Speciation of heavy metals in recent sediments of three coastal ecosystems in the Gulf of Cadiz, Southwest Iberian Peninsula
2833Environmental Toxicology and Chemistry, Vol. 22, No. 12, pp. 28332839, 2003q 2003 SETACPrinted in the USA0730-7268/03 $12.00 1 .00Environmental ChemistrySPECIATION OF HEAVY METALS IN RECENT SEDIMENTS OF THREE COASTALECOSYSTEMS IN THE GULF OF CA DIZ, SOUTHWEST IBERIAN PENINSULAVERONICA SA ENZ, JULIA N BLASCO,* and ABELARDO GO MEZ-PARRADepartmento de Oceanografa, Instituto de Ciencias Marinas de Andaluca (C.S.I.C.), Campus Universitario de Ro San Pedro,11510 Puerto Real, Cadiz, SpainDepartmento de Qumica-Fsica, Facultad de Ciencias del Mar de Universitario de Cadiz, Campus Universidad de Ro San Pedro s/n,11510 Puerto Real, Cadiz, Spain(Received 23 September 2002; Accepted 10 March 2003)AbstractA five-step sequential extraction technique was used to determine the partitioning of Cr, Mn, Fe, Cu, Zn, Cd, and Pbamong the operative sedimentary phases (exchangeable ions, carbonates, manganese and iron oxides, sulfides and organic matter,and residual minerals) in coastal sediment from three locations in the southwest Iberian Peninsula. Two sites are located close toindustrial areas, the salt marshes of the Odiel River and Bay of Cadiz, and one in a nonindustrial area, the Barbate River saltmarshes. The Odiel River salt marshes also receive the drainage from mining activities in the Huelva region. In the sediments fromthe Bay of Cadiz and Barbate River salt marshes, Cr, Cu, Fe, and Zn were extracted from the residual fraction at percentages higherthan 60%. In the sediments from the Odiel River salt marshes, concentrations of all the metals, except Cu, Zn, and Cd, exceeded60% in the residual fraction as well. In the sediments from the Bay of Cadiz and Barbate River salt marshes, the main bioavailablemetals were Mn and Cd; in those from the Odiel River salt marshes, the main bioavailable metals were Zn and Cd, respectively.The environmental risk was determined by employing the environmental risk factor (ERF), defined as ERF 5 (CSQV 2 Ci/CSQV),where Ci is the heavy metal concentration in the first four fractions and CSQV is concentration sediment quality value (the highestconcentration with no associated biological effect). Our results showed that the sediments from the Cadiz Bay and Barbate Riversalt marshes do not constitute any environmental risk under the current natural conditions. In contrast, in the Odiel River saltmarshes, Cu, Zn, and Pb yielded ERFs of less than zero at several sampling stations and, consequently, pose a potential threat forthe organisms in the area. This is a consequence of the high levels of metals in the area derived from the mining activity (pyrite)and industrial activities and the association of these heavy metals with more labile fractions of the sediments.KeywordsSpeciation Sediment Heavy metals Salt marshesINTRODUCTIONThroughout history, estuaries have been favored sites forhuman settlements and their associated industrial activities andhave therefore received heavy contaminant inputs. Estuarinesediments show a strong tendency to accumulate contaminants,especially heavy metals; thus, analysis of these sediments con-stitutes a rapid means of obtaining time-integrated informationconcerning a range of sedimentological variables . In ad-dition, estuaries often include wetland sites of internationallyrecognized ecological importance as well as economic im-portance for their shellfish production. Heavy metals are eco-logically relevant, mainly because of their toxicity and theirnonbiodegradable character .Sediments act as sinks for trace metals; thus, they becomea more or less permanent part of the ecosystem. Remobilizationprocesses reintroduce them into the ecosystem in bioavailableforms . Metals can be released into the water phase whenconditions such as pH, redox potential, ionic strength, and theconcentration of organic complexing agents are appropriate;therefore, sediments also act as sources of the metals .The biogeochemical and ecotoxicological significance of agiven element is determined not only by its concentration ina natural or terrestrial system but also by its chemical form,which in turn is controlled by the physical, chemical, andbiological characteristics of that system . Knowledge re-garding the chemical speciation of heavy metals in sediments* To whom correspondence may be addressed(email@example.com).is important for assessing the capacity of those sediments toact as sources of the potentially toxic metals. The determi-nation of the total concentrations of metals is not, on its own,sufficient for prediction of such behavior .Currently, the methods used most frequently to study thedistribution of metals in a solid phase are based on partitiontechniques using different chemical extractants in sequence(i.e., the use of selective chemical extraction is widespread). It is generally recognized that the partitioning of tracemetals achieved by such procedures is operational, and theresults are determined by several experimental factors, suchas the nature of the reagents used, time, temperature, mixingrates, the ratio of reagent to sediment, and the physicochemicalcharacteristics of the sample (especially its chemical compo-sition). The chemical composition of a sample could causeheterogeneous chemical reactions, leading to changes in thedistribution of metals during extraction, such as the precipi-tation of newly formed compounds and readsorption of re-leased metals .Speciation is very useful, not only for determining the de-gree of association of the metals to the sediments and the extentto which they might be remobilized into the environment butalso for differentiating metals with a lithogenic origin fromthose with an anthropogenic origin . Metals associatedwith any of the following four fractionsthe adsorptive orexchangeable fraction, the carbonate fraction, the reduciblefraction, and the organic fractionare considered to be moremobile and, therefore, more easily released from the sediment2834 Environ. Toxicol. Chem. 22, 2003 V. Saenz et al.Fig. 1. Map of the Odiel River salt marshes (AG), Cadiz Bay (CB),and Barbate River (BR) salt marshes (Andalucia, southwest IberianPeninsula). Locations of sampling stations are shown.Table 1. Granulometric distribution and organic carbon (OC) and total carbon (TC) in the three sampling ecosystemsaOdiel River salt marshesA B C D E F G CB BR% Clay% Silt% Sand% OC% TC8.5341.5149.961.971.529.1737.6353.201.291.1213.0476.6210.342.692.1718.5769.0112.422.732.0411.3635.4053.241.6188.8.131.5298.070.3184.108.40.2067.571.140.335.1124.8370.061.653.0716.7649.4733.771.712.37a Sampling stations (AG) are located in the Odiel River salt marshes (Huelva, Spain) and Bay of Cadiz (CB) and Barbate River (BR) in CadizBay and Barbate River salt marshes (Cadiz, Spain).and potentially more dangerous. These four fractions covermetals from anthropogenic sources and, therefore, give infor-mation regarding the type and severity of the pollution to whicha particular environment has been exposed. Metals reportingto the inert fraction may be interpreted as having originatedfrom geochemical or natural sources (i.e., of lithogenic origin).In the present study, we investigated the partitioning ofseven metals (Cr, Mn, Fe, Cu, Zn, Cd, and Pb) among differentfractions in superficial sediments from three coastal areas inthe southwest (SW) Iberian Peninsula: The salt marshes of theOdiel River (OR), the Bay of Cadiz (CB), and the BarbateRiver (BR). All three areas are of considerable ecological rel-evance, and the aim of this work is to provide informationconcerning the distributions of heavy metals in the sedimentssampled in these three valuable ecosystems to predict the en-vironmental risks associated with these metals.MATERIALS AND METHODSStudy areaThe salt marshes of the OR, CB, and BR are located onthe SW coast of the Iberian Peninsula (Fig. 1). The OR wasdeclared a biosphere reserve because of its large populationof migratory birds. The CB hosts a wide diversity of aquaticspecies, and many areas have been transformed for the de-velopment of aquaculture activities . (It is also catalogedas a nature park.) The BR is also cataloged as a nature parkand has commercial activities of bivalve cultivation (mainlyshellfish and oysters). Despite their status as administrativelyprotected areas, these zones are exposed to different types andlevels of contamination. The OR marshes are affected by inputsof contaminants from the Huelva industrial area and the ORitself, which flows through a well-known mining area calledthe Iberian Pyrite Belt. The CB includes an international portwith heavy maritime traffic and for decades has supported amajor industrial activity in the marine construction sector; inrecent years, this industrial activity has been diversified by theincorporation of other industries using metals, such as themanufacture of vehicle and aircraft components. The BR is anarea relatively free from domestic and industrial contaminants,and its pollution inputs are derived more from agriculturalactivities. Therefore, it is included in the present study as areference against which the zones subjected to metal pollutioncan be evaluated.Sampling and sample pretreatmentSediments were collected from June through July 1996 fromnine sampling stations: A, B, C, D, E, F, and G in the OR(zone 1), CB, (zone 2), and BR (zone 3). Their locations areshown in Figure 1. A Van Veen grab was used to collectapproximately 1 kg of superficial sediment (05 cm) at eachof the sampling points. Samples were transferred to polyeth-ylene bags and stored under a N2 atmosphere at 248C. Aportion of each sediment sample was air-dried at 708C to aconstant weight and passed through a 500-mm sieve. Thosesubsamples of dried sediment were subjected to a selectivechemical extraction. The sediment characteristics (percentagesof sand, silt, clay, organic carbon, and total carbon) of samplingstations are shown in Table 1. The grain-size distribution inthe fine fraction less than 63 mm was carried out in a CoulterChannelyzer C1000 (Coulter Electronics, Hertfordshire, UK).Organic carbon was determined by the method of Gaudette etSpeciation of heavy metals in salt-marsh sediments Environ. Toxicol. Chem. 22, 2003 2835Table 2. Reagents and conditions for fractioning the sediment simplesFraction Reagents and conditions Extracted sediment componentsF1: Exchangeable metals 1 M MgCl2, 1 h of shaking at room temperature Exchangeable ionsF2: Metals bound to carbonates 1M NaOAc, pH 5 (HOAc), 5 h of shaking at room tempera-tureAdsorbed ions, carbonates, andsome oxyhydroxidesF3: Metals bound to Fe-Mn oxides 0.04 M NH2OHHCl in 25% (v/v) HOAc, 6 h of occasionalagitation at 968CAmorphous and oxyhydroxides ofFe and MnF4: Metals bound to organic matter 0.02 M HNO3, 30% H2O2, pH 2 (HNO3), 5 h of occasionalagitation, extracted with 3.2 M NH4OAc in 20% (v/v)HNO3Sulfides and organic matterF5: Residual metals Microwave acid digestion with aqua regia-HF Lithogenic crystalline materialsTotal digestion Microwave acid digestion with aqua regia-HF All sediment componentsTable 3. Comparison between the heavy metal concentrations in the sediment of the studied stations obtained by adding the concentrations inthe five fractions obtained by the sequential extraction procedure and the metal levels obtained by the sediment total digestion procedure ofLoring and Rantala aOdiel River salt marshesA B C D E F G CB BRCrMnFeCuZnCdPb99.9985.63103.21110.0290.31112.4798.4098.2677.96102.66117.1786.8680.7182.2699.7287.57117.9695.9086.35118.8390.8099.1091.13106.38103.4582.8198.1188.6093.4684.3580.5796.3292.3690.3997.7882.87102.1289.96107.3794.9883.4184.31102.9774.6488.84103.17103.37104.3883.5497.3598.29111.0877.64108.86104.38101.3092.2490.7592.9496.03102.0289.4387.66a Results are expressed as the percentage with respect to total digestion procedure. Sampling stations (AG) are located in the Odiel River saltmarshes (Huelva, Spain) in Cadiz Bay (CB) and Barbate River (BR) salt marshes (Cadiz, Spain).al. , and total carbon was determined with an elementalanalyzer (Carlo Erba 1106, Milano, Italy).Chemical analysisThe extraction procedure used in the present study is amodification of the sequential extraction procedure proposedby Tessier et al. . The modification involves determiningthe inert or residual fraction using the method described byLoring and Rantala . Extraction with a mixture of aquaregia (HNO3:HCl, 1:3 [v/v]) and hydrogen fluoride (HF) pro-vides an estimate of the residual and total metal content in thesample. This procedure is necessary to ensure the completesolution of aluminum. This mixture has also been shown tobe effective in breaking down organic matter, and HF decom-poses silicates [26,27]. Volumes of 0.5 ml of aqua regia and3.0 ml of HF were added to the aliquot of oven-dried sedimentin a 120-ml Teflont bomb, and the mixture was heated in a700-W microwave oven (Panasonic NN 6200, Mississauga,ON, Canada) and further treated with boric acid . A de-scription of the five fractions obtained with this method aswell as the operative conditions used for each extraction aregiven in Table 2. This procedure was carried out in triplicate.The sample:reagent ratios were 1:40 (w/v) in fraction (F)1,F2, and F3, 1:80 (w/v) in F4, and 1:20 (w/v) in F5. Reactiveblank and reference sediment (estuarine sediment 277 CRMcertified by the Bureau Community of Reference [BCR], Brus-sels, Belgium) were used to ensure quality control. Trace met-als were determined by graphite furnace atomic absorptionspectrometry or by flame atomic absorption spectrometry (Per-kin-Elmer 4100 ZL and Perkin-Elmer 3110, respectively, Nor-walk, CT, USA), depending on the trace metal concentrationand the fraction analyzed.All reagents employed were from Merck (Darmstadt, Ger-many) and of analytical grade or Suprapur quality. Standardworking solutions of the different elements analyzed were pre-pared from the corresponding 1,000 mg/L of Merck Titrisolsolutions.Comparisons between sampling stations were performed bycluster analysis using the biomedical statistical package. Thealgorithm used was the normalized euclidean distance betweenthe centered clusters.RESULTS AND DISCUSSIONTable 3 shows the sum of the five fractions of the sequentialextraction procedure and the heavy metal concentration foundby applying the total digestion procedure . When the sed-iment samples undergo the sequential extraction procedure, ahigh recovery for all the heavy metals is obtained. The resultsare particularly satisfactory for Cr and Cu, whereas the re-covery observed for Mn and Pb is not so high.Heavy metal concentrations for the five fractions of thesediment at each of the nine stations studied is presented inFigure 2. The concentration is expressed in mg/g, except forFe, which is expressed in mg/g. The concentrations of theheavy metals (Cu, Zn, Cd, Pb, and Fe) from anthropogenicsources (corresponding to the first four fractions) are higherat OR than at CB or BR.In Figure 3, the partitioning patterns for Cr, Mn, Fe, Cu,Zn, Cd, and Pb corresponding to each sampling site are pre-sented as bar diagrams. The quantity of metal extracted in eachstep of the leaching scheme is given as a percentage of totalmetal content. The behavior of Cr and Fe was similar in allsamples. Both metals were most abundant in the residual frac-tion and in the most highly polluted samples (OR). The thirdfraction was approximately 16.5% for Cr and 12 to 17% for2836 Environ. Toxicol. Chem. 22, 2003 V. Saenz et al.Fig. 2. Speciation of trace metals in the sediments of the Odiel River(AG), Cadiz Bay (CB), and Barbate River (BR) salt marshes (seeFig. 1). Results are expressed as mg/g dry sediment, except for Fe(mg/g dry sediment). Fraction (F) 1 to F5 correspond to fractions ofthe Tessier sequential procedure.Fig. 3. Speciation of trace metals in the sediments of the Odiel River(AG), Cadiz Bay (CB), and Barbate River (BR) salt marshes (seeFig. 1). Results are expressed as a percentage with respect to totaldigestion. Fraction (F) 1 to F5 correspond to fractions of the Tessiersequential procedure.Fe. This behavior is in agreement with the partitioning foundin other coastal ecosystems (Table 4).The partitioning patterns of Mn, Cu, and Zn at CB and BRare similar and differ from those observed at OR. At the firsttwo sites, Mn was extracted mainly in the second fraction,related to metal bound to carbonates, and the third fraction,related to metal bound to Fe and Mn oxyhydroxides. In thesame two areas, substantial amounts of Mn were also extractedin the exchangeable (F1) and residual (F5) fractions. In thesetwo areas, Mn speciation coincides with the results reportedin the Acheloos River estuary  and in CB . The Mnhas an affinity to carbonates in both lacustrine and coastalsediments . At OR, Mn was extracted mainly in the re-sidual fraction, but F1, F2, and F3 (in that order) were alsowell represented. This partitioning pattern is similar to thatfound in the Huelva estuary . The high percentage of Mnpreviously reported, associated with the residual fraction (F5)in the sediments of OR, is in agreement with previous obser-vations  that in sediments contaminated by mine drainagewaters, as is the case with OR, Mn is mainly concentratedwithin the crystalline structure of the primary and secondaryminerals.Regarding Cu, in the samples from CB and BR the residualfraction (F5) was approximately 62.9 to 72.3%. This indicatesthe mineralogical origin of this metal in these zones and itsreduced mobility from the watersediment interface. However,in the samples from OR, copper was extracted mainly in F4(21.487.9%) and was related to the solubilization of oxidiz-able phases from sediments (organic matter and sulfide). Theamount of residual metal (F5) was low in comparison to thetotal metal content (8.244.1%). This indicates the anthro-pogenic source of this metal as a consequence of the industrialand mining activities as well as from moderate urban pollution.In the CB and BR sediments, Zn was mostly concentratedin the residual fraction (64.869%). The distribution of non-residual metal species was between the fractions dissolved byreducing or oxidizing reagents (F3 and F4, respectively). Onthe contrary, in all the samples from OR, the percentages oftotal metal extracted from the residual minerals (F5) werelower and ranged from 12.5 to 52.4%. The distribution ofnonresidual Zn in decreasing order was in F3, F2, and F4. Thedistribution pattern in the OR coincides with that reported byother authors in this area, and the speciation pattern found inCB and BR is also similar to that presented for CB (Table 4).The Cd behaved similarly in all samples. Being a mobileheavy metal, it was present in F1 (2070% in most samples)and was mainly distributed among the first three fractions(.70% of total Cd). At OR, it was principally extracted in F1and, at CB and BR, in F2 and F3. Small amounts of metalremained in F4 and F5.The Pb was mainly present in the residual fraction in allsamples, although the percentage of this metal bound to re-Speciation of heavy metals in salt-marsh sediments Environ. Toxicol. Chem. 22, 2003 2837Table 4. Speciation of the analyzed heavy metals in the sediment of different coastal systemsaMetal Ecosystem F1 F2 F3 F4 F5 ReferencesCr Acheloos River estuary, GreeceCoastal sediment, Baja California, USAHuelva estuary, SpainOdiel River salt marshes, SpainCadiz BayOdiel River salt marshesCadiz Bay salt marshesBarbate River salt marshes20.250000.3001.50.26006.90.70.50.67.15.31506.4220.127.116.11.18.104.22.168.22.214.171.1248.39074.391.278.479.092.689.4Present studyPresent studyPresent studyMn Acheloos River estuary, GreeceHuelva estuary, SpainCadiz BayOdiel River salt marshesCadiz Bay salt marshesBarbate River salt marshes1.19.311.75.311.114.572.1041.112.942.344.611.225126.96.36.1992.413.458.622.86323.712.613.458.622.86323.712.6Present studyPresent studyPresent studyFe Acheloos River estuary, GreeceCoastal sediment, Baja California, USAHuelva estuary, SpainOdiel River salt marshes, SpainOdiel River salt marshesCadiz Bay salt marshesBarbate River salt marshes0.3000,0.1,0.1,0.15.20.40.188.8.131.52.812.26143417.311.6184.108.40.206.9220.127.116.11.878.19284.55177.386.479Present studyPresent studyPresent studyCu Humber estuary, UKMersey estuary, UKVigo estuary, SpainCoastal sediment, Baja California, USAHuelva estuary, SpainOdiel River salt marshesCadiz Bay salt marshesBarbate River salt marshes18.104.22.16822.214.171.124.126.96.36.19907.56.86.98.188.8.131.52.9148.28.853.137.4581660.958.911.320.330.637.4388022.918.272.362.9Present studyPresent studyPresent studyZn Vigo estuary, SpainHuelva estuary, SpainCadiz BayOdiel River salt marshesCadiz Bay salt marshesBarbate River salt marshes015.73.57.9001018.99.5184.108.40.206428.82035.112.615.41112.9712.315.916.13924.36025.26964.8Present studyPresent studyPresent studyCd Humber estuary, UKMersey estuary, UKHuelva estuary, SpainOdiel River salt marshesCadiz Bay salt marshesBarbate River salt marshes31.9220.127.116.115.59.937.729.19.910.538.527.626.122.816.317.714.6304.36.318.104.22.168.707.612.824.922.624.8Present studyPresent studyPresent studyPb Mersey estuary, UKVigo estuary, SpainHuelva estuary, SpainOdiel River salt marshesCadiz Bay salt marshesBarbate River salt marshes5.3000.91.41.116.89107.621.713.8553110.717.32018.99.222.214.171.12413.75377.171.55059.2Present studyPresent studyPresent studya Results are expressed as the percentage of total metal concentration. Fraction (F) 1 to 5 correspond to the fractions of the sequential extractiontechnique of Tessier et al. .sidual minerals (F5) differed widely among stations (5090%).After the residual fraction, F3 and F2 had the greatest abun-dances of Pb (626% for F3 and 222% for F2). In all samples,the exchangeable metal content was very low (,2% of thetotal metal concentration). In the sediments from OR, the per-centages of Pb associated with the residual fraction (F5) arethe same as those found in the sediments of CB and BR exceptfor locations C, D, and E, where the percentages are higher.Despite qualitative similarities, at all the OR sites Pb contentwas high and characteristic of contaminated sediments (Fig.2). Samples from OR showed that Pb is present in the residualfraction . At sites C, D, and E, where the percentages ofthis metal in the residual fraction (F5) are relatively high, thePb origin in the sediment is mainly lithogenic. In anthropo-genic polluted sediments , Pb is principally associated withF3 (ironmanganese oxyhydroxides); therefore, CB and BRand locations A, B, F, and G in OR represent the areas withthe greatest severity of Pb pollution.A cluster analysis was carried out to classify the samplingstations according to their heavy metal content and speciation.The dendogram (Fig. 4) allows three groups to be established.Group 1 corresponds to all the stations of CB and BR as wellas to sampling stations A and B of OR; all of these show thelowest values for heavy metals. Group 2 includes stations Fand G of OR, both of which display high levels of metals asa consequence of a chronic pyritic contamination from themining activities of this area. Group 3 comprises stations C,D, and E of the OR salt marshes, which are moderately con-taminated by mining activities and also affected by the chem-ical industries of Huelva.The degree of environmental risk can be predicted by theuse of the environmental risk factor (ERF), defined as CSQV2838 Environ. Toxicol. Chem. 22, 2003 V. Saenz et al.Fig. 4. Dendogram showing clustering of sampling sites (AG) forOdiel River, Cadiz Bay (CB), and Barbate River (BR) salt marshes(see Fig. 1).2 Ci/CSQV , where Ci is, in our case, the heavy metalconcentration corresponding to the first four fractions (reactivefractions) and CSQV is the concentration sediment qualityvalue, which corresponds to the highest concentration with noassociated biological effect. The SQV data selected in the pre-sent study correspond to site-specific values for the Gulf ofCadiz [36,37]. For Cd, a value of 1.2 mg/kg, obtained fromthe biological effects databasesediments, was selected as theSQV. This value corresponds to effects range low, becausesite-specific values were not available. The definition of F1 toF4 as the bioavailable fractions was made following previouslyreported criteria . Other authors  define F1 to F3 asthe reactive fractions. However, we prefer the first definition,because the metal of the first four fractions can be mobilized.In any case, the ERF values were similar for most cases, re-gardless of whether the first three or four fractions are con-sidered. F4 is included in our case to calculate Ci. All samplingstations of OR showed an ERF of less than zero for Cu andZn, for Pb at stations E to G, and for Cd at stations C, D, E,and F. The stations of CB showed an ERF of greater than zerofor all metals analyzed. The Fe and Mn were not consideredbecause of their very limited toxicity when they are associatedwith sediments. Although the parameter SQV has limitations, the use of site-specific values allows us to avoid someof the drawbacks in their application for ecological risk as-sessment. The different operative fractions obtained by theprocedure of Tessier et al.  or other proposed speciationschemes, such as the BCR  or acid-volatile sulfide ,can also be useful for measuring the bioavailability of con-taminants. In short, the ERF values calculated as the sum ofthe different operative fractions from the speciation procedurecan be employed to predict the potential adverse effects ofcontaminated sediment on the ecosystem.In summary, the highest proportions of metals in the sed-iments of CB and BR salt marshes occurred in the residualfraction, which means that they cannot be solubilized and,therefore, do not pose a threat to their environment. Althoughanthropogenic inputs are higher in CB than in BR salt marshes,the similarity in the heavy metal concentrations is related todifferent granulometric distribution; thus, the siltclay sizepercentage represents approximately 30% in CB and 60% inBR salt marshes. However, the metals found in sediments ofthe OR salt marshes showed not only a considerably highertotal metal content but also lower percentages associated withthe residual fraction because of the mining activities (copperand pyrites) in this area since the times of the Tartesians andRomans and the nearby industrial area of Huelva. In general,this level of metal pollution does represent an environmentalrisk.AcknowledgementThis work has been funded by the Comision In-terministerial de Cienca y Technologia (AMB94-0291) and the Min-istry of Education (Research Fellowship to V. Saenz). The authorsare grateful to I. Fernandez, Mara F. Osta, and P. Vidal.REFERENCES1. Carral E, Puente X, Villares R, Carballeira A. 1995. 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