Effects of ph on cd and zn uptake by plant - Tài li?u text
Effects of ph on cd and zn uptake by plant
Aquatic Toxicology 49 (–157Effects of pH on cadmium and zinc uptake by the midgelarvae Chironomus ripariusL. Bervoets *, R. BlustDepartment of Biology, Uni6ersity of Antwerp(RUCA), Groenenborgerlaan171,2020Antwerp, BelgiumReceived 15 October 1998; received in revised form 6 July 1999; accepted 26 July 1999AbstractWe studied the effect of pH on the uptake of cadmium and zinc by fourth instar larvae of the midge Chironomusriparius within the pH range 5.5 –10.0, using chemically defined solutions. The effect of prior acclimation on metaluptake was examined for four pH levels, i.e. pH 5.5, 7.0, 8.0 and 9.5. At least three factors were important indetermining the effect of pH on the cadmium and zinc uptake by midge larvae. The effect of pH on metal uptake isthe combined result of changes in free metal ion activity, changes in pH of exposure and changes in pH ofacclimation, the latter representing a physiological effect. Within each acclimation group metal uptake in larvaeincreased with increasing pH of exposure in the range 5.5–9.0 but decreased between pH 9.0 and 10.0. Taking intoaccount the decreased free metal ion activity, metal uptake was still high at pH 10.0. A possible explanation for thisis that an increase in pH alters the metal uptake process by decreasing the protonation of the binding sites. That is,the biological availability of the free metal ion increases with increasing pH. Among the different pH exposuregroups, acclimation had a positive effect up to pH 9.0 but a negative effect between 9.0 and 10.0. Two differentuptake models were applied to describe the observed variation in metal uptake. With a non-linear, semi-empiricalmodel, the integration of the different pH effects for the pooled data described no more than 38% of the totalvariation in cadmium uptake and 36% of the total variation in zinc uptake by midge larvae. When the model wasfitted to the uptake data of larvae acclimated to the exposure conditions, 78 and 69% respectively of the variation wasdescribed. The second model, a biological ligand model, was not able to discriminate between effects of pH inacclimated and non-acclimated exposure groups. Only for the data of larvae acclimated to the exposure conditions themodel could describe a significant amount of the observed variation in metal uptake, R2values being comparable tothose of the first model. The remaining high undescribed variation could be ascribed to the high natural variation inmetal uptake by midge larvae. (C) 2000 Elsevier Science B.V. All rights reserved.Keywords:U pH C C Zinc/locate/aquatox1. IntroductionThe bioavailability of trace metals to aquaticorganisms largely depends on the speciation of themetals in the solution (Campbell and Stokes,* Corresponding author. Tel.: +32-3-2180349; fax: +32-3-2180497.E-mail address:bervoets@ruca.ua.ac.be (L. Bervoets)/$ - see front matter (C) 2000 Elsevier Science B.V. All rights reserved.PII: S)00066-1L. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 1571461985; Campbell, 1995). Earlier studies haveshown that the bioavailability of cadmium andzinc from solutions is the function of the freemetal ion activity which is the most prevalentspecies in freshwater (Sunda et al., 1978; Engeland Flower, 1979; Allen et al., 1980; De Lisle andRoberts, 1988; Blust et al., 1992). One of themost important environmental factors, which infl-uences the bioavailability of metals to aquaticorganisms, is the pH of the solution. In severalstudies an increase in uptake or toxicity of certainmetals with increasing pH was observed in avariety of aquatic organism (Cusiamo et al.,1986; Krantzberg and Stokes, 1988; Blust et al.,1991; Schubauer-Berigan et al., 1993; Odin et al.,1996; Croteau et al., 1998). In contrast, in someother studies or for other metals an increaseduptake or toxicity of metals was observed withdecreasing pH (Krantzberg and Stokes, 1988;Palawski et al., 1989; Taylor et al., 1994; Ger-hardt, 1994; Odin et al., 1995).Changes in pH will influence the partitioningof many metals between the sediment and theaqueous phase and will alter the speciation of themetals in the water. Acidification generally willresult in an increased metal transfer from thesolid to the liquid phase with higher free metalion concentrations in the water (Palawski et al.,1989; Odin et al., 1995; Lucan-Bouche? et al.,1997a,b; Playle, 1998). However, decreasing pHalso results in an increasing amount of competingions, i.e. hydrogen ions, for the same bindingsites. As a consequence, pH may influence theuptake of metals in two antagonistic ways. Adecrease in pH will result in an increase in freecadmium or zinc ion activity but also in protona-tion of the binding sites at the cell surface(Campbell and Stokes, 1985; Campbell, 1995;Simkiss and Taylor, 1995; Hare and Tessier,1996; Croteau et al., 1998). Apart from the chem-ical effects, pH might have an effect on the bio-logical (behavioural and/or physiological)processes and also indirectly alter metal uptake(Knutzen, 1981; Wildi et al., 1994).In most freshwater ecosystems, chironomid lar-vae belong to the most common invertebrates.Larvae of the non-biting midge Chironomusriparius can be found in both lentic (e.g. Parmaand Krebs, 1977; Jernelo¨v et al., 1981) and loticenvironments (e.g. Bendell-Young and Harvey,1991; Timmermans et al., 1992; Postma etal., 1995). Chironomid larvae can be found inwaters with very low pH conditions (Jernelo¨vet al., 1981; Bervoets et al., 1994; Cranston et al.,1997), and the species C. riparius can toleratepH of less than 4 (Jernelo¨v et al., 1981; Lohnerand Fisher, 1990; Bruner and Fisher, 1993) andpH of more than 10 (Bervoets, unpublisheddata).Since pH has a combined effect on both chemi-cal and biological processes it was the aim ofthis study to determine the separate and com-bined effect of these processes on metal uptake.The effect of changing pH conditions on thecadmium and zinc uptake by fourth instar larvaeof the midge C. riparius (Meigen) (Diptera, Chi-ronomidae) was studied, in relation to the accli-mation conditions (biological effect) and the freemetal ion activity (chemical effect). In these ex-periments only exposure via the water was con-sidered.2. Materials and methods2.1. Test organismEgg ropes of the midge C. riparius (Meigen)used in the experiments were obtained from acontrolled laboratory culture at the Royal Bel-gian Institute for Natural Sciences (KBIN, Brus-sels, Belgium). Larvae were cultured in 10-lplastic aquaria containing a paper towel sub-strate. Chironomids were maintained at a temper-ature of 21°C and a 6:18 h light– dark regime in aclimate chamber and fed with a suspension ofground commercial fish food (TetraMin(R), Melle,Germany) (Vermeulen et al., 1997). Culture waterwas replaced weekly. When the fourth larvalstage (instar 4) was reached the larvae wereplaced at 15°C in the dark and held in aquaria athigh densities (1 larvae per cm2) to retard pupa-tion while maintaining them in normal physiolog-ical state (Mackey, 1977; Ineichen et al., 1979;Bangenter and Fischer, 1989).L. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 1571472.2. Experimental proceduresIn the culture, and in all acclimation and exper-imental conditions the medium was artificialRiver Water (RW). The composition of1lofthischemically defined freshwater was 0.096 gNaHCO3, 0.004 g KCl, 0.123 g MgSO4.7H2O and0.06 g CaSO4.2H2O, resulting in a pH of 7.8 atroom temperature. The media were prepared bydissolving the analytical grade reagents (Merckp.a.) in deionized water. The solutions were aer-ated for at least 24 h before the experiments werestarted, to promote equilibration with the atmo-sphere. Dissolved oxygen was measured with apolarographic electrode system (WTW OXI91/EO90) and hydrogen ion activity with a glasselectrode (Ingold ).Stocks of cadmium and zinc, containing 100mM Cd and 1000 mM Zn, were prepared. Theradioisotopes109Cd and65Zn (Amersham Interna-tional, UK) were used as tracers, 46.2 MBq/lofeach tracer being added to the metal stock solu-tions. In all experimental exposure solutions theresulting metal concentrations were 0.1 mMCdand 1 mM Zn. These concentrations were chosenbecause of their environmental relevance. The re-sulting radioactivity of both tracers was 46.2KBq/l.Six days before an experiment was performed,larvae were collected from the culture and accli-mated to four different pH values, i.e. pHaccl5.5,7.0, 8.0 and 10.0. Solutions were adjusted to thedesired test pH using analytical-grade HCl orNaOH. The pH during the acclimation periodwas controlled using a pH-stat system (Consort,Belgium). With this system, pH and temperaturewere controlled continuously. Water pH generallydrifted from the target value by B 0.3 units.Resulting pH ranges were 5.2–5.6, 6.7–7.1, 7.8 –8.2, and 9.5 –9.8; with the pH stat system it wasnot possible to maintain a pH of 10.0. For sim-plification purposes, pHacclvalues will be referredto as pH 5.5, 7.0, 8.0, and 9.5, respectively. Alllarvae were of the same age and came from thesame batch culture, and at the end of the acclima-tion periods larvae from all acclimation groupswere fourth instars and body weight did not differsignificantly among groups. This indicates that atthe start of the experiments the condition of thetest organisms was equal among all acclimationgroups.For all experiments, 50 midge larvae of com-parable size were placed in a series of plasticvessels containing 50 ml test solution. These ves-sels were placed in a thermostatic water bath at15°C. Both cadmium and zinc uptake by thechironomid larvae were linear over time for atleast 8 h during exposure to a total concentrationof 0.1 mM Cd and 1 mM Zn (Bervoets, 1996).Therefore accumulation was measured after6hofexposure. After exposure, the 50 individuals werecollected on a 250 mm sieve and rinsed with 50 mlof deionized water (Baudin and Nucho, 1992).For each treatment group four to eight replicateswere taken.In a preliminary experiment the effect of rinsingwith deionized water was compared to rinsingwith a solution of 1 mM of 8-hydroxyquinoline-5-sulfonic acid, a strong ligand that has been usedto remove cadmium bound to the external sur-faces of brine shrimp (Blust et al., 1995). Bothsolutions removed the same amount of cadmiumand zinc so that rinsing with deionised watersuffices to remove metals adsorbed to the externalsurfaces. Larvae were blotted dry and in groupsof 50 transferred to counting vials for gammaspectrometry.The radioactivity of the samples was measuredin a Minaxi-Auto-gamma 5530 spectrometer fittedwith a thallium-activated sodium iodine well crys-tal (Canberra Packard). Sample counts were cor-rected for background and the correspondingcadmium and zinc activities were calculated usingthe following equation:Muptake2+=ACTmidge60.CE.Wmidge.t.SAin which M2+uptakeis the cadmium or zinc uptake,ACTmidgeis the65Zn or109Cd activity of thelarvae after correction for background radiation(counts/min), CE is the counting efficiency (CPM/0.178 for Zn and CPM/0.575 for Cd), Wmidgeisthe dry weight of the larvae (g), t is the incubationtime (h) and SA is the specific activity of thewater (46.2 Bq65Zn/nmol total Zn and 462 Bq109Cd/nmol Cd). The counted larvae were driedL. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157148for 24 h at 60°C and weighed on a Mettler H54balance to the nearest 0.1 mg. The cadmium andzinc uptake was expressed on a dry weight basisin nmol/g.To determine the effect of the pH of exposureand acclimation on metal uptake, all acclimationgroups were exposed to metal containing solu-tions of six different pH, i.e. 5.5, 6.0, 7.0, 8.0, 9.0and 10.0. To control the pH during the experi-ments, 4 inert biological buffers were used: MES(2-(N-morpholino)ethanesulphonic acid, pKa=6.1) was used to control the pH at 5.5 and 6.0;MOPS (3-(N-Morpholino)propanesulfonic acidpKa=7.2) was used to control the pH at 7.0;EPPS (N-(2-Hydroxyethyl)piperazine-N%-(3-pro-panesulfonic acid), pKa=8.0) was used to controlthe pH at 8.0, and CHES (2-(N-cyclohexy-lamino)ethane-sulfonic acid, pKa=9.3) was usedto control pH at 9.0 and at 10.0. In general,biological pH buffers have very low metal -stabil-ity constants and complexation is negligible at theconcentration of 10 mmol l-1of buffer that wasused to buffer the solutions (Good et al., 1966).Solutions were further adjusted to the desired testpH using analytical-grade HCl or NaOH. Thedissolved oxygen concentration and pH were mea-sured at the beginning and the end of each exper-iment. Generally, all measured oxygen valuesremained within 10% of the initial values, anddifferences in pH before and after the experimentswereB 0.1 pH unit. Cadmium and zinc in theexperimental solutions were measured by an axialinductively coupled plasma atomic emission spec-trometer (ICP-AES, Liberty Series II, Varian).Metal levels in filtered (through a membrane filter0.22 mm pore size (Acrodisc(R), Gelman)) andunfiltered samples were compared.2.3. Chemical speciation modellingThe equilibrium concentrations of the chemicalspecies considered were calculated using the com-puter program SOLUTION (Blust and Van Gin-neken 1998), an adaptation of the programCOMPLEX (Ginzburg, 1976). This speciationmodel allows the calculation of the compositionof solutions in equilibrium with the atmosphere.A thermodynamic stability data base for zinc andcadmium was built based on the data of Smithand Martell (1976), Martell and Smith (1982) andSmith and Martell (1989). The thermodynamicand conditional stability constants for the mostprevalent cadmium and zinc species considered inthe chemical speciation model are given in Table1. Case specific input comprises the total concen-trations of the metals and ligands in the solution,the free hydrogen ion concentration (pH), redoxpotential (pE), temperature, and the gas phasethat is maintained in equilibrium with the solu-tion. Results of the chemical speciation calcula-tions are expressed on the molar concentrationscale. Activities were obtained by multiplying theconcentrations of the chemical species with theappropriate activity coefficients. Activity coeffi-cients were calculated using the estimationmethod of Helgeson (Birkett et al., 1988).Table 1Thermodynamic and conditional stability constants for thecadmium and zinc species considered in the chemical specia-tion modelaLog QLog KSpeciesCdOH+3.91 3.74Cd(OH)207.64 7.38Cd(OH)3-8.68 8.42CdCl+1.97 1.802.59 2.34CdCl20CdCl3-2.40 2.141.331.47CdCl42-2.11CdSO402.453.103.44Cd(SO4)22-4.35CdCO304.014.824.99ZnOH+10.20Zn(OH)209.9413.6513.90Zn(OH)3-15.3415.50Zn(OH)42-0.360.53ZnCl+0.69 0.43ZnCl20ZnCl3-0.450.700.32 0.15ZnCl42-1.982.32ZnSO403.26Zn(SO4)22-2.92Zn(SO4)34-2.032.034.76ZnCO305.10ZnHCO3+11.03 10.69aK=thermodynami Q=conditionalstability constant, valid at the calculated freshwater ionicstrength of 0.009 M.L. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157149Fig. 1. Metal speciation in function of pH (t°=15°C) A, B, zinc.methods used are outlined in Sokal and Rohlf(1981).3. Results3.1. Chemical speciationIn Fig. 1A and B the results of the modelcalculations in function of pH are summarised forrespectively cadmium and zinc. For cadmium thefree metal ion activities remain nearly constantover the pH range 5.5–8.0 (decreasing from 67.6to 63.2 nM). Between a pH 8.0 and 10.0 the freecadmium ion activities drop from 63.2 to 0.11nM. At pH of 9.0 however free cadmium ionactivity is still 10.1 nM. For zinc the free metalion activities remain constant over a narrower pHrange i.e. 5.5 to 7.4, decreasing from 702 to 685nM. Between a pH 7.4 and 10.0 the free zincactivities drop from 685 to 0.19 nM. At theexposure pH of 8.0 and 9.0 the free zinc ionactivities are respectively, 497 and 12.6 nM.In all experimental solutions the measured totalmetal concentrations were 0.11 (9 0.01) mMCdand 1.07 (9 0.03) mM Zn. No significant differ-ences were measured between filtered andunfiltered samples, indicating that precipitation ofcertain metal species (e.g. CdCO30, ZnCO30) wasnot significant.3.2. Effect of pH on metal uptakeThe effect of pH on metal uptake was com-pared for four different pH acclimation groups(pHaccl) which were exposed to six different pHvalues (pHexp). This made it possible to separatethe effect of pH of acclimation from pH of expo-sure on the uptake of the metals by the larvae.Fig. 2 shows the results of the effect of the pHof exposure on Cd uptake in the different acclima-tion groups. Within each acclimation group cad-mium uptake increases with increasing pH ofexposure with the exception of pHexp10.0 in theacclimation groups pHaccl5.5, 8.0 and 9.5. In theacclimation groups pHaccl5.5 and pHaccl8.0 nosignificant difference between uptake at pHexp9.0and 10.0 was observed (pHaccl5.5: t =0.24, df 5,2.4. Statistical analysisAnalysis of variance and non-linear regressionswere used to analyse the data. All data weretested for homogeneity of variance by the log-anova test and for normality by the Kol-mogorov– Smirnov test for goodness of fit.Significance levels of tests are indicated by aster-isks according to the following probability ranges:* P5 0.05; ** P5 0.01; *** P5 0.001. StatisticalL. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157150Fig. 2. Uptake of cadmium by midge larvae in function ofexposure pH for the different pH acclimation groups(Cdtotal=0.1 mmol l-1, temp 1591°C). Means with standarddeviation are given.Fig. 3. Uptake of zinc by midge larvae in function of exposurepH for the different pH acclimation groups (Zntotal=1.0 mmoll-1, temp 159 1°C). Means with standard deviation are given.9.0 to 1.5 nmol g-1at pH110.0 was observed(t= 3.06, df =9, PB 0.05). The highest increasein cadmium uptake was measured in the pHaccl8.0group, where the mean uptake increased from 1.4nmol g-1atapHexpof 5.5 to 4.3 nmol g-1at apHexpof 9.0. In all cases prior acclimation had asignificant effect on the uptake of cadmium by themidge larvae, the highest uptake being observedat the acclimation of pHaccl8.0. A two-way analy-sis of variance of the data showed that both theeffect of the pH of exposure and the pH ofacclimation on Cd uptake are highly significant(Table 2a).Fig. 3 shows the results of the effect of the pHof exposure on zinc uptake in the different accli-mation groups. Generally, the results were similarto those for Cd. In acclimation group pHaccl5.5,no significant differences in zinc uptake at thedifferent pH of exposure were observed. In theother acclimation groups zinc uptake increasesP= 0.81; pHaccl8 t= 0.72, df 16, P =0.50) and atacclimation group pHaccl9.5, a significant de-crease in Cd uptake from 3.2 nmol g-1at pHexpTable 2Two-way analysis of variance for the effect of pH of exposureand the pH of acclimation on metal uptake by midge larvae(24 treatment groups with four replicates)FsMean of squaresSource of variation df(a)Cadmium uptakeExposure pH 45.613 33.81*Acclimation pH 5 18.22 13.51*Interaction 15 1.73 1.29a(b) Zinc uptakeExposure pH 3 *9.74*71335Acclimation pHInteraction 2.85*208815ans,* P50.001.L. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157151with increasing pH of exposure, with a decrease inuptake at pHexp10.0 for the acclimation groupspHaccl8.0 and 9.5. In the latter cases a significantdecrease was measured (pHaccl8 t= 3.39, df=17,PB 0.005; pHaccl10 t= 2.75, df=9, PB0.05).Again the highest increase in zinc uptake wasmeasured in the pHaccl 8.0 group, where themean uptake increased from 15.7 nmol/gatanexposure of 5.5 –19.0 nmol/g at an exposure pHexpof 9.0. As for cadmium, prior acclimation had asignificant effect on the uptake of zinc by themidge larvae, the highest uptake being observedat the pHaccl8.0.Two-way analysis of variance showed that boththe effect of the pH of exposure and the pH ofacclimation on Zn uptake are highly significant(Table 2b). The combined effect is highly signifi-cant as well.In many cases the variation in metal uptakewithin the exposure groups was high to very high.Relative standard deviations within groups of upto 58% for zinc uptake and up to 67% for cad-mium uptake were calculated.3.3. Modelling metal uptakeTo determine the relative importance of thedifferent factors contributing to the variation inmetal uptake by the midge larvae, two differentmodels to describe the observed variation in metaluptake were compared:3.3.1. Empirical modelAn empirical non-linear regression model wasconstructed (Blust et al., , 1994;Bervoets et al., 1996a). Metal uptake was relatedto the product of three nth-power terms thatdescribe the effect of the change in the free metalion activity (Mact), pH of exposure (pHexp) andpH of acclimation (pHaccl) on metal uptake. Acoefficient of proportionality (Cf) was introducedto relate the activity of the metal ion in thesolution, to the metal uptake by the midge larvae.The equation for both metals becomes:Meupt=Cf*(Mekact *pHlexp*pHmaccl)The relative importance of the different termswas determined for the pooled results by a for-ward selection procedure. This was done by start-ing with the free metal ion activity as the soleindependent variable and stepwise adding theother terms to evaluate whether their contributionto the amount of variation described wassignificant.3.3.2. The biological ligand modelThis semi-empirical model considers the organ-ism as another ligand with metal ions and protonscompeting for the same biological uptake site (X)(Hare and Tessier, 1996; Croteau et al., 1998;Playle, 1998):Me2++X =XMe;KMeX=[XMe]/[Me2+][X] (1)XH= H++X;Ka=[X][H +]/[XH] (2)concentration of uptake sites is given by:[X]T=[XH] +[X]+ [XMe] (3)which, if combined with the expressions for theequilibrium constants in Eq. (1) and (2) and as-suming that only a small fraction of the sites isoccupied by Cd or Zn (i.e. [XMe]BB[X]T), gives[XMe]= (KMeXKHX[X]T/H++KHX) [Me2+] (4)If it is assumed that metals taken up by C.riparius is proportional to [ ? XMe], that isMeupt[? XMe], combining this relation with Eq.(4) gives:Meupt=F([Me2+]/(H++Ka)) (5)Where F(=kKKa[X]T) is a constant specific toC. riparius.Table 3 gives the results of the non-linear re-gression analysis for cadmium uptake by midgelarvae. Relating cadmium uptake to the free cad-mium ion activity describes only 6% of the totalvariation in cadmium uptake. When the term wasadded which accounts for the effect of the pH ofexposure (pHexp), 26% of the variation was de-scribed. Adding the term which accounts for theeffect of the pH of acclimation (pHaccl) described38% of the variation in cadmium uptake. Consid-ering only the results of the cadmium uptakeexperiments performed at the pH of acclimation(i.e. pH of exposure=pH of acclimation) 78% ofthe variation in cadmium uptake was described.L. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157152Considering other cadmium species as bioavailableand including them in the uptake model did notincrease the amount of variation described.Table 4 gives the results of the non-linear regres-sion analysis for the zinc uptake by midge larvaeusing the empirical model. Relating zinc uptake tothe free zinc ion activity, almost none of theobserved variation in zinc uptake could be de-scribed. When the term was added which accountsfor the effect of pH of exposure, 24% of thevariation was described. Adding the term whichaccounts for the effect of pH of acclimation de-scribed 36% of the variation in zinc uptake. Consid-ering only the results of the zinc uptake experimentsperformed at the pH of acclimation describes 64%of the variation in zinc uptake. Considering otherzinc species as bioavailable and including them inthe uptake model did not increase the amount ofvariation described.With the semi-empirical model it was not possi-ble to describe any of the variation in metal uptakeusing the pooled data for either cadmium or zinc.Considering only the results of the metal uptakeexperiments performed at the pH of acclimation79% of the variation in cadmium uptake and 68%of the variation in zinc uptake was described.Calculated values of F were 0. and2.819 0.66 nmol/g, for cadmium and zinc, respec-tively and Kavalues were 4.-5and2.389 1.19 10-5m for both cadmium and zincuptake (means9 S.E.).Fig. 4A and B summarise the results of the metaluptake by midge larvae exposed to the pH ofacclimation for respectively cadmium and zinc. Forcadmium significant differences were found amongthe different uptake groups (ANOVA: F3,17=23.7,PB 0.001). With a Duncan post hoc test it wasshown that all groups differed significantly fromeach other (PB 0.001) with the exception of pH 7.0compared to pH 5.5. Also for zinc significantdifferences were found among the different uptakegroups (ANOVA: F3,17=15.1, PB 0.001). With aDuncan post hoc test it was shown that group pH8 differed significantly from all other groups (PB0.001) and the other groups differed significantlyonly from group pH 8.0 (P B 0.001).4. DiscussionIn this study the effect of pH on the uptake ofTable 3Cadmium uptake by C. riparius: non-linear regression modelfor the pooled dataaBSEL1Variable L2(1) Cdupt=Cf*(Cdactk)(R2=0.06**, n=154)0.80.357*Coefficient-0.088***k-exponent -0..062(2) Cdupt=Cf*(Cdactk?pHexpl)(R2=0.26***, n=154)Coefficient 0.011c0.128*** 0.045k-exponent 0.083 0.1732.889 4.305l-exponent 3.597*** 0.708(3) Cdupt=Cf*(Cdactk?pHexpl?pHacllm)(R2=0.38***, n=154)Coefficient 0.001bk-exponent 0.00.145***2..***l-exponent1.165m-exponent 1.*** 0.345aB: partial reg SE: standard error forpartial reg L1, L2: confidence limits forpartial regression coefficientsbCadmium uptake in midge larvae in nmol/g.cns,* P50.05;*** P50.001Table 4Zinc uptake by C. riparius: non-linear regression model for thepooled dataaVariable L2L1SEB(1) Znupt=Cf*(Znactk)(R2B0.01ns, n=154)Coefficient 30.02* 15.04 14.98 45.06-0.008nsk-exponent(2) Znupt=Cf*(Znactk?pHexpl)(R2=0.24***, n=154)Coefficient 0.135ns0.222*** 0.048k-exponent 0.174 0.2704.480*** 0.889 3.591l-exponent 5.369(3) Znupt=Cf*(Znactk?pHexpl?pHacllm)(R2=0.36***, n=154)0.517nsCoefficient0.228*** 0.042k-exponent 0.186 0.270l-exponent 4.476*** 0.774 3.702 5.250m-exponent 1.609*** 0.391 1.218 2.000aB: partial reg SE: standard error forpartial reg L1,L2: confidence limits forpartial regression coefficients. Zinc uptake in midge larvae innmol/g.ns * P50.05; *** P50.001L. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157153Fig. 4. Metal uptake rate by C. riparius at the pH of acclima-tion (Cdtotal=0.1 mmol l-1, temp 1591°C). Means withstandard deviation are given. (A) C (B) Zinc. a,b,c,d:significant different (PB0.001) from pH 5.5; 7; 8 and 10,respectively.4.1. Effect of the free metal ion and pH ofexposureGenerally the free metal ion is considered asthe biologically most available species. For bothmetals the free ion activity remains nearly con-stant between 5.5 and 8.0 and decreases from 8.0to 10.0, reaching very low levels at this pH. Whenmetal uptake was related to the free metal ionactivity, a negligible part of the variation in up-take could be described. Most likely this is theresult of the combined effect of pH on metalspeciation (decreasing free metal ion activity withincreasing pH) and on the competition betweenprotons and metal ions for the same uptake sites.In all cases the uptake of both metals increaseswith increasing exposure pH with the exceptionof pH 9.0 and 10.0. In most cases, metal uptakeeven decreased at pH 10.0 compared to uptake atpH 9.0. In the pH range 5.5 –9.0 our results agreewith findings for other aquatic organisms exposedto cadmium or zinc. Schubauer-Berigan et al.(1993) found an increase of the toxicity of Cdand Zn with increasing water pH (pH 6.3, 7.3and 8.3) for three aquatic invertebrate species.The same trend in toxicity was found by Cusiamoet al. (1986) who exposed steelhead trout at cad-mium, copper and zinc at pH 4.7, 5.0 and 7.0.They found an increase in metal toxicity withincreasing pH for all tested metals. These findingsare consistent with theoretical considerations. Ahypotheses put forward in literature is that thefree metal ions (i.e. Cd2+and Zn2+) are incompetition with the hydrogen ions at the mem-brane level and therefore restrict uptake underacid conditions (Campbell and Stokes, 1985;Blust et al., 1991; Hare and Tessier, 1996;Croteau et al., 1998). In the pH range we used,the hydrogen ion activity decreased from 2.79 mMat pH 5.5–0.07 nM at pH 10.0.We could find in the literature only one studywhere organisms were exposed to pH higher than9.0 in combination with metals (Belanger andCherry, 1990). In that study impaired reproduc-tion and mortality of Ceriodaphnia dubia wasobserved below pH 6 and above pH 9 whendaphnids were exposed to pH only. Howevercadmium and zinc by larvae of the midge C.riparius was examined using chemically definedsolutions. At least three factors are important indetermining the effect of pH on cadmium andzinc uptake by midge larvae. The effect of pH onmetal uptake is the combined result of (1)changes in the free metal ion activity: this deter-mines the fraction of the metal in solution whichis available for uptake, (2) changes in pH ofexposure and (3) changes in pH of acclimation.These two latter factors influence the permeabilityof the exchange surfaces for metal ions and otherphysiological processes.L. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157154when the organisms were exposed to zinc andcopper at pH 6, 8 and 9 an inverse relationshipbetween pH and effect was observed, regardless ofacclimation conditions.The decreased uptake at pH 10.0 in our studyprobably is the result of the decrease in metal ionactivity of both cadmium and zinc. Althoughmetal ion activities were very low at pH 10.0 (0.11and 0.19 nM, respectively for cadmium and zinc)uptake is still relatively high. An explanation forthis relative high metal uptake at pH 10.0 mightbe that an increase in pH alters the metal uptakeprocess by decreasing the protonation of the bind-ing sites. That is, the biological availability of thefree metal ion increases with increasing pH.Another possible explanation could be that oneor more of the inorganic metal species, which aredominant at the highest pH, are available to themidge larvae. However, adding these species inthe uptake model, could not increase the de-scribed variation in metal uptake. Moreover it isunlikely that the carbonate species are available toaquatic organisms (Blust et al., 1991; Campbell,1995).4.2. Effect of acclimationThe effect of pH on the uptake of metals by themidge larvae is not only determined by the effecton chemical speciation but also by physiologicaleffects. At all exposure conditions acclimationhad a remarkable but inconsistent effect on up-take of both metals. The marked effect of accli-mation on cadmium and zinc uptake by the midgelarvae is a strong indication that pH has not onlyan effect on the speciation of the metals or proto-nation of the binding sites but also alters thephysiological condition of an organism and thusindirectly affects metal uptake. Previous acclima-tion to different salinities also resulted in a signifi-cant effect on cadmium uptake by larvae of C.riparius (Bervoets et al., 1995) but not on zincuptake (Bervoets et al., 1996b). A possible hy-pothesis for the acclimation effect is a pH depen-dent behaviour of the larval C. riparius. Wildi etal. (1994) found an increase in larval mucus secre-tion at lower pH, which could result in a retardeddiffusion of the metals along the concentrationgradient. Another possibility is an effect of pH onrespiration. Alibone and Fair (1981) observed anincrease of respiration rate in Daphnia magna withincreasing pH. No behavioural or physiologicaldata in literature were found on the effect of pHhigher that 9.0.4.3. Modelling metal uptakeWith the empirical non-linear model for thepooled data no more than 26% and 24% of thevariation in cadmium and zinc respectively uptakecould be described. An increase of described vari-ation up to 38% and 36% respectively was ob-served when the factor that accounts for pH ofacclimation was added. The high proportion ofundescribed variation is largely due to the naturalvariation in metal uptake by the midge larvae.Also in other studies on cadmium uptake bymidge larvae, a high variation in metal uptakewithin a treatment was observed (Seidman et al.,1986; Timmermans et al., 1992; Bervoets et al.,a). Moreover, when the non-linear up-take models were fitted to the mean uptake valuesup to 63% of the cadmium uptake and 54% of thezinc uptake could be described.Another possible explanation for the high pro-portion of undescribed variation is that the pH ofacclimation has an inconsistent effect on metaluptake. From the modelling of the metal uptake itwas obvious that pH of acclimation had a positiveeffect on the metal uptake (Table 3, Table 4) witha coefficient of 1.51 for cadmium and 1.61 forzinc. However, metal uptake increases with in-creasing pH of acclimation between pHaccl5.5 and8.0 and decreases at pHacclof 10.0 in all exposuregroups and for both metals. When using only thedata of larvae acclimated to the exposure condi-tions it was possible to describe a relatively highproportion of the variation in metal uptake (78and 64% of the variation, respectively for cad-mium and zinc uptake).With the empirical non-linear model it was notpossible to take into account the non-consistenteffect of metal uptake in function of pH. Howeverwith the model of Hare and Tessier (1996) it wasnot possible to describe any of the observed varia-tion in metal uptake using the pooled data. This isL. Ber6oets, R. Blust/Aquatic Toxicology49 ( – 157155probably due to the fact that the model does notdiscriminate between effects of pH in acclimatedand non-acclimated exposure groups. This be-came clear when only the data of uptake at thepH of acclimation were included in the model. Aswith the non-linear model it was possible to de-scribe 79 and 68% in uptake of respectively cad-mium and zinc.These semi-empirical models provide an attrac-tive way to incorporate effects of chemical specia-tion and interactions at the biological inter-face. However, the effect of pH is more complexthan a simple competition between metal ionsand protons as implied in the biological ligandmodel.Although the described variation increases forboth models when only the acclimated larvae areconsidered, still more than 20 and 30% of thevariation in uptake of respectively cadmium andzinc remains undescribed. As stated before, this ismainly due to the high natural variation in metaluptake, especially apparent in short term uptakestudies and at low environmental metal levels.Evidence for this was found by calculating, usingAnalysis of Variance, the relative magnitudes ofthe variance components (Sokal and Rohlf,1981). 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