autumnof 1999, a year and a half after the mine spill.

‘Total’ concentration values(extracted withaqua regia; this fraction is also called ‘quasi total’)of S, As and Pb in soil samples are shown inTable 3. There are three pollution levels for theseelements, as also shown by the PLI values:(1)the control soils are ‘non-polluted’;(2) the reme-diated soils are ‘fairly polluted’; and(3) thesludge-covered soil is ‘highly polluted’.

The available concentration values(extractedwith EDTA) of Cd, Cu and Zn in soil samplesshowed a similar pattern, with three pollutionlevels (Table 4). However, the available(EDTA-extracted) concentration values of As and Pbshowed only two pollution levels because thevalues in the sludge-covered soil were not different


Table 3Concentration of S, As and Pb extracted with ‘aqua regia’ andPLI (pollution load index) values for the soils of theCynodonsampling sites(mean"S.E.;ns3); for soil type see Table 2

Site Soil S As Pb PLItype (%) (mg kg )y1 (mg kg )y1

1 C 0.01"0.001 8.59"0.62 15.1"0.6 0.692 C 0.03"0.004 4.40"0.66 14.6"3.3 0.673 R 2.35"0.14 417"90 634"171 11.934 R 0.35"0.14 79.3"22.4 159"42 4.865 R 0.99"0.40 175"68.7 357"137 7.656 S 7.07 929 2300 22.21Concentration in – – 6 35 1

‘normal’ soils (median)a

According to Bowen(1979).a

Table 4Available (EDTA) concentrations of trace elements in soils ofCynodon sites(mean values"S.E.,ns3); for soil type see Table 2

Site Soil As Cd Cu Fe Mn Ni Pb Zntype (mg kg )y1 (mg kg )y1 (mg kg )y1 (mg kg )y1 (mg kg )y1 (mg kg )y1 (mg kg )y1 (mg kg )y1

1 C 0.28"0.05 0.01"0.00 1.40"0.21 42.4"6.7 57.7"4.0 0.27"0.04 3.24"0.22 2.21"0.212 C 0.41"0.05 0.03"0.003 3.76"0.14 35.7"2.4 34.9"1.4 0.41"0.05 3.20"0.23 2.03"0.313 R 4.73"1.28 1.50"0.38 51.4"11.3 56.8"7.8 52.5"4.6 0.50"0.02 66.9"8.0 167"27.24 R 2.15"0.28 0.96"0.09 38.3"7.3 70.7"5.8 61.2"4.6 0.32"0.03 28.6"3.6 74.9"7.15 R 1.65"0.48 1.50"0.65 56.6"14.2 64.7"16.2 71.9"5.2 0.52"0.04 50.2"6.9 112"39.36 S 2.05 3.4 154 1850 174 2.05 14.4 286

from those in the remediated soils(Table 4), anddespite being much higher the ‘total’ concentration(929 mg kg of As under sludge vs. 224 mgy1

kg in the average remediated soil, and 2300 mgy1

kg of Pb vs. 383 mg kg in remediated soils).y1 y1

Available (EDTA-extracted) concentration of Fe,Mn and Ni also showed two pollution levels, butin this case values were similar in the control andin the remediated soils(Table 4), and all werelower than in the sludge-covered soil. Thus theavailability (after EDTA extraction) of a particularelement in the soil not always corresponds withits ‘total’ (after extraction with aqua regia) con-centration. In some cases(e.g. for Cu and Pb), theavailable concentration can be overestimated usingthe extraction with EDTA, due to the complexequilibrium involved(Vidal et al., 1999).

The rapid removal of the sludge was importantto reduce the soil pollution level, moreover, takinginto account the dynamics of the trace elements inthe sludge–soil interphase. Thus the soil pollutionin site 6(covered with sludge) increased in avail-

able As from 2 mg kg in autumn 1999 to 33y1

mg kg in spring 2000(Madejon, unpublished).y1 Ín general, soils of the Guadiamar floodplain

affected by the spill still remain polluted by traceelements and heavy metals(Moreno et al., 2001;Ayora et al., 2001), and the sites here studiedreflect this general situation. The clean-up andremediation operations, despite being absolutelynecessary, only partially alleviated pollution by thetrace elements. In many cases, cleaning machineryoperations buried part of the sludge(previouslyconfined to the topsoil) and increased the concen-tration of metals(Moreno et al., 2001). The resultis a patchy and irregular distribution of the metalpollution along the Guadiamar and Agrio valleys.

3.2. Effects on grass macronutrients

Soil pollution by the mine spill induced signif-icant changes in the concentration of macronutri-ents(N, P, K, Ca and Mg) in Cynodon, and to alesser extent(not statistically significant) in Sor-


Fig. 2. Nutrient concentration(mean values on dry matter and S.E. bars) in Cynodon dactylon (in grey) and Sorghum halepense(in white) growing in the three soil types(control,ns6; sludge,ns3; and remediated,ns9). For each species, bars with the sameletters do not differ significantly(P-0.05).

ghum (Fig. 2). Cynodon plants growing in the soilcovered with sludge(site 6) had lower concentra-tion values of N, P and K than in the control soils,whereas concentration values of S, Ca and Mgwere higher.Cynodon plants growing in remedi-ated soils had no significant differences fromcontrol in concentration of macronutrients.Sor-ghum plants growing in the soil covered withsludge had no significant differences in concentra-tion of macronutrients from plants in control orremediated soils(despite being the means of Pand K lower, the variability is very high; Fig. 2),with the exception of S which was significantlyhigher.

The high pollution by heavy metals and traceelements in soils covered with sludge(site 6)seems to negatively affect the uptake of N, P and

K by Cynodon. Not only the uptake, but also themetabolic activity of these mineral nutrients maybe inhibited by high pollution of trace elements insoil (Kabata-Pendias and Pendias, 1992). Howev-er, in the remediated soils(sites 3–5), the nutri-tional equilibrium ofCynodon was much recoveredand did not differ significantly from plants growingin non-contaminated soils(Fig. 2).

Sorghum plants were less affected by the severesoil pollution in terms of concentration of N, Pand K. These plants probably have some mecha-nisms to regulate the internal concentration ofmineral nutrients in their tissues in spite of thehigh concentration of heavy metals and traceelements in the soil solution.

The concentration of S, Ca and Mg inCynodontissues had an opposite response to the high pol-


lution in the sludge-covered soil(Fig. 2), i.e. therewas an increasing trend; although this effect wasnot significant in the remediated soils. Thisincrease of Ca levels in plant tissues can be relatedto the protective action of Ca against the toxicityof metals and metalloids(Mengel and Kirkby,1987; Carbonell et al., 1998).

Murillo et al. (1999) documented a higherconcentration of N and K(but no significant effectfor Ca, Mg and P) in leaves of cultivated sorghum(Sorghum bicolor) recently covered with sludge(samples were taken 2 months after the mine spill)than in adjacent control soils: in that case, theaffected plants responded to a potential ‘fertilising’effect of the water and the sludge flood.

The slurry was composed of approximately2 000 000 m of tailings(solid phase) and 3–43

million cubic metres of polluted, acid waters,which penetrated the soil deeper than tailings(Ayora et al., 2001). Tailings tended to diminishafter 10 cm in depth(Simon et al., 1999). Acid´waters could then dip the bulk root system of thesorghum crop, sown before the accident to feedthe cattle(Murillo et al., 1999). Moistening andacidification caused by these acid waters in therhizosphere of the cultivatedSorghum could haveenhanced the nutrient uptake mechanism to acertain extent.

The situation considered here is completelydifferent, which could explain the nutritional dif-ferences found for theSorghum plants in bothscenarios. In the first case, a short-time response(2 months after the spill) was studied for cultivatedSorghum already grown in the affected area(Murillo et al., 1999). While in the second case(this study), a middle-time response(18 monthafter the spill) has been studied for wildSorghumcolonising (or resprouting in) the affected area.Early growth of these new coloniser plants(suchas theSorghum studied here) had to occur underan important residual pollution by heavy metals inthe soil. This polluted environment should affectthe nutrient uptake and growth of the youngrootlets. Also, possible differences in the nutrientuptake mechanisms between the wildSorghum andthe cultivated, more vigorous, hybridSorghumshould be considered.

Sulfur is a very abundant element in the ‘pyritic’sludge and has significantly high values in bothCynodon and Sorghum grasses growing in thesludge-covered soil(Fig. 2). A high concentrationof sulfur may produce an important imbalance ofmajor nutrients(Ernst, 1988). This comparativelyhigh concentration of S(especially inCynodon)indicates a noticeable pollution level of the soil–plant system, derived from the ‘pyritic’ sludge.

The results above show that these wild grasseshad no problem for major nutrient attainment inthe remediated soils of the affected area, where upto 20 t ha of organic amendments have beeny1

applied. Managers of the ‘green corridor’ shouldconsider examples of fertilisation with major nutri-ents(N, P, K and Ca) enhancing plant developmentin the presence of trace element pollution(Smithand Bradshaw, 1979; Nagy and Proctor, 1997).Such interactions help to understand plantresponses to stress factors under natural conditions(Hagemeyer, 1999).

3.3. Effects on grass micronutrients

Cu, Fe, Mn and Zn have important physiologicalroles as micronutrients in plants. However, theirexcessive concentration in plant tissues may causetoxic symptoms. For example, concentrations ofCu greater than 40 mg kg of dry matter mayy1

induce toxicity in plants, and may also cause toxiceffects in animals(i.e. sheep) feeding on them(Annenkov, 1982a).

In general, the concentration of Cu, Fe, Mn andZn in both grasses was higher in the highlypolluted, sludge-covered soils(Table 5), althoughnot significantly for Cu and Mn inSorghum.Below we discuss the response for each elementseparately.

3.3.1. CopperThe concentration of Cu in tissues ofCynodon

growing in the sludge-covered soil averaged 28mg kg , exceeding the maximum level toleratedy1

by sheep(25 mg kg , Chaney, 1989), whereasy1

Sorghum had a lower Cu concentration, not signif-icantly different from the control(Table 5). How-ever, the Cu levels in unwashedCynodon andSorghum plants (as they would be ingested by


Table 5Micronutrient concentrations(mg kg of dry matter) in decontaminated above ground parts ofCynodon and Sorghum plantsy1

(values for unwashed samples in parentheses); for soil type see Table 2

Species Soil Cu Fe Mn Zn

Cynodon C 11.4 b(13.1) 402 b(982) 55.8 c(79.5) 53.6 b(45.8)S 28.4 a(48.4) 4180 a(8323) 210 a(186) 179 a(247)R 13.5 b(24.3) 1124 b(2744) 103 b(117) 130 a(158)

Sorghum C 8.28 a(10.2) 149 b(202) 37.9 a(50.6) 48.4 b(146)S 13.7 a(50.6) 2446 a(6390) 56.7 a(99.3) 167 a(284)R 12.2 a(16.3) 596 b(718) 44.0 a(47.2) 162 a(193)

Values followed by the same letter in the same column, for each species, do not differ significantly(P-0.05).

herbivores) was approximately 50 mg kg in they1

polluted, sludge-covered soil, clearly above toxiclevel (see Tables 5 and 7 and Fig. 3). These grasspollution figures support the adequacy of theurgent cleaning up operations of spill affected soilscarried out in 1998 immediately after the minedisaster.

An excessive intake of Cu(e.g. above 25 mgkg in the case of sheep; Chaney, 1989) cany1

induce a sharp decrease of haemoglobin contentand increase of methemoglobin; in general, intra-vascular hemolysis occurs with subsequent anae-mia (Gupta and Gupta, 1998). However, a parallelhigh S intake can counteract Cu effect by itsconversion to sulfide(CuS) in the rumen(Annen-kov, 1982a).

The remediated soils remain polluted by Cu(seeabove), however, Cu concentration in grasses(both Cynodon and Sorghum) growing in thesesoils is not significantly different from control(Table 5). These Cu values are within the normalrange for plants(3–20 mg kg , see Tables 5 andy1

7).There is no significant correlation between Cu

concentration in soil(values for EDTA extraction)and in grasses(rs0.22 forCynodon, ns15, usingdata from control and remediated soils). Below acertain Cu pollution level in the soil,Cynodon andSorghum grasses seem able to regulate the Cuconcentration in their tissues(Table 5).

3.3.2. IronSoil pollution in sludge-covered soils induced a

high concentration of Fe in grasses; averages of4180 mg kg inCynodon and 2446 mg kg iny1 y1

Sorghum, well above the toxic level for livestock(1000 mg kg , see Tables 5 and 7). In remediatedy1

soils, Fe level in grasses decreased, in particularfor Sorghum (Table 5). There was a high variabil-ity among samples and although statistically Felevels inCynodon were not different from control,some samples reached toxic levels(up to maxi-mum levels of 2930 mg kg ).y1

Characteristics signs of chronic Fe toxicosis formost animals are reduced growth rate, and reducedfeed intake and efficiency(Gupta and Gupta,1998). A concentration greater than 2400 mgkg may be toxic for cattle, although it shouldy1

be borne in mind that undesirable effects canappear even at lower subtoxic doses(Annenkov,1982b).

There is a significant positive correlationbetween Fe concentration in soils(EDTA-values)and Fe in grasses(rs0.64; P-0.01; ns15 forCynodon, excluding site 6). The increasing soil Fe(at least the available fraction) seems to enhanceto a certain extent Fe uptake by this plant.

The maximum levels found inCynodon andSorghum exceed the critical toxicity concentrationfor total Fe reported by Romheld and Marschner¨(1991); 400–1000 mg kg in plant tissues. Iny1

soil conditions of high Fe solubility, plants mayuptake and accumulate a very large amount of Fe;e.g. grasses from serpentine soils may contain upto 3580 mg kg (Kabata-Pendias and Pendias,y1

1992). On the other hand, the precise definition ofcritical toxicity concentration for Fe is difficult, asthe proportion of Fe(III ) which precipitate pref-erentially in the apoplast, vs. the highly toxicFe , which freely circulates in the cytoplasm and2q


Fig. 3. As, Cd, Cu and Pb concentrations(mean values on dry matter) in plant tissue ofCynodon dactylon in the three soil types(control, ns6; sludge,ns3; and remediated,ns9). The interval between maximum and minimum values is shown; dotted linedepicts maximum level tolerated by livestock according to Chaney(1989).

cell organelles, is not known.(Romheld and Mar-¨schner, 1991).

3.3.3. ManganeseSoil pollution in the sludge-covered soils

induced a higher Mn concentration inCynodongrass (but not in Sorghum) although remainingbelow the toxic levels for livestock(Tables 5 and7).

In remediated soils,Cynodon plants had higherMn concentration than in control soils, and therewas a significant correlation between Mn levels insoil (EDTA-values) and in grasses(rs0.76; P-0.01; andns15).

Manganese was not one of the polluting heavymetal in the mine spill; in fact its concentration inthe sludge(393–954 mg kg ) was in the samey1

range as that of the unaffected soils of the area(398–939 mg kg ) (Cabrera et al., 1999). More-y1

over, Mn availability(EDTA extracted) in reme-diated soils were not significantly different fromcontrol soils(Table 4). However, it is remarkablethe sensitivity of grasses to such small changes insoil levels of Mn, showing a significant correlationand significant differences from control.

This pattern could be related to the passiveabsorption of Mn. Despite there being ample evi-dence that Mn uptake is metabolically controlled,


Table 6Trace element concentrations(mg kg of dry matter) in decontaminated above ground parts ofCynodon and Sorghum plantsy1

(values for unwashed samples in parentheses)

Species Soil As Cd Ni Pb Tl

Cynodon C 0.78 c(0.68) 0.03 b(0.24) 4.67 a(9.95) 1.55 c(0.49) 0.01 cS 75.0 a(168) 0.72 a(2.18) 7.42 a(9.79) 148 a(270) 0.73 aR 7.55 b(20.3) 0.62 a(0.63) 4.49 a(11.06) 15.6 b(38.4) 0.14 b

Sorghum C 0.23 b(0.20) 0.02 a(0.03) 1.25 b(0.96) 0.62 b(0.60) 0.01 bS 25.1 a(86.6) 0.76 a(1.50) 2.49 a(3.74) 47.4 a(196) 0.29 aR 2.60 b(3.84) 0.55 a(0.55) 2.09 ab(1.50) 4.95 b(8.10) 0.05 b

Values followed by the same letter in the same column, for each species, do not differ significantly(P-0.05).

passive absorption of Mn is also likely to occur,especially in the high and toxic range of this metalin solution (Kabata-Pendias and Pendias, 1992).Most pasture grasses are tolerant to have very highMn concentration in their leaves(e.g.)1500 mgkg ; see Foy et al., 1988).y1

3.3.4. ZincSoil pollution in sludge-covered soils induced a

significant increase of Zn concentration in tissuesof both grasses; averages of 179 mg kg iny1

Cynodon and 167 mg kg inSorghum, but iny1

both cases below toxic levels for herbivores(seeTables 5 and 7). However, the maximum levelrecorded for the unwashed material(389 mgkg in Sorghum) was greater than the thresholdy1

level tolerated by sheep.In remediated soils significant pollution of Zn

remained(see Table 4) which was reflected inhigher Zn levels in grasses, bothCynodon andSorghum, than in control soils(Table 5). Therewas also a significant positive correlation betweenZn levels in soil(EDTA-values) and in Cynodonplants (rs0.845; P-0.01; andns15). In thesesoils, the maximum level recorded for theunwashed material(281 mg kg inCynodon) arey1

very close to the sheep tolerance(Table 7).Although acute Zn toxicity in animals is uncom-mon, excessive intake can derive in more or lesssubtle negative effects(reaction with red cells andhepatocytes, lowered activities of some enzymesand of levels of HDL cholesterol, Gupta andGupta, 1998).

3.4. Trace elements in grasses

The high pollution of heavy metals and traceelements in soils covered with sludge induced ahigh concentration of As, Cd, Ni Pb and Tl ingrasses(Table 6); all values(except Ni forCyno-don) were significantly higher than control. Inremediated soils,Cynodon plants still accumulatedhigher level of As, Cd, Pb and Tl than in controlsoils (Table 6); whereasSorghum plants did notshow significant differences in trace elements fromplants in control soils.

In general, As and Pb were the elements show-ing greatest pollution in grasses; in relation to non-contaminated soils, the increments for As were96= in Cynodon and 109= in Sorghum, and forPb were 95= in Cynodon and 76= in Sorghum.In remediated soils, grasses also showed higher(8–11 times) As and Pb concentration than incontrol soils. The pollution levels of As and Pb ingrasses reflect the marked differences between thethree soil pollution levels: control, remediated andsludge-covered(as described above). The accu-mulation of Tl in Cynodon plants also showed thethree pollution levels, being 73= (in sludge-covered) and 14= (in remediated) higher than incontrol soils.

The pollution of Cd in grasses was similar inboth remediated and sludge-covered soils, and theincrement with relation to control was less acute(24= in Cynodon and 38= in Sorghum). Soilpollution of Ni was relatively low, given its scar-city in the mine sludge(10–20 mg kg , Cabreray1

et al., 1999); only Sorghum plants in sludge-


Table 7Normal ranges in plants, phytotoxic concentrations and toxic levels for livestock of several trace elements(from Chaney, 1989 andother authors, see table footnotes); levels in parentheses were estimated(by NRC) by extrapolating between animal species

Element Normal levels(mg kg dry foliage)y1

Phytotoxic levels(mg kg dry foliage)y1

Maximum levels tolerated by livestock(mg kg dry diet)y1

Cattle Sheep Swine Chicken

Asinorg. 0.01–1 3–10 50 50 50 50Cd 0.1–1 5–700 0.5 0.5 0.5 0.5Cu 3–20 25–40 100 25 250 300

10–70a 300–500b

Fe2q 30–300 – 1000 500 3000 10005000b

Mn 15–150 400–2000 1000 1000 400 20001000b

Ni 0.1–5 50–100 50 (50) (100) (300)Pb 2–5 – 30 30 30 30Zn 15–150 500–1500 500 300 1000 1000

1000 2000b

Toxic levels for crops according to Gupta and Gupta(1998).a

Toxic levels according to Annenkov(1982c).b

covered soils showed significant increment(2=)in relation to control. In this section we discussseparately the response for each element(exceptfor Ni) and possible toxicity effects.

3.4.1. ArsenicThe high concentration of As in the sludge, up

to 4000 mg kg (Cabrera et al., 1999), causedy1

some social alarm due to its known toxicity forhuman and livestock; high doses of this elementcan cause dehydration, weakness and lethargy(Gupta and Gupta, 1998). Actually, we documenthere thatCynodon plants growing in the sludge-covered soil accumulated an average of 75 mgkg ; this value exceeds the toxic level(50 mgy1

kg ) recommended for livestock forage(this wasy1

not the case ofSorghum accumulating 25 mgkg ). Moreover, the unwashed grass samples ofy1

both Cynodon and Sorghum, as they would beingested directly by herbivores, reached As con-centrations(168 and 87 mg kg , respectively)y1

above toxic level(see Tables 6 and 7). These twowild grass species seem relatively tolerant to Aspollution, growing in sludge-covered soils withapproximately 900 mg kg of total As concentra-y1

tion and 2 mg kg of available As(EDTA-y1

extracted); the accumulation of As in these grasstissues is higher than the values considered as

phytotoxic (3–10 mg kg , Table 7), probablyy1

recommended for crop plants. Smith et al.(1998)documented thatCynodon plants can reach con-centrations up to 3000 mg kg in heavily contam-y1

inated soils.In remediated soils, As concentration in the

aboveground part ofCynodon (8 mg kg ) andy1

Sorghum (3 mg kg ) were far below the maxi-y1

mum level tolerated by livestock. This result sup-ports the adequacy of the urgent cleaning upoperations carried out in the area affected by themine spill. However, the monitoring of As levelsin soils and grasses should be continued. Althoughnot reaching the critical level of 50 mg kgy1

recommended for livestock, some grasses withhigh As concentration(e.g. unwashedCynodonplants reached up to 40 mg kg) can be harmfuly1

for more sensitive wild animals.There is a significant positive correlation

between As concentration in soils(EDTA-extract-ed) and in Cynodon grass(rs0.84; P-0.01; andns15). That is, the increasing As availability insoil induces a higher uptake and accumulation bygrasses(at least, byCynodon).

It is predictable that the risk of As toxicity inwild animals living and colonising the spill-affect-ed area will decrease. We think that the externalsoil contamination on grass surface(main As


source) will decrease progressively with increasingsoil stabilisation and vegetation cover.

3.4.2. LeadLead was the metal(after Fe and Zn) with the

highest concentration in the sludge(up to 9700mg kg , Cabrera et al., 1999), and has beeny1

emphasised as one of the main sources of toxicityby the mine spill(Prat et al., 1999). Lead poison-ing is the most frequently diagnosed toxicologicalcondition in veterinary medicine. Clinical sighs ofPb toxicity entail loss of appetite, weight loss,depression, muscular weakness, stiffness of joints,diarrhoea and often anaemia(Gupta and Gupta,1998).

We document here(Tables 6 and 7 and Fig. 3)that Cynodon and Sorghum grasses growing insludge-covered soils reached Pb concentration(148 and 47 mg kg , respectively) well abovey1

the toxic level (30 mg kg ) recommended fory1

livestock. Moreover, the unwashed samples(270and 196 mg kg , respectively) greatly exceededy1

that toxic level.Soil remediation was not very effective reducing

Pb availability in soils (Table 4), but greatlyreduced Pb concentration in grasses(see Fig. 3).However, someCynodon samples in the remedi-ated soils still have Pb concentration in theirtissues(up to 56 mg kg ) above toxic levels;y1

more worrying, the majority of unwashedCynodonsamples were contaminated(average of 38 mgkg ) exceeding toxic levels. The urgent cleaningy1

up operations of soil was much needed, but stillthere is remarkable Pb pollution in grasses and acontinuous monitoring should be kept.

There is a positive significant correlationbetween Pb levels in soil(EDTA-values) and inCynodon plants(rs0.63; P-0.05; andns15).

The risk of Pb toxicity for animals is expectedto decrease(as discussed for As). Partly becauseexternal concentration of Pb in grass surface willbe progressively lower due to soil stabilisation andplant growth and renewal(to be corroborated byfurther studies). Additionally, most Pb uptaken byplants remains in the root system(Adriano, 1986)and the soil–plant barrier(according to Chaney,1989), may act protecting the food chain againstPb toxicity. In any case, given the present results

on Pb (and As) concentration in grasses near orabove toxic levels, the restraint for grazing thearea affected by the mine spill is completelyjustified.

3.4.3. CadmiumPlants can tolerate a relatively high content of

Cd in soils, but if they accumulate large quantitiesin their tissues(up to 700 mg kg ; Chaney,y1

1989), may cause harmful effects on humans andanimals feeding on them. An excessive intake ofCd can cause hypertension, cancer, immune dis-orders, severe gastric, cramps, vomiting, diarrhoea,cough, headache, brown urine, and renal failure(Gupta and Gupta, 1998). But see Beyer(2000)who argues that Cd toxicity levels for wildlife hasbeen exaggerated.

Soil pollution induced a significantly higher Cdconcentration in grasses(Table 6). Cynodon andSorghum plants growing in the sludge-coveredsoils had approximately 0.7 mg kg of Cd,y1

exceeding the toxic level(0.5 mg kg ) recom-y1

mended for livestock(Chaney, 1989). This toxicityof Cd was even more surpassed by the unwashedsamples(2.2 mg kg for Cynodon and 1.5 mgy1

kg for Sorghum). On the other hand, the rathery1

high Ca and Zn concentration in these grasses(Fig. 2 and Table 5) can be beneficial for ahypothetical animal consumer, as both elementscompete with Cd in the intestinal absorption proc-ess(Chaney, 1989).

In remediated soils, the Cd availability(seeEDTA values in Table 4) was reduced in compar-ison with the sludge-covered soils, however, Cdconcentration in grasses was little affected(Fig. 3and Table 6). In fact, average values for Cdconcentration in grasses, bothCynodon and Sor-ghum, were still near or above toxic level(Table6 and Fig. 3). These results for Cd also supportthe legal measures taken to forbid animal grazingin the area affected by the mine spill.

There is a highly significant positive correlationbetween Cd levels in soil(EDTA values) and inCynodon plants (rs0.95 andP-0.01; excludingthe sludge-covered site). It is expected that adecreasing soil pollution by Cd will be reflectedin lower concentration of Cd in grasses. Addition-ally, the concentration of Ca and Zn in grasses,


reasonably high in these soils(Fig. 2), will be apositive feature for animal nutrition. A continuousmonitoring of trace elements and mineral nutrientsin grasses(and other palatable plants) in theaffected area is needed.

3.4.4. ThalliumDespite a noticeable Tl content in the sludge

(up to 60 mg kg , according to Cabrera et al.,y1

1999), the concentration in grasses was relativelylow, partly explained by the very low mobility ofthis trace element under present conditions(Vidalet al., 1999), and in contrast with general data inliterature(Kabata-Pendias and Pendias, 1992).

Soil pollution induces a higher accumulation ofTl in grasses(Table 6). In sludge-covered soils,the concentration of Tl in plant tissues averaged0.7 mg kg for Cynodon and 0.3 mg kg fory1 y1

Sorghum. These values are much higher than thosein control soils but far lower than the safe valuefor the trophic web(2.5 mg kg of dry matter,y1

according to Makridis and Amberger(1996)), andthan the excessive or toxic level of 20 mg kgy1

reported by Kabata-Pendias and Pendias(1992)for mature leaf of different plants. In remediatedsoils, Cynodon plants had lower Tl concentrationthan in the sludge-covered soil, but higher than incontrol (Table 6).

Despite the relatively low values of Tl found insoil and grasses, there is a possible increase of Tlavailability in these remediated soils and the con-sequent Tl uptake by plants. Tl is easily absorbedby the roots; and because the geochemical behav-iour of Tl is analogous to that of the essentialelement K, its uptake and distribution in plantsmaybe expected to be similar to that of K. Thisrisk makes the continuous monitoring of Tl levelsadvisable in the area affected by the mine spill, toavoid possible toxic effects. Negative effects of Tlin humans include abdominal pain, hair loss, tach-ycardia and cardiac arrhythmia. Tl is also a neu-rotoxin that causes tremor, ataxia, ptosis of theeyelids and painful lower extremities. Death mayresult from cardiac failure or respiratory failure(Leung and Ooi, 2000).

4. Conclusions

After the Aznalcollar mine spill(in April 1998),án emergency soil clean up procedure was imme-diately carried out. Mechanical removal of thesludge (extremely contaminated by several traceelements) and of the soil layer underneath(10–20-cm-thick) reduced the soil pollution in theaffected area. However, the concentration of traceelements and heavy metals measured in soil sam-ples and wild grasses still indicates a fairly highpollution level.

Plants ofCynodon dactylon and Sorghum hale-pense growing in sludge-covered soils(left asreference of maximal pollution) have high concen-tration of As, Cd, Cu, Fe and Pb, above toxiclevels. This result supports the urgency of soilcleaning up operations to remove the sludge assoon as possible.

The pollution of soil and plants is less severe inthe remediated soils, although it is still worrying.Wild grasses growing in these remediated soilsaccumulated toxic levels of Cd(both species) andPb (only Cynodon) in their tissues. Moreover,unwashed samples of grasses(as they are ingestedby animals) had toxic levels of Cd, Cu and Pb.These results support the restraint of animal graz-ing in the spill-affected area, and a continuousmonitoring of the pollution levels in grasses andother palatable plants to evaluate possible conse-quences of trace elements entering the trophicweb.

Revegetation of the riverbanks and floodplainwould complete the metal immobilisation task.Native grasses, which have successfully colonisedspill-affected areas, such asCynodon dactylon andSorghum halepense, can be used for this purpose.

Acknowledgments

This study was carried out under the frameworkof the ‘Programa de investigacion del corredor´verde(picover)’, supported by funds of the ‘Con-sejerıa de Medio Ambiente de la Junta de Anda-´lucıa’, Seville, Spain. We thank M.H. Hurtado and´J.M. Alegre for their help in sampling and Dr C.Romero for plant determination. Trace elementconcentrations in plants were determined in the


Centre for Scientific Instrumentation(Universityof Granada).

References

Adriano DC. Trace elements in the terrestrial environment.New York: Springer-Verlag, 1986. (533 pp).

Annenkov BN. Mineral feeding of sheep. In: Georgievskii VI,Annenkov BN, Samokhin VI, editors. Mineral nutrition ofanimals London: Butterworths, 1982:321–354.

Annenkov BN. Mineral feeding of cattle. In: Georgievskii VI,Annenkov BN, Samokhin VI, editors. Mineral nutrition ofanimals London: Butterworths, 1982:285–320.

Annenkov BN. Mineral feeding of pigs. In: Georgievskii VI,Annenkov BN, Samokhin VI, editors. Mineral nutrition ofanimals London: Butterworths, 1982:355–389.

Ayora C, Barettino D, Carrera J, Manzano M, Mediavilla C.Las aguas y los suelos tras el accidente de Aznalcollar.´Boletın Geologico y Minero 2001;112(special number):1–´ ´294.

Bargagli R. Trace elements in terrestrial plants: An ecophy-siological approach to biomonitoring and biorecovery. Ber-lin: Springer-Verlag, 1998. (324 pp).

Beyer WN. Hazards to wildlife from soil-borne cadmiumreconsidered. J Environ Qual 2000;29:1380–1384.

Bowen HJM. Environmental chemistry of the elements. Lon-don: Academic Press, 1979. (333 pp).

Cabrera F, Clemente L, Dıaz Barrientos E, Lopez R, Murillo´ ´JM. Heavy metal pollution of soils affected by the Guadia-mar toxic flood. Sci Total Environ 1999;242:117–129.

Carbonell AA, Aarabi MA, DeLaune RP, Gambrell RP, PatrickWH. Arsenic in wetland vegetation: availability, phytotox-icity, uptake and effects on plant growth and nutrition. SciTotal Environ 1998;217:189–199.

Chaney RL. Toxic element accumulation in soils and crops:protecting soil fertility and agricultural food-chains. In: Bar-Yosef B, Barrow NJ, Goldshmid J, editors. Inorganic con-taminants in the vadose zone Berlin: Springer-Verlag,1989:140–158.

Cherney JH, Robinson DL, Kappel LC, Hembry FG, IngrahamRH. Soil contamination and elemental concentration offorages in relation to grass tetany. Agron J 1983;75:447–451.

Demolon A, Leroux D. Guide pour l’etude experimental des´sols. Paris: Gautier Villars, 1952. (251 pp).

Ernst WHO. Response of plants and vegetation to mine tailingand dredged materials. In: Salomons W, Forstner U, editors.¨Chemistry and biology of solid waste. Dredged material andmine tailings Berlin: Springer-Verlag, 1988:55–69.

Foy CD, Scott BJ, Fisher JA. Genetic differences in planttolerance to manganese toxicity. In: Graham RD, HannamRJ, Uren NC, editors. Manganese in soils and plantsDordrecht: Kluwer Academic Publishers, 1988:293–307.

Gee GW, Bauder JW. Particle-size analysis. In: Klute A, editor.Methods of soil analysis. Part 1. Physical and mineralogicalmethods. Agronomy no. 9, 2nd editionASA, 1986:383–411.

Griepink B, Muntau H. The certification of the contents(massfractions) of As, B, Cd, Cu, Hg, Mn, Mo, Ni, Pb, Sb, Seand Zn in rye grass. CRM 281, report no. EUR 11839 EN.Luxembourg, 1987.

Griepink B, Muntau H. The certification of the contents(massfractions) of arsenic, cadmium, copper, lead, selenium andzinc in a sea lettuce(Ulva lactuca). CRM no. 279, reportno. 11185 EN. Luxembourg, 1988.

Grimalt JO, Ferrer M, Macpherson E. The mine tailing accidentin Aznalcollar. Sci Total Environ 1999;217:3–11.

Gupta UC, Gupta SC. Trace element toxicity relationships tocrop production and livestock and human health: implica-tions for management. Commun Soil Sci Plant Anal1998;29:1491–1522.

Hagemeyer J. Ecophysiology of plant growth under heavymetal stress. In: Prasad MNV, Hagemeyer J, editors. Heavymetal stress in plants. From molecules to ecosystems Berlin:Springer-Verlag, 1999:157–181.

IGME. Contribucion al establecimiento del fondo geoquımico´ ´previo a la rotura de la balsa minera en Aznalcollar, en eláluvial del rıo Guadiamar. Madrid: Instituto Tecnologico´ ´Geominero de Espana, Ministerio de Medio Ambiente, 1998.˜(31 pp).

Junta de Andalucıa. La estrategia del Corredor Verde. Conclu-´siones del Seminario Internacional sobre Corredores Ecolo-´gicos. Consejerıa de Medio Ambiente de la Junta deÁndalucıa. Sevilla, 1999:61 pp.´

Kabata-Pendias A, Pendias H. Trace elements in soils andplants. Boca Raton, Florida: CRC Press, 1992. (365 pp).

Leung KM, Ooi VEC. Studies on thallium toxicity, its tissuedistribution and histopathological effects in rats. Chemo-sphere 2000;41:155–159.

Makridis CH, Amberger A. Thallium concentration in soilsand crops and critical values with respect to food chain. In:Rodrıguez-Barrueco C, editor. Fertilizers and environment´Dordrecht: Kluwer, 1996:443–448.

McLaughlin MJ, Hamon RE, McLaren RG, Speir TW, RogersSL. Review. A bioavailability-based rationale for controllingmetal and metalloid contamination of agricultural land inAustralia and New Zealand. Aus J Soil Res 2000;38:1037–1086.

Mengel K, Kirkby EA. Principles of plant nutrition. Bern:International Potash Institute, 1987. (687 pp).

Moreno F, Cabrera F, Fernandez JE, Giron IF. Propiedades´ ´hidraulicas y concentracion de metales pesados en los suelos´ ý en las aguas de drenaje de dos zonas afectadas por elvertido. Boletın Geologico y Minero 2001;112:178–184.´ ´

Murillo JM, Maranon T, Cabrera F, Lopez R. Accumulation˜ ´ óf heavy metals in sunflower and sorghum plants affectedby the Guadiamar spill. Sci Total Environ 1999;242:281–292.

Nagy L, Proctor J. Plant growth and reproduction on a toxicalpine ultramafic soil: adaptation to nutrient limitation. NewPhytol 1997;137:267–274.

Prat N, Toja J, Sola C, Burgos MD, Plans M, Rieradevall M.Éffect of dumping and cleaning activities on the aquatic


ecosystems of the Guadiamar River following a toxic flood.Sci Total Environ 1999;242:231–248.

Romheld V, Marschner H. Function of micronutrients in plants.Ïn: Mortvedt JJ, Cox FR, Shuman LM, Welch RM, editors.Micronutrients in agriculture. SSSA book series no. 4Madison, WI, USA: Soil Science Society of America,1991:297–328.

Simon M, Ortiz I, Garcıa I, Fernandez E, Fernandez J,´ ´ ´ ´Dorronsoro C, Aguilar J. Pollution of soils by the toxic spillof a pyrite mine(Aznalcollar, Spain). Sci Total Environ´1999;242:105–115.

Smith RAH, Bradshaw AD. The use of metal tolerant plantpopulations for the reclamation of metalliferous waste. JAppl Ecol 1979;16:595–612.

Smith E, Naidu R, Alston AM. Arsenic in the soil environment:a review. Adv Agron 1998;64:149–195.

SPSS. SPSS for Windows. 1999.�SPPS Inc.,(1989–1999),Chicago, Illinois, USA.

Tomlinson DL, Wilson JG, Harris CR, Jeffrey DW. Problemsin the assessments of heavy metal levels in estuaries andformation of a pollution index. Helgol Meeresunters1980;33:566–575.

Valdes B, Talavera S, Fernandez-Galiano E. Flora vascular de´ Ándalucıa occidental. Barcelona: Ketres, 1987. (1680 pp).´

Vidal M, Lopez-Sanchez JF, Sastre J, Jimenez G, Dagnac T,´ ´ ´Rubio R, Rauret G. Prediction of the impact of the Aznal-collar toxic spill on the trace element contamination ofágricultural soils. Sci Total Environ 1999;242:131–148.

Documents

Bioaccumulation of As, Cd, Cu, Fe and Pb in wild grasses affected by the Aznalcóllar mine spill (SW Spain