Bioavailability Metals

7/30/2019 Bioavailability Metals

1/14

www.scilet.com

INTRODUCTION

Sediment contamination in estuaries and its biologicaleffects need to be properly and fully assessed. Recentpapers recommend conducting specific studies to deter-

mine the effects of pH and salinity on bioavailability ofcontaminants bound to estuarine sediments (Chapmanand Wang, 2001). Trace metals are among the mostcommon contaminants bound to estuarine sediments.The bioavailability of these metals to aquatic organismsis dependent on the physical and chemical forms of themetal (Luoma and Bryan, 1983; Vercauteren and Blust,1996).

Many metal species show little or no bioavailability,e.g. metals adsorbed on particulate matter, carbonatecomplexes and metal complexes with strong chelatingagents (EDTA) although still some debate about it(Allen and Hansen, 1996). Some authors have proposed

that the bioavailability of a dissolved metal is relatedto its free ion activity (e.g., Morel, 1983). The first stepin the uptake of metals by an organism comprises thetransport of the cation through the plasmatic mem-brane. For many dissolved metals, entry into mollusksinvolves passive diffusion, perhaps facilitated by carriermolecules, and passage along a gradient maintained byintracellular ligands of increasing binding strength(Langston et al., 1998). This lipidic membrane is abarrier to the simple diffusion of ions due to its hydro-phobic nature, and this transport is usually assumed tobe mediated by transmembrane proteins with complex-

Chemical Speciation and Bioavailability (2003), 15(4) 101

Bioavailability of heavy metals bound to estuarine

sediments as a function of pH and salinity values

I. Ribaa,b, E. Garca-Luquea, J. Blascob and T.A. DelVallsa*aDpto de Qumica Fsica, Facultad de Ciencias del Mar y Ambientales, Universidad de Cdiz, 11510 PuertoReal, Cdiz, Spain

bInstituto de Ciencias Marinas de Andaluca (CSIC), Polgono Ro San Pedro s/n, 11510 Puerto Real, Cdiz,

Spain

ABSTRACT

The role of two key-variables such as pH and salinity in chemical speciation and bioavailability of heavy metalsoriginated by mining activities bound to sediments was evaluated under estuarine conditions. Two sedimentsamples collected in two estuaries in southern Spain (Ra of Huelva and Guadalquivir estuary) together withdilution of toxic mud from the Aznalcllar mining spill (April, 1998) were used to determine their chemicalspeciation and bioavailability at different pH (6.5, 7.5, 8.5) and salinity (10, 20, 35) values using the estuarineclam Ruditapes philippinarum. The chemical speciation was established by means of measurements of themobilization of heavy metals from sediments to waters and determining in it pH, salinity, alkalinity and heavymetal concentration. The geochemical model MINTEQA2 was used to establish the thermodynamic species inthe assays. To assess the bioavailability of the heavy metals the concentration of metallothioneins in the clamtissues and the mortality of this organism was measured at different pH and salinity values. The influence of bothsalinity and pH was detected in the chemical behavior of metals and in their associated biological responsesestablished by metallothioneins and the percentage of mortality. At low values of both variables (pH=6.5 andS=10), the biological effects were highest, and it was related to the free ion Zn2+.

Keywords: sediment quality, Gulf of Cdiz, estuaries, metallothionein, Ruditapes philippinarum.

*To whom correspondence should be addressed:E-mail: [email protected]

CSB/Riba 26/1/04 1:33 pm Page 101


2/14

ing groups that bind the metals (Simkiss and Taylor,1995). Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins which have

been shown to occur in most zoological taxa (Langstonet al., 1998). It is generally considered that these proteinsplay a primary role in the homeostasis of essentialmetals such as Cu and Zn. However, non-essentialmetals, particularly Cd, are also able to induce MTssynthesis and according to their respective affinities forthis ligand (Hg>Ag>Cu>Cd>Zn), to displace essentialmetals from pre-existing metallothioneins (Stillman etal., 1999).

The Free Ion Activity Model (FIAM) assumes, first,the diffusion of the cation from the bulk solution to themembrane surface and that the kinetic of the reactionbetween the ligand and the metal are rapid and non-limiting compared with the transport process through

the membrane; and secondly, that the carrier is in excessand there is thus no saturation (Campbell, 1995). Takinginto account these assumptions and the chemical reac-tions between the metal ion and the ligands present bothin the solution and in the carrier protein, it can be demon-strated that the uptake is related only to the free ionactivity of the metal. Nevertheless, changes in the com-position and conditions of the medium may modify thestructure of the plasmatic membrane and the number ofavailable ligand groups present in the carrier proteins,hence affecting their chemical speciation and theirbioavailability (Van Ginneken et al., 1999).

Because estuaries provide an interface between fresh

and salt waters, they have strong gradients in manyphysical and chemical variables, including salinity, pH,dissolved oxygen, temperature, nutrients and amount andcomposition of particles. Unlike freshwaters, where pHis the controlling factor, in estuaries salinity is the con-trolling factor for the partitioning of contaminantsbetween sediments and overlying or interstitial watersand both are the key-variables to control the bioavail-ability, and the toxicity of heavy metals bound tosediments.

The main objective of this paper is to assess theinfluence of pH and salinity values on the chemicalspeciation and bioavailability of heavy metals (Zn, Pb, Cd,Cu) bound to estuarine sediments using a true estuarine

species such asRuditapes philippinarum simulatingthe typical values recorded for pH and salinity in mostof the estuaries (pH ranged between 6 and 8 and salinityranged between 10 and 35). Besides, this paper is a con-tinuation of previously reported results (Riba et al.,2002a) about differences in toxicity using the samespecie of clams and related to the total concentration ofheavy metals in sediments and waters. In this sense, thispaper addresses the influence of the chemical speciationof metals on the toxicity ofRuditapes philippinarumusing a complementary and sublethal biological responsesuch as metallothionein (MT).

METHODOLOGY

Sampling and experimental design

To establish the mobilization of heavy metals bound tosediments and to determine their chemical speciationin water and their bioavailability to clams we have useddifferent nature sediment samples: different percentagesof toxic mud collected near a pyrite-zinc mine werediluted using a clean sediment located in the Bay ofCdiz (0.3 and 7.9% dry weight of toxic mud), theprocedures of dilution were previously reported by Ribaet al. (2002b); two environmental stations collected inestuaries from the Gulf of Cdiz affected by differentheavy metal contamination, Ra de Huelva (H) with achronic origin of contamination and Guadalquivirestuary (GR) impacted by an acute event of contami-nation (Aznalcllar mining spill, April, 1998) (Riba et

al., 2002c). Figure 1 shows the location of the sitesselected to collect the sediment samples. The sedimentsamples were collected using a 0.025 m2 Van Veen graband transferred to a cooler where it was homogenizeduntil no textural differences were detected.

These sediment samples were used to expose indi-viduals of the clamRuditapes philippinarumas describedbelow.

Two sediment bioassays were developed using theclamRuditapes philippinarum. One based on differentvalues of salinity (10, 20, 35) and the other one basedon the use of different values of pH (6.5, 7.5, 8.5). Theoverlaying water was adjusted at the desired salinityand pH values as described by Riba et al. (in press).The test using the clamRuditapes philippinarum wascarried out in whole sediment. The sediment toxicitytests were developed using 15 L vessels containing 2 Lof sediment overlaid with filtered seawater adjustedpreviously at the different salinities and pH and usinga 1:4 v/v sediment:water relation. The pH, salinity,temperature (20C) and the concentration of dissolvedoxygen (>5 mg L1, 60% saturation) were measuredand controlled every day.

Forty clams per tank previously acclimated to thedifferent salinity and pH values (15 days) were usedduring 10 days at the assay conditions. Two differentendpoints were selected to assess the toxic effects

associated with the mobilization of metals (Zn, Pb, Cd,Cu): the percentage of mortality and the metallothioneinlevels in the soft body of the survival individuals at theend of the experiment.

Chemical analysisSpecimens ofRuditapes philippinarum of each tankwere dissected out, and the soft body (pools of tenclams per replicate of each station) were taken andstored at 80C until their processing. Samples werehomogenized in a PotterElverhjem at a ratio 3-5 w/vin ice cold buffer 100 mM Tris-HCl/Base pH 8.1 (at

Bioavailability of heavy metals bound to estuarine sediments as a function of pH and salinity values102

www.scilet.com

CSB/Riba 26/1/04 1:33 pm Page 102


3/14

4C) with 1 mM DTT. The homogenate was centri-fuged at 50.000 g at 4C for 120 min. The supernatantwas heat-treated at 95C for 4 minutes transfer immedi-ately to a cool on ice. The samples were centrifuged at10,000g for 15 min at 4C. The supernatant was

employed to quantify metallothionein levels usingdifferential pulse polarography, according to the pro-cedure described by Olafson and Olsson (1991). In theabsence of a clam MT Standard, quantification of MTconcentration in the cytosol of the soft body was based

I. Riba, E. Garcca-Luque, J. Blasco and T.A. DelValls 103

www.scilet.com

Figure 1Map of the studied area in the south of Spain showing the location of the environmental samples selected in theestuarine ecosystem, Ra de Huelva (H) and Guadalquivir estuary (GR).

CSB/Riba 26/1/04 1:33 pm Page 103


4/14

on rabbit liver MT, (MT-I), levels of MT are expressedas microgram per gram of protein. Total protein con-centrations were measured by the Bradford method

(1976) using BSA(bovine serum albumin) as referencestandard material.Water samples were taken of both, pH and salinity

bioassays to determine the speciation of the metals insolution as a result of competition between differentmetal complexes, metal chelates and free metals ionsand to relate this processes to metal bioavailability.

The total concentration of heavy metals (Cd, Pb, Zn,Cu) was carried out in water samples at different salinityand pH values using a differential pulse anodic strippingvoltametry (DPASV). Measurements were taken withstatic drop mercury electrode (SMDE), using theMetrohm 693 processor as reported by Ponce et al.(2000). Briefly, the water samples were filtered through

nuclepore 0.4m polycarbonate filters (Millipore,HTTP047) and acidified using HNO3 (Merck, Suprapur)to pH < 2, then samples were digested using UV radi-ation (6 h) at 85C (Metrohm, 705UV) and finallyanalyzed by DPASV. The heavy metal speciation insolution was determined at the different conditionsusing the geochemical equilibrium model MINTEQA2(Allison et al., 1991). The modeling was based onchemical analysis of the next parameters in the watersolutions: metals, temperature, salinity, pH, alkalinityand estimated concentrations of majority elements(SO4

2, Cl, K+, Na2+, Mg2+, Br-, F) from the two dif-ferent bioassays and for each station.

The salinity was determined by means of an inductionsalinometer (Beckman RS-10). The total alkalinity andthe inorganic carbon speciation in the samples wasdetermined using a potentiometric analyzer (Metrohm670) with glass combination electrodes (Metrohm, ref.6.0210.100) as described by Forja et al. (in press).Briefly, the water quantities analyzed were 100 0.001 g. The analyses were performed at constant tem-perature in a water jacketed open cell and they werecross-checked using an alkalinity reference materialprovided by Dr Andrew Dickson at Scripps Institutionof Oceanography (Batch #33). The results for sixsamples gave an average value of 2010.62.9 M fortotal inorganic carbon, while the certified value was

2009.85 0.85M. This accuracy and precision is goodenough for the inorganic carbon calculation in the usedsystems. The titration was carried out with 0.2M HCl.The inorganic carbon speciation has been calculatedwith the pH (NBS scale) and the total alkalinity valuesand using the dissociation constant reported byMehrbarch et al. (1973).

The total concentration of SO42, Cl-, K+, Na2+, Mg2+,

Br-, F has been estimated by the salinity values and con-sidering the stoichiometry of the major solute componentsand based on the assumption that the relative compositionof sea water is constant (Millero and Sohn, 1992).

Data treatmentThe biological adverse responses (percentage of mor-tality and the induction of the metallothioneins) from

the replicates of the tests and the controls were com-pared using ANOVA and Scheffes Ftests to identifysignificant differences in sensitivity between mediumsand controls (P


5/14

mobility of heavy metals from the sediment to the waterwhen the salinity varies will depend on the relativeimportance of the two counteractive processes (Chapmanand Wang, 2000): (1) desorption due to increase com-plexation with sea water anions (Cl and SO4

2) and/orincreasing competition for particle sorption sites withsea water cations (Na+, K+, Ca2+, Mg2+); (2) coagulation,

precipitation and flocculation. In this sense, s formetals may decrease, increase or be constant depend-ing on the nature of the sediments and the estuarineconditions. The values of for Cd decrease for allstations when salinity decrease so this metal tends to bemore efficiently trapped by low-salinity sediments andits concentration in water is higher at high salinity thanat low salinity values. For this metal the highest value(198) was derived at the highest salinity value (35) inthe highest toxic mud dilution (7.9). The mobilizationfor Pb, Cu and Zn are similar to Cd in control sediments(, Figure 3) and in sediments from the Ria of Huelva,

being different in the dilutions of toxic mud and in thesediments from the Guadalquivir estuary (GR), inwhichs increase when salinity decrease. In this sense,metals from those sediments are more easily mobilizedto the water than in the other stations.

In general, high values of are calculated for heavymetal concentrations measured at low salinity (10) and

low pH (6.5) tested in the bioassay, except for Cd in thesalinity bioassay. The bioavailability of these metalscould be higher at those low values than at higher valuesof salinity and pH.

Chemical speciationComputer speciation modeling allows insights into allaspects of a system dealing with the effects of numerousmetals and ligands present and is able to optimize theeffects of concentration and other environmentalvariables such as pH and salinity (Davidge et al., 2001).The environmental effect of the heavy metals is strongly


www.scilet.com

Figure 2 (103) values defined as the ratio of the total concentration of a determined metal in water divided by the totalconcentration in sediment of all the stations, Bay of Cdiz (G), toxic mud 0.3% (M), toxic mud 7.9% (L), Ra of Huelva(G) and Guadalquivir estuary (I), at different pH values used in the bioassay. (are dimensionless).

CSB/Riba 26/1/04 1:33 pm Page 105


6/14

dependent on the chemical speciation since it will affectthe bioavailability and, hence, the mortality provokedby the metals on the clams used in the bioassays.

The output of the computer modeling, using thewater-ligand model MINTEQA2 are shown in theFigure 4 related to the four heavy metals at different pH

and for the control (C), toxic mud dilutions (0.3% and7.9%) and the two environmental sediment samples(H and GR). The chemical species that associates themobilized heavy metal are for cadmium: Cd2+,CdOH,CdCl,CdSO4,CdCO3; for the heavy metal copper:Cu2+,CuOH,CuCl,CuSO4,CuCO3, Cu2(OH)2

2+,for the heavy metal Zn: Zn2+, ZnOH, ZnCl,ZnSO4, ZnCO3, ZnOHCl and for the heavy metallead: Pb2+, PbOH, PbCl, PbSO4, PbCO3. All thechemical species affected by the symbol are referredto the sum of all the associations of the metal with thecharges available for each ligand.

In general, acidification of the assay provokes anincrease of the free ion for all the metals at all thestation, except for Cd and Pb in sediments collected inthe Guadalquivir estuary. Furthermore, the increase ofthe free ion specie is specially detected in Zn and Cu inthe highest dilution of toxic mud, being the predomi-

nant chemical specie for Zn. A difference between thespeciation of Zn and Cu is related to the trend in thepercentage of free ion with the increase of the toxicmud amount in the dilutions. The free ion formation ofZn increases with the amount of toxic mud diluted,whereas for Cu it decreases. In this sense, the chemicalspeciation of copper is more related to the mobilizationof Cu from sediments in the Bay of Cdiz than thatdetermined by the toxic mud. For the heavy metal Cdand Pb the chemical specie predominant in all thesamples was CdCl and PbCl.

Summarized results of the output of the computer


www.scilet.com

Figure 3 (103) values defined as the ratio of the total concentration of a determined metal in water divided by the totalconcentration in sediment of all the stations, Bay of Cdiz (G), toxic mud 0.3% (M), toxic mud 7.9% (L), Ra of Huelva(G) and Guadalquivir estuary (I),at different salinity values used in the bioassay. (are dimensionless).

CSB/Riba 26/1/04 1:33 pm Page 106


7/14

modeling are shown in Figure 5 related to the fourheavy metals at different salinity values for the control(C), toxic mud dilutions (0.3% and 7.9%) and the twoenvironmental sediment samples (H and GR). Thechemical species that associates the mobilized heavymetal are for cadmium: Cd2+,CdOH,CdCl,CdSO4,CdCO3; for the heavy metal copper: Cu

2+, CuOH,CuCl,CuSO4, CuCO3, Cu2(OH)2

2+, for the heavymetal Zn: Zn2+, ZnOH, ZnCl, ZnSO4, ZnCO3,ZnOHCl and for the heavy metal lead: Pb2+, PbOH,PbCl, PbSO4, PbCO3. All the chemical species

affected by the symbol are referred to the sum ofall the associations of the metal with the charges avail-able for each ligand.

In general, the concentration of the free ion for allthe metals increases with the decrease of the salinityvalues, except for copper in all the station but the highesttoxic mud dilution. In this dilution, the percentage ofthis specie still low and about the 18% of the totalcopper in solution at a value of 10 for salinity. Thehighest amount of the free ion is associated with theheavy metal Zn in the highest percentage of toxic mud


www.scilet.com

Figure 4. Diagrams of chemical speciation of heavy metals Cd, Cu, Zn and Pb bound to sediments studied, Bay of Cdiz(C), toxic mud dilution (0.3% and 7.9%), Ra of Huelva (H) and Guadalquivir estuary (GR) as a function of pH values.Chemical species (GFree metal,GMOH,MMCl;MMSO4; IMCO3; ICu(OH)2

2+ and ZnOHCl) are expressedas percentage of the total dissolved concentration of the metals mobilized from the sediments assayed.

CSB/Riba 26/1/04 1:33 pm Page 107


8/14

diluted being higher than 80% of the total dissolved Zn.Besides, the concentration of the free ion of this metalincreases with the toxic mud dilution. Concentrationsof this free ion in control, lowest toxic mud dilution(0.3%), and the two environmental sediment samplesare in a similar range and about 70% of the total con-centration of dissolved Zn. Asimilar behavior is detectedfor the heavy metals Pb and Cd but as in the case ofcopper they are in a percentage lower than 20% appear-ing as free ions. The chemical species associated with

these two metals are predominantly related to thespecies CdCl and PbCl.

Bioavailability of metalsThe bioavailability of the mobilized heavy metals wasdetermined by means of the induction of the metal-bind-ing proteins (MTs) and by the percentage of the mortal-ity in the two bioassays after ten days of exposure time.

The percentage of survival in the different sedimentbioassays at different pH and salinity values is shown


www.scilet.com

Figure 5Diagrams of chemical speciation of heavy metals Cd, Cu, Zn and Pb bound to sediments studied, Bay of Cdiz(C), toxic mud dilution (0.3% and 7.9%), Ra of Huelva (H) and Guadalquivir estuary (GR) as a function of salinity values.Chemical species (GFree metal; GMOH;MMCl;MMSO4; IMCO3; ICu(OH)2

2+ and ZnOHCl) are expressedas percentage of the total dissolved concentration of the metals mobilized from the sediments assayed.

CSB/Riba 26/1/04 1:33 pm Page 108


9/14

in Figure 6. The mortality at highest dilution of toxicmud was always significantly different compared tothose measured in control at any pH and salinity values.

Significant differences are associated with the toxicmud dilution of 0.3% for pH values of 6.5 and forsalinity values of 10. For pH and salinity values higherthan 6.5 and 10 respectively the mortality at this dilutionwas not significantly different to control.

In Figure 7 shows the summarized results of the con-centration of MTs measured in the total body of theindividuals exposed to control (C), toxic mud dilution(0.3% and 7.9%) and the two sediments from the estu-aries (Ria of Huelva (H) and Guadalquivir (GR)).Significant differences in the induction of the MTs were

measured among treatments and control and among pHand salinity assays. Results obtained in the pH bioassayshow that the induction of MTs are significantly dif-

ferent at any pH treatment related to the individualsexposed to the highest toxic mud dilution. For this treat-ment the induction of MTs increases with the increaseof the proton concentration, being highest at the lowestpH value assayed (6.5). The other toxic mud dilution(0.3%) shows only significantly different in the inductionof MTs measured in clams compared to those measuredin control associated with the lowest pH value (6.5).The environmental stations were not significantly dif-ferent than the results obtained in the control at any pHassayed.


www.scilet.com

Figure 7Histogram of concentration of metallothioneins expressed as microgram per gram of total protein (mg) measuredin the pH and in the salinity bioassays. Asterisks show those induction of metallothioneins significantly different (P


10/14

In general, the induction of MTs in the differentsalinity assays (Figure 7) shows a similar pattern thanthat mentioned for pH although with total concentration

of inducted MTs lower than in the pH bioassay. In thissense, the highest toxic mud dilution was significantlydifferent to the induction measured at the control treat-ments at salinity values of 10 and 20 and increasingwith the decrease of the salinity values, being highestat the lowest salinity value assayed (10). The rest of thetreatments show induction of MTs not significantlydifferent among salinity values neither to the controltreatments.

In summary, the highest toxic mud dilution (7.9%)shows an induction of MTs increasing with the decreaseof salinity and pH values. Besides, the lowest concen-tration of toxic mud (0.3%) shows induction of MTs atlowest value of pH. None of the environmental stations

show significant induction of MTs for any of the pHand salinity assays. These results are in concordancewith the percentage of mortality measured in bothbioassays, especially for both dilutions of toxic mud(0.3% and 7.9% dry weight of toxic mud) in which acorrelation (r2) higher than 0.89 was found betweeninduction of MTs and measured mortality. It informsabout the cause of the mortality, which is related to theconcentrations of heavy metals that previously hadinduced the MTs.

A central aspect of the role of MT as a defensesystem/stress response thus concerns its rate of syn-thesis, turnover and affinity by metals: if the protein is

to be effective as a detoxifying agent, synthesis mustbe sufficiently rapid, and binding sufficiently strong, toprevent excessive binding to more sensitive molecules(Langston et al., 1998). Metallothionein can be alsoinduced besides by heavy metals by other stress agents,such as strong variations of temperature, oxygen andsalinity that can increase the cellular concentrations ofMT (Viarengoet al., 1988). The toxicity of some metals,as Cu and Fe, is related to their prooxidant effect; inmarine organisms these metals can induce oxyradicalproduction leading to lipid peroxidation. In mussels ithas been verified that MT play a role as defense mech-anism to oxidative stress (Viarengo et al., 1999).Although it is not absolutely addressed to link change

in MT levels to injury at cellular organism levels (Oostet al., 2003) the correlations found in this work betweeninduction of MTs and mortality in the clam used in theassay demonstrate that once the concentration of themetals have reached the binding capacity of the detoxi-fication agent (MTs) the mortality of the clam ismeasured.

Linking biological responses and chemicalspeciationFrom the obtained results in the association of theheavy metals compared to the biological responses

measured in the different bioassays it can be identifiedthe chemical specie responsible for the effect measured.The total concentration of heavy metals measured in

water can not explain alone all the effects measured inthe assays. Although the effects determined in thehighest dilution of toxic mud could be related to the dis-solved concentration of the metals in this treatment (see, Figures 2 and 3) it can not easily discriminates whichis the metal responsible of the effects. Furthermore, thetotal concentration of some metals measured in waterexposed to sediments from the estuary located in theRia of Huelva (H) at lowest values of pH and salinityare higher or similar than those measured in the lowesttoxic mud dilution (0.3%) but with absence of biologicaleffect. To better explain the effect measured in bothdilutions of toxic mud but not detected in the waterexposed to sediments from treatment Hit is important

to note different pattern in the speciation of some metalsbetween both treatments. From Figures 4 and 5 it canbe observed that the percentage of Zn2+ at both treat-ments of salinity and pH reaches values of percentagehigher than those measured in the treatment of Huelvaand in the control sediments. It could be appreciated thesame pattern for the heavy metal Cu but in this case,the free ion does not reaches higher percentages thanthose obtained in the control treatments (64%) for anyof the toxic mud dilutions neither in the Ria of Huelvatreatment.

In order to compare the set of data from the chemicalconcentrations and speciation of metals and the two

biological effects measured, a multivariate analysisapproach was applied. The analysis uses the valuesobtained of these 10 original variables in 15 differentcases for each pH and salinity bioassays in the negativecontrol of toxicity, two toxic mud dilutions (0.3% and7.9%) and two environmental stations located in theRia of Huelva (H) and in the Guadalquivir estuary(GR).

The application of PCA to the original 10 variablesat the pH bioassay indicates that they can be groupedin two new factors. These new variables explain morethan 96% of the total variance in the original data set.In the present study, we selected to interpret a group ofvariables as those associated with a particular compo-

nent where loadings were 0.35 or higher (Table 1),corresponding to an associated explained variance ofmore than 30%. This approximates Comreys cut-off of0.6 for a good association between an original variableand a component and also takes into account discontin-uities in the magnitudes of the loadings of the originalvariables. The loadings following varimax rotation forthe two components are given in Table 1. Each factoris described according to the dominant group of variablesand based on their scores for each of the cases studiedand shown in Table 2. Both factors, group the heavymetal concentrations (total and free ion) except in factor


www.scilet.com

CSB/Riba 26/1/04 1:33 pm Page 110


11/14


12/14

on their scores for each of the cases studied and shownin Table 2. Only factors 1 and 2 include association ofboth chemical and biological variables. The first factor

includes both biological effects with highest loadingsthan in any of the other new factors. These biologicaleffects were related to the concentrations of metals Znand Pb and the highest loading was obtained for theirconcentration in the form of free ions (Zn2+, 0.81; Pb2+,0.80). The second factor only associates mortality andwith lower loading than in the first factor. This factorrelates the biological effect to the concentration ofmetals, both total and free ion although the loadings oftotal concentrations in this factor were higher than theconcentrations of free ion metals. The third factor onlyassociates chemical concentrations in it and was notdiscussed here.

The factor score obtained for factor 1 shows that the

free ion concentration of Zn can explain the differentbioavailability of metals based on the free ion concen-trations that provokes the biological effect measuredat lowest salinity value for the toxic mud dilution of0.3%. In this sense, the concentration of free ion Zn2+

could be associated with the mortality of the individu-als ofRuditapes philippinarum exposed to this toxicmud dilution. Although the free ion of lead wasincluded in the factor its low total dissolved concen-trations can neglects its effect compared to that associ-ated with the metal Zn. The score of factor 2 was pos-itive in the toxic mud dilution and confirms the biologicaleffect measured in these treatments. In this case, the

biological effect is related to the total dissolved con-centrations of heavy metals that were mobilized fromthe sediments.

From these results, it is confirmed that the toxicityidentified in the bioassays was related to the mobiliza-tion of metals from sediment to water then related tothe total concentration of metals (specially Zn) in water.It is also shown that the free ion can explain the dif-ferences in the toxicity of the same sample when valuesof salinity change. Nevertheless, the total concentrationof metals in water is the main responsible of the toxicitymeasured. The environmental significance of theseresults can be related to the different toxicity that canbe measured associated with the same sample in dif-

ferent areas of an estuary. Then, in the areas with lowsalinity and pH values the mobilization of some metals(Zn, Pb and Cu) can be toxic if the threshold level ofthe organisms (clam in this study) is overlapped.However, the same sample can be not toxic if we are inthe marine influenced area of the estuary with highervalues of pH (7.5) and salinity (20).

CONCLUSIONS

The mobilization of heavy metals from sediment towater assayed shows a different pattern for the pH and

the salinity bioassays. Whereas in the salinity assay theheavy metal mobilization varies from metal to metaldepending on the relative importance of two counter-

active processes, desorption from sediments to wateror coagulation, flocculation, and precipitation; in thepH assays the acidification of the medium provoked anincreases of the mobility of metals with the decrease ofthe pH values.

The chemical speciation of the different heavy metalsvaries depending on the metal and the assay using dif-ferent values of pH and salinity. Acidification of thesediment samples produces high percentages of free ionfor all the metals. Salinity affects highly the speciationof all the metals and specially related to cadmium andlead for the environmental stations located in the estu-aries of the Gulf of Cdiz. This pattern could be associ-ated with a decrease of the bioavailability of these

metals at higher values of salinity and in the marineareas of the estuary (S about 35).

Low values of pH and salinity show mortality andinduction of MTs associated with heavy metals in the dif-ferent dilutions of toxic mud with clean sediments. In thissense, the decrease in the pH and salinity values increasesthe toxicity associated with the heavy metals bound tosediments assayed. The values of these two key-variablesin the estuarine systems such as those assayed determineboth the chemical speciation of the heavy metals and thetoxicity associated with them in two different ways: (a)affecting to the chemical speciation of the metals pro-ducing more bioavailable species at lower values of pH

and salinity; and (b) influencing to the sensitivity of theorganisms through the increase of diffusion of some met-als through the protein membranes and to the organism.Both, low values of salinity and pH increase the concen-tration of metal in water in the form of free ions, speciallyZn2+. This increase permits a more easily transportedthrough the plasmatic membrane producing an increaseof the MTs. Once the threshold of binding to MTs isexceeded at low values of salinity and pH and in the sta-tion of toxic mud dilution, the metal produces the mor-tality observed in the clams.

The heavy metals from the mining spill bound to thetoxic mud are more bioavailable at low pH and salinityvalues and it could explain previous results that identi-

fied toxicity in low hydrodynamic areas of the estuarywith similar values of pH and salinity as those tested inthis study (Riba et al., in press). Furthermore, it canexplain that although the concentrations of heavymetals in the dilutions of toxic mud are much lowerthan those monitored in the Ra of Huelva they areassociated with biological responses such as inductionof metallothionein and lethal toxicity. It gives an inter-esting indication of the fundamental difference betweenthe long-term effects of continuous heavy metal dis-charge over centuries and the effect of an isolated,albeit very large, single discharge.


www.scilet.com

CSB/Riba 26/1/04 1:33 pm Page 112


13/14

ACKNOWLEDGEMENTS

We thank Dr. John Roitz for critically reading an earlierversion of this manuscript, also Rosa Vzquez and the

staff of the aquaculture facility at the Faculty of Marineand Environmental Sciences, University of Cdiz. Thisresearch was supported by grant REN2002-01699 fromthe Spanish National Plan for research, innovation anddevelopment (Ministerio de Ciencia y Tecnologa).Inmaculada Riba thanks the Consejera de Educaciny Ciencia de la Junta de Andaluca for her Doctoralfellowship (F.P.D.I.).

REFERENCES

Allen, H.E. and Hanssen, D.L. 1996. The importance of tracemetal speciation to water quality criteria. Water. Environ.

Res., 68, 4254.

Allison, J.D., Brown, D.S. and Novo-Gradac, K.J. 1991.MINTEQA2/PRODEFA2. A geochemical assessmentmodel for environmental systems: version 3.0 usersmanual. U.S. EPA, Environmental Research Laboratory.EPA/600/3-91/021.

Bradford, M.M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizingthe principle of proteindye binding.Anal. Biochem., 72,248254.

Campbell, P.G.C. 1995. Interactions between trace metalsand aquatic organims, a critique of the free ions activitymodel. In: Tessier, A. and Turner, D.R. (eds.), MetalSpeciation and Bioabailavility in Aquatic Systems IUPACSeries on Analytical and Physical Chemistry of

Environmental Systems, pp. 45102. Wiley, New York.

Chapman, P.M. and Wang, F. 2001. Assessing sedimentcontamination in estuaries.Environ. Toxicol. Chem., 20,322.

Davidge, J., Thomas, C.P. and Williams, D.R. 2001.Conditional formation constants or chemical speciationdata? Chem. Spec. Bioavail., 13, 129134.

DelValls, T.A. and Chapman, P.M. 1998. Site-specificsediment quality values for the Gulf of Cdiz (Spain) andSan Francisco Bay (USA) using the sediment quality triadand multivariate analysis. Cienc. Mar., 24, 313336.

Forja, J.M., Ortega, T., DelValls, T.A. and Gmez-Parra, A.2003. Benthic fluxes of inorganic carbon in shallow

coastal ecosystems of the Iberian Peninsula.Mar. Chem.,(in press).

Frane, J., Jennrich, R. and Sampson, P. 1985. Factor Analysis.In: Dixon, W.J. (ed.), BMDP Statistical Software, pp.480500. University of California Press, Berkeley.

Langston, W.J., Bebianno, M.J. and Burt, G.R. 1998. Metalhandling strategies in molluscs. In: Langston, W.J. andBebianno, M.J. (eds.), Metal Metabolism in Aquatic

Environments, pp. 219283. Chapman and Hall, London.

Luoma, S.N. and Davis, J.A. 1983. Requirements formodelling trace metal partitioning in oxidized estuarinesediments.Mar. Chem., 12, 159181.

Mehrbach, C., Culberson, C.H., Hawley, J.E. and Pytkowicz,R.M. 1973. Measurement of the apparent dissociationconstant of carbonic acid in seawater at atmospheric pres-sure.Limnol. Oceanogr., 18, 897907.

Millero, F.J. and Sohn, M.L. 1992. Chemical Oceanography.CRC Press, Florida.

Morel, F.M.M. 1983. Principles of Aquatic Chemistry.Wiley, New York.

Olafson, R.W. and Olsson, P.E. 1991. Electrochemicaldetection of metallothionein. Methods. Enzymol., 205,205213.

Oost van der, R. Beyer, J. Nico, P.E. and Vermeulen, N.P.E.2003. Fish bioaccumulation and biomarkers in environ-mental risk assessment: a review. Environ. Toxicol.Pharmacol., 13, 57149.

Ponce, R., Forja, J.M. and Gmez-Parra, A. 2000. Influenceof anthropogenic activity on the vertical distribution of

Zn, Cd, Pb and Cu in intersticial water and coastal marinesediments (Cdiz Bay, SW Spain). Cienc. Mar., 26,479502.

Riba, I., Forja, J.M., Gmez-Parra, A. and DelValls, T.A.2002a. pH and salinity-dependent toxicity of heavymetals bounded to sediments in littoral ecosystems (SW,Spain). In: Proceedings of the SETAC Europe 12th

Annual Meeting, 1216 May 2002, pp.212.Vienna.

Riba, I., Forja, J.M., DelValls, T.A., Guerra, R. andIacondini, A. 2002b. Determining Sediment Toxicityassociated with the Aznalcllar mining spill (SW, Spain)using a bacterial bioassay. In: Pelli, M., Porta, A. andHinchee, R.E. (eds.), Characterization of ContaminatedSediments S1-1, pp. 93100. Venice.

Riba, I., DelValls, T.A., Forja, J.M. and Gmez-Parra, A.2002c. Monitoring the impact of the Aznalcllar MiningSpill on recent sediments from the Guadalquivir estuary,Southwest Spain.Bull. Environ. Contam. Toxicol., 69,129138.

Riba, I., DelValls, T.A., Forja, J.M. and Gmez-Parra, A.The influence of the pH and salinity in the toxicity ofheavy metals bound to sediments from the Gulf of Cdiz.

Environ. Tox. Chem. (in press).

Riba, I., Conradi, M., Forja, J.M., DelValls, T.A. Sedimentquality in the Guadalquivir estuary: lethal effects associatedwith the Aznalcllar mining spill. Mar Pollut Bull (inpress).

Shin, P.K.S., Ng, A.W.M. and Cheung, R.Y.H. 2002.

Borrowing responses of the short-neck clamRuditapesphilippinarum to sediment contaminants. Mar. Pollut.Bull., 45, 133139.

Simkiss, K. and Taylor, M.G. 1995. Transport of metalsacross membranes. In: Tessier, A. and Turner, D.R.(eds.),Metal Speciation and Bioabailavility in AquaticSystems IUPAC Series on Analytical and PhysicalChemistry of Environmental Systems, pp. 244. Wiley,New York.

Stillman, M.J., Green, A.R., Gui, Z., Fowle, D. and Presta,P.A. 1999. Circular dichroism, emission and exafs studiesof Ag(I), Cd(II), Cu(I), and Hg(II) binding to metallo-


www.scilet.com

CSB/Riba 26/1/04 1:33 pm Page 113


14/14

thioneins and modeling the metal binding site. In:Klaassen, C. (ed.), Metallothionein IV, pp. 2335.Birkhauser Verlag, Basel.

Van Ginneken, L., Chowdhury, M.J. and Blust, R. 1999.Bioavailability of cadmium and zinc to the common carp,Cyprinus carpio, in complexing environments: a test forthe validity of the free ion activity model. Environ.Toxicol. Chem., 18, 22952304.

Vercauteren, K. and Blust, R. 1996. Bioavailability of dis-solved zinc to the common musselMytilus edulis in com-plexing environments.Mar. Ecol. Prog. Ser., 137, 123132.

Viarengo, A. Mancinelli, M. Orunesu, M. Martino, G.Faranda, F. and Mazzuccotelli, A. 1988. Effects of sub-lethal copper concentrations, temperature, salinity andoxygen levels on calcium content and on cellular distri-

bution of copper in the gills ofMytilus galloprovincialis,Lam.: a multifactorial experiment.Mar. Environ. Res.,24, 227231.

Viarengo, A. Burlando, B. Cavaletto, M. Marchi, B.Ponzano, E. and Blasco, J. 1999. Role of metallothioneinagainst oxidative stress in the mussel Mytilus gallo-

provincialis. Am. J. Physiol., 277, 16121619.


www.scilet.com

CSB/Riba 26/1/04 1:33 pm Page 114

Documents

Bioavailability Metals