Effects of ph on cd and zn uptake by plant

The effect of pH on metal uptake is the combined result of changes in free metal ion activity, changes in pH of exposure and changes in pH of acclimation, the latter representing a physi

Effects of pH on cadmium and zinc uptake by the midge

larvae Chironomus riparius

L Bervoets *, R Blust

Department of Biology, Uni 6ersity of Antwerp(RUCA), Groenenborgerlaan171 , 2020Antwerp, Belgium

Received 15 October 1998; received in revised form 6 July 1999; accepted 26 July 1999

Abstract

We studied the effect of pH on the uptake of cadmium and zinc by fourth instar larvae of the midge Chironomus

riparius within the pH range 5.5 – 10.0, using chemically defined solutions The effect of prior acclimation on metal

uptake was examined for four pH levels, i.e pH 5.5, 7.0, 8.0 and 9.5 At least three factors were important in determining the effect of pH on the cadmium and zinc uptake by midge larvae The effect of pH on metal uptake is the combined result of changes in free metal ion activity, changes in pH of exposure and changes in pH of acclimation, the latter representing a physiological effect Within each acclimation group metal uptake in larvae increased with increasing pH of exposure in the range 5.5 – 9.0 but decreased between pH 9.0 and 10.0 Taking into account the decreased free metal ion activity, metal uptake was still high at pH 10.0 A possible explanation for this

is 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 exposure groups, acclimation had a positive effect up to pH 9.0 but a negative effect between 9.0 and 10.0 Two different uptake models were applied to describe the observed variation in metal uptake With a non-linear, semi-empirical model, the integration of the different pH effects for the pooled data described no more than 38% of the total variation in cadmium uptake and 36% of the total variation in zinc uptake by midge larvae When the model was fitted to the uptake data of larvae acclimated to the exposure conditions, 78 and 69% respectively of the variation was described The second model, a biological ligand model, was not able to discriminate between effects of pH in acclimated and non-acclimated exposure groups Only for the data of larvae acclimated to the exposure conditions the

model could describe a significant amount of the observed variation in metal uptake, R2values being comparable to those of the first model The remaining high undescribed variation could be ascribed to the high natural variation in metal uptake by midge larvae © 2000 Elsevier Science B.V All rights reserved

Keywords:Uptake; pH effects; Chironomus riparius; Cadmium; Zinc

1 Introduction

The bioavailability of trace metals to aquatic organisms largely depends on the speciation of the metals in the solution (Campbell and Stokes,

* Corresponding author Tel.: + 2180349; fax: +

32-3-2180497.

E-mail address: bervoets@ruca.ua.ac.be (L Bervoets)

0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V All rights reserved.

PII: S 0 1 6 6 - 4 4 5 X ( 9 9 ) 0 0 0 6 6 - 1

1985; Campbell, 1995) Earlier studies have

shown that the bioavailability of cadmium and

zinc from solutions is the function of the free

metal ion activity which is the most prevalent

species in freshwater (Sunda et al., 1978; Engel

and Flower, 1979; Allen et al., 1980; De Lisle and

Roberts, 1988; Blust et al., 1992) One of the

most important environmental factors, which

infl-uences the bioavailability of metals to aquatic

organisms, is the pH of the solution In several

studies an increase in uptake or toxicity of certain

metals with increasing pH was observed in a

variety 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 some

other studies or for other metals an increased

uptake or toxicity of metals was observed with

decreasing 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 partitioning

of many metals between the sediment and the

aqueous phase and will alter the speciation of the

metals in the water Acidification generally will

result in an increased metal transfer from the

solid to the liquid phase with higher free metal

ion concentrations in the water (Palawski et al.,

1989; Odin et al., 1995; Lucan-Bouche´ et al.,

1997a,b; Playle, 1998) However, decreasing pH

also results in an increasing amount of competing

ions, i.e hydrogen ions, for the same binding

sites As a consequence, pH may influence the

uptake of metals in two antagonistic ways A

decrease in pH will result in an increase in free

cadmium 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 Chironomus

riparius can be found in both lentic (e.g Parma

and Krebs, 1977; Jernelo¨v et al., 1981) and lotic environments (e.g Bendell-Young and Harvey, 1991; Timmermans et al., 1992; Postma et al., 1995) Chironomid larvae can be found in waters with very low pH conditions (Jernelo¨v

et al., 1981; Bervoets et al., 1994; Cranston et al.,

1997), and the species C riparius can tolerate

pH of less than 4 (Jernelo¨v et al., 1981; Lohner and Fisher, 1990; Bruner and Fisher, 1993) and

pH of more than 10 (Bervoets, unpublished data)

Since pH has a combined effect on both chemi-cal and biologichemi-cal processes it was the aim of this study to determine the separate and com-bined effect of these processes on metal uptake The effect of changing pH conditions on the cadmium and zinc uptake by fourth instar larvae

of the midge C riparius (Meigen) (Diptera,

Chi-ronomidae) was studied, in relation to the accli-mation conditions (biological effect) and the free metal ion activity (chemical effect) In these ex-periments only exposure via the water was con-sidered

2 Materials and methods

2.1 Test organism Egg ropes of the midge C riparius (Meigen)

used in the experiments were obtained from a controlled laboratory culture at the Royal Bel-gian Institute for Natural Sciences (KBIN, Brus-sels, Belgium) Larvae were cultured in 10-l plastic 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 a climate chamber and fed with a suspension of ground commercial fish food (TetraMin®, Melle, Germany) (Vermeulen et al., 1997) Culture water was replaced weekly When the fourth larval stage (instar 4) was reached the larvae were placed at 15°C in the dark and held in aquaria at high 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)

2.2 Experimental procedures

In the culture, and in all acclimation and

exper-imental conditions the medium was artificial

River Water (RW) The composition of 1 l of this

chemically defined freshwater was 0.096 g

NaHCO3, 0.004 g KCl, 0.123 g MgSO4.7H2O and

0.06 g CaSO4.2H2O, resulting in a pH of 7.8 at

room temperature The media were prepared by

dissolving the analytical grade reagents (Merck

p.a.) in deionized water The solutions were

aer-ated for at least 24 h before the experiments were

started, to promote equilibration with the

atmo-sphere Dissolved oxygen was measured with a

polarographic electrode system (WTW OXI91/

EO90) and hydrogen ion activity with a glass

electrode (Ingold 104573002)

Stocks of cadmium and zinc, containing 100

mM Cd and 1000 mM Zn, were prepared The

radioisotopes109Cd and65Zn (Amersham

Interna-tional, UK) were used as tracers, 46.2 MBq/l of

each tracer being added to the metal stock

solu-tions In all experimental exposure solutions the

resulting metal concentrations were 0.1 mM Cd

and 1 mM Zn These concentrations were chosen

because of their environmental relevance The

re-sulting radioactivity of both tracers was 46.2

KBq/l

Six days before an experiment was performed,

larvae were collected from the culture and

accli-mated to four different pH values, i.e pHaccl 5.5,

7.0, 8.0 and 10.0 Solutions were adjusted to the

desired test pH using analytical-grade HCl or

NaOH The pH during the acclimation period

was controlled using a pH-stat system (Consort,

Belgium) With this system, pH and temperature

were controlled continuously Water pH generally

drifted from the target value by B0.3 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 was

not possible to maintain a pH of 10.0 For

sim-plification purposes, pHaccl values will be referred

to as pH 5.5, 7.0, 8.0, and 9.5, respectively All

larvae were of the same age and came from the

same batch culture, and at the end of the

acclima-tion periods larvae from all acclimaacclima-tion groups

were fourth instars and body weight did not differ

significantly among groups This indicates that at

the start of the experiments the condition of the test organisms was equal among all acclimation groups

For all experiments, 50 midge larvae of com-parable size were placed in a series of plastic vessels containing 50 ml test solution These ves-sels were placed in a thermostatic water bath at 15°C Both cadmium and zinc uptake by the chironomid larvae were linear over time for at least 8 h during exposure to a total concentration

of 0.1 mM Cd and 1 mM Zn (Bervoets, 1996) Therefore accumulation was measured after 6 h of exposure After exposure, the 50 individuals were collected on a 250 mm sieve and rinsed with 50 ml

of deionized water (Baudin and Nucho, 1992) For each treatment group four to eight replicates were taken

In a preliminary experiment the effect of rinsing with deionized water was compared to rinsing with a solution of 1 mM of 8-hydroxyquinoline-5-sulfonic acid, a strong ligand that has been used

to remove cadmium bound to the external sur-faces of brine shrimp (Blust et al., 1995) Both solutions removed the same amount of cadmium and zinc so that rinsing with deionised water suffices to remove metals adsorbed to the external surfaces Larvae were blotted dry and in groups

of 50 transferred to counting vials for gamma spectrometry

The radioactivity of the samples was measured

in a Minaxi-Auto-gamma 5530 spectrometer fitted with a thallium-activated sodium iodine well crys-tal (Canberra Packard) Sample counts were cor-rected for background and the corresponding cadmium and zinc activities were calculated using the following equation:

Muptake2 + = ACTmidge

60.CE.Wmidge.t.SA

in which M2 +

uptake is the cadmium or zinc uptake, ACTmidge is the 65Zn or 109Cd activity of the larvae after correction for background radiation (counts/min), CE is the counting efficiency (CPM/

0.178 for Zn and CPM/0.575 for Cd), Wmidge is

the dry weight of the larvae (g), t is the incubation

time (h) and SA is the specific activity of the water (46.2 Bq 65Zn/nmol total Zn and 462 Bq

109Cd/nmol Cd) The counted larvae were dried

for 24 h at 60°C and weighed on a Mettler H54

balance to the nearest 0.1 mg The cadmium and

zinc uptake was expressed on a dry weight basis

in nmol/g

To determine the effect of the pH of exposure

and acclimation on metal uptake, all acclimation

groups were exposed to metal containing

solu-tions of six different pH, i.e 5.5, 6.0, 7.0, 8.0, 9.0

and 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 acid

pKa= 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 control

the pH at 8.0, and CHES

(2-(N-cyclohexy-lamino)ethane-sulfonic acid, pKa= 9.3) was used

to 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 the concentration of 10 mmol l− 1of buffer that was used to buffer the solutions (Good et al., 1966) Solutions were further adjusted to the desired test

pH using analytical-grade HCl or NaOH The dissolved oxygen concentration and pH were mea-sured at the beginning and the end of each exper-iment Generally, all measured oxygen values remained within 10% of the initial values, and differences in pH before and after the experiments wereB0.1 pH unit Cadmium and zinc in the experimental solutions were measured by an axial inductively coupled plasma atomic emission spec-trometer (ICP-AES, Liberty Series II, Varian) Metal levels in filtered (through a membrane filter 0.22 mm pore size (Acrodisc®, Gelman)) and unfiltered samples were compared

2.3 Chemical speciation modelling

The equilibrium concentrations of the chemical species considered were calculated using the com-puter program SOLUTION (Blust and Van Gin-neken 1998), an adaptation of the program COMPLEX (Ginzburg, 1976) This speciation model allows the calculation of the composition

of solutions in equilibrium with the atmosphere

A thermodynamic stability data base for zinc and cadmium was built based on the data of Smith and Martell (1976), Martell and Smith (1982) and Smith and Martell (1989) The thermodynamic and conditional stability constants for the most prevalent cadmium and zinc species considered in the chemical speciation model are given in Table

1 Case specific input comprises the total concen-trations of the metals and ligands in the solution, the free hydrogen ion concentration (pH), redox potential (pE), temperature, and the gas phase that is maintained in equilibrium with the solu-tion Results of the chemical speciation calcula-tions are expressed on the molar concentration scale Activities were obtained by multiplying the concentrations of the chemical species with the appropriate activity coefficients Activity coeffi-cients were calculated using the estimation method of Helgeson (Birkett et al., 1988)

Table 1

Thermodynamic and conditional stability constants for the

cadmium and zinc species considered in the chemical

specia-tion model a

Log Q Log K

Species

CdCl2

1.33 1.47

CdCl42−

2.11

3.10 3.44

Cd(SO4)22−

4.35

4.82 4.99

ZnOH +

10.20

13.65 13.90

Zn(OH)3−

15.34 15.50

Zn(OH) 42−

0.36 0.53

ZnCl +

ZnCl 2

ZnCl42−

1.98 2.32

ZnSO4

3.26

Zn(SO4) 3

4.76

aK = thermodynamic stability constant; Q = conditional

stability constant, valid at the calculated freshwater ionic

strength of 0.009 M.

Fig 1 Metal speciation in function of pH (t° = 15°C) A,

cadmium; B, zinc.

methods used are outlined in Sokal and Rohlf (1981)

3 Results

3.1 Chemical speciation

In Fig 1A and B the results of the model calculations in function of pH are summarised for respectively cadmium and zinc For cadmium the free metal ion activities remain nearly constant over the pH range 5.5 – 8.0 (decreasing from 67.6

to 63.2 nM) Between a pH 8.0 and 10.0 the free cadmium ion activities drop from 63.2 to 0.11

nM At pH of 9.0 however free cadmium ion activity is still 10.1 nM For zinc the free metal ion activities remain constant over a narrower pH range i.e 5.5 to 7.4, decreasing from 702 to 685

nM Between a pH 7.4 and 10.0 the free zinc activities drop from 685 to 0.19 nM At the exposure pH of 8.0 and 9.0 the free zinc ion activities are respectively, 497 and 12.6 nM

In all experimental solutions the measured total metal concentrations were 0.11 (90.01) mM Cd and 1.07 (90.03) mM Zn No significant differ-ences were measured between filtered and unfiltered samples, indicating that precipitation of certain metal species (e.g CdCO3, ZnCO3) was not significant

3.2 Effect of pH on metal uptake

The effect of pH on metal uptake was com-pared for four different pH acclimation groups (pHaccl) which were exposed to six different pH values (pHexp) This made it possible to separate the 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 pH

of exposure on Cd uptake in the different acclima-tion groups Within each acclimaacclima-tion group cad-mium uptake increases with increasing pH of exposure with the exception of pHexp 10.0 in the acclimation groups pHaccl 5.5, 8.0 and 9.5 In the acclimation groups pHaccl 5.5 and pHaccl 8.0 no significant difference between uptake at pHexp9.0 and 10.0 was observed (pH 5.5: t = 0.24, df 5,

2.4 Statistical analysis

Analysis of variance and non-linear regressions

were used to analyse the data All data were

tested 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:

* P 50.05; ** P50.01; *** P50.001 Statistical

Fig 2 Uptake of cadmium by midge larvae in function of

exposure pH for the different pH acclimation groups

(Cdtotal= 0.1 mmol l − 1 , temp 15 91°C) Means with standard

deviation are given.

Fig 3 Uptake of zinc by midge larvae in function of exposure

pH for the different pH acclimation groups (Zntotal= 1.0 mmol

l − 1 , temp 15 91°C) Means with standard deviation are given. 9.0 to 1.5 nmol g− 1 at pH1 10.0 was observed

(t = 3.06, df = 9, PB0.05) The highest increase

in cadmium uptake was measured in the pHaccl8.0 group, where the mean uptake increased from 1.4 nmol g− 1 at a pHexpof 5.5 to 4.3 nmol g− 1 at a

pHexpof 9.0 In all cases prior acclimation had a significant effect on the uptake of cadmium by the midge larvae, the highest uptake being observed

at the acclimation of pHaccl8.0 A two-way analy-sis of variance of the data showed that both the effect of the pH of exposure and the pH of acclimation on Cd uptake are highly significant (Table 2a)

Fig 3 shows the results of the effect of the pH

of exposure on zinc uptake in the different accli-mation groups Generally, the results were similar

to those for Cd In acclimation group pHaccl 5.5,

no significant differences in zinc uptake at the different pH of exposure were observed In the other acclimation groups zinc uptake increases

P = 0.81; pHaccl8 t = 0.72, df 16, P = 0.50) and at

acclimation group pHaccl 9.5, a significant

de-crease in Cd uptake from 3.2 nmol g− 1at pHexp

Table 2

Two-way analysis of variance for the effect of pH of exposure

and the pH of acclimation on metal uptake by midge larvae

(24 treatment groups with four replicates)

Fs

Mean of squares Source of variation df

(a)Cadmium uptake

(b) Zinc uptake

9.74*

7133 5 Acclimation pH

a ns, not significant;

* P50.001.

with increasing pH of exposure, with a decrease in

uptake at pHexp 10.0 for the acclimation groups

pHaccl 8.0 and 9.5 In the latter cases a significant

decrease was measured (pHaccl8 t = 3.39, df = 17,

PB0.005; pHaccl 10 t = 2.75, df = 9, PB0.05)

Again the highest increase in zinc uptake was

measured in the pHaccl 8.0 group, where the

mean uptake increased from 15.7 nmol/g at an

exposure of 5.5 – 19.0 nmol/g at an exposure pHexp

of 9.0 As for cadmium, prior acclimation had a

significant effect on the uptake of zinc by the

midge larvae, the highest uptake being observed

at the pHaccl 8.0

Two-way analysis of variance showed that both

the effect of the pH of exposure and the pH of

acclimation on Zn uptake are highly significant

(Table 2b) The combined effect is highly

signifi-cant as well

In many cases the variation in metal uptake

within the exposure groups was high to very high

Relative standard deviations within groups of up

to 58% for zinc uptake and up to 67% for

cad-mium uptake were calculated

3.3 Modelling metal uptake

To determine the relative importance of the

different factors contributing to the variation in

metal uptake by the midge larvae, two different

models to describe the observed variation in metal

uptake were compared:

3.3.1 Empirical model

An empirical non-linear regression model was

constructed (Blust et al., 1991, 1992, 1994;

Bervoets et al., 1996a) Metal uptake was related

to the product of three nth-power terms that

describe the effect of the change in the free metal

ion activity (Mact), pH of exposure (pHexp) and

pH of acclimation (pHaccl) on metal uptake A

coefficient of proportionality (Cf) was introduced

to relate the activity of the metal ion in the

solution, to the metal uptake by the midge larvae

The equation for both metals becomes:

Meupt= C *(Mef k act *pH l

exp*pH m

accl) The relative importance of the different terms

was 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 sole independent variable and stepwise adding the other terms to evaluate whether their contribution

to the amount of variation described was significant

3.3.2 The biological ligand model

This semi-empirical model considers the organ-ism as another ligand with metal ions and protons

competing for the same biological uptake site (X)

(Hare and Tessier, 1996; Croteau et al., 1998; Playle, 1998):

Me2 ++ X = XMe;K MeX = [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 the equilibrium constants in Eq (1) and (2) and as-suming that only a small fraction of the sites is occupied by Cd or Zn (i.e [XMe]B B[X]T), gives

[XMe] = (K MeX K HX [X]T/H++ KHX) [Me2 +] (4)

If it is assumed that metals taken up by C.

riparius is proportional to [

Meupt[ (4) gives:

Meupt= F([Me2 +]/(H++ Ka)) (5)

Where F( = kKKa[X] T) is a constant specific to

C riparius.

Table 3 gives the results of the non-linear re-gression analysis for cadmium uptake by midge larvae Relating cadmium uptake to the free cad-mium ion activity describes only 6% of the total variation in cadmium uptake When the term was added which accounts for the effect of the pH of exposure (pHexp), 26% of the variation was de-scribed Adding the term which accounts for the effect of the pH of acclimation (pHaccl) described 38% of the variation in cadmium uptake Consid-ering only the results of the cadmium uptake experiments performed at the pH of acclimation (i.e pH of exposure = pH of acclimation) 78% of the variation in cadmium uptake was described

Considering other cadmium species as bioavailable

and including them in the uptake model did not

increase the amount of variation described

Table 4 gives the results of the non-linear regres-sion analysis for the zinc uptake by midge larvae using the empirical model Relating zinc uptake to the free zinc ion activity, almost none of the observed variation in zinc uptake could be de-scribed When the term was added which accounts for the effect of pH of exposure, 24% of the variation was described Adding the term which accounts 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 experiments performed at the pH of acclimation describes 64%

of the variation in zinc uptake Considering other zinc species as bioavailable and including them in the uptake model did not increase the amount of variation described

With the semi-empirical model it was not possi-ble to describe any of the variation in metal uptake using the pooled data for either cadmium or zinc Considering only the results of the metal uptake experiments performed at the pH of acclimation 79% of the variation in cadmium uptake and 68%

of the variation in zinc uptake was described Calculated values of F were 0.66590.116 and 2.8190.66 nmol/g, for cadmium and zinc,

respec-tively and Ka values were 4.4891.94 10− 5 and 2.3891.19 10− 5 m for both cadmium and zinc uptake (means9S.E.)

Fig 4A and B summarise the results of the metal uptake by midge larvae exposed to the pH of acclimation for respectively cadmium and zinc For cadmium significant differences were found among the different uptake groups (ANOVA: F3,17= 23.7,

PB0.001) With a Duncan post hoc test it was shown that all groups differed significantly from

each other (PB0.001) with the exception of pH 7.0 compared to pH 5.5 Also for zinc significant differences were found among the different uptake groups (ANOVA: F3,17= 15.1, PB0.001) With a

Duncan post hoc test it was shown that group pH

8 differed significantly from all other groups (PB 0.001) and the other groups differed significantly

only from group pH 8.0 (PB0.001)

4 Discussion

In this study the effect of pH on the uptake of

Table 3

Cadmium uptake by C riparius: non-linear regression model

for the pooled data a

(1) Cdupt= Cf *(Cdactk ) (R2= 0.06**, n = 154)

0.535 0.179

0.178 0.357*

Coefficient

−0.088***

(2) Cdupt= Cf *(Cdactk pH expl ) (R2= 0.26***, n = 154)

Coefficient 0.011 c

0.128*** 0.045

2.889 4.305

l-exponent 3.597*** 0.708

(3) Cdupt= Cf *(Cdactk pH expl pH acllm ) (R2= 0.38***, n = 154)

Coefficient 0.001 b

2.943

3.552***

l-exponent

1.165

a B: partial regression coefficients; SE: standard error for

partial regression coefficients; L1, L2: confidence limits for

partial regression coefficients

b

Cadmium uptake in midge larvae in nmol/g.

c ns, not significant;

* P50.05;

*** P50.001

Table 4

Zinc uptake by C riparius: non-linear regression model for the

pooled data a

(1) Znupt= Cf *(Znactk ) (R2B0.01ns, n=154)

Coefficient 30.02* 15.04 14.98 45.06

−0.008 ns

k-exponent

(2) Znupt= Cf *(Znactk pH exp

l ) (R2

= 0.24***, n = 154)

Coefficient 0.135 ns

0.222*** 0.048

4.480*** 0.889 3.591

(3) Znupt= Cf *(Znactk pH expl pH acllm ) (R2= 0.36***, n = 154)

0.517 ns

Coefficient

0.228*** 0.042

a B: partial regression coefficients; SE: standard error for

partial regression coefficients; L1, L2: confidence limits for

partial regression coefficients Zinc uptake in midge larvae in

nmol/g nsnot significant; * P 50.05; *** P50.001

Fig 4 Metal uptake rate by C riparius at the pH of

acclima-tion (Cdtotal= 0.1 mmol l − 1 , temp 15 91°C) Means with

standard deviation are given (A) Cadmium; (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 of

exposure

Generally the free metal ion is considered as the biologically most available species For both metals the free ion activity remains nearly con-stant between 5.5 and 8.0 and decreases from 8.0

to 10.0, reaching very low levels at this pH When metal uptake was related to the free metal ion activity, a negligible part of the variation in up-take could be described Most likely this is the result of the combined effect of pH on metal speciation (decreasing free metal ion activity with increasing pH) and on the competition between protons and metal ions for the same uptake sites

In all cases the uptake of both metals increases with increasing exposure pH with the exception

of pH 9.0 and 10.0 In most cases, metal uptake even decreased at pH 10.0 compared to uptake at

pH 9.0 In the pH range 5.5 – 9.0 our results agree with findings for other aquatic organisms exposed

to cadmium or zinc Schubauer-Berigan et al (1993) found an increase of the toxicity of Cd and Zn with increasing water pH (pH 6.3, 7.3 and 8.3) for three aquatic invertebrate species The same trend in toxicity was found by Cusiamo

et 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 with increasing pH for all tested metals These findings are consistent with theoretical considerations A hypotheses put forward in literature is that the free metal ions (i.e Cd2 + and Zn2 +) are in competition with the hydrogen ions at the mem-brane level and therefore restrict uptake under acid 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 2.79mM

at pH 5.5 – 0.07 nM at pH 10.0

We could find in the literature only one study where organisms were exposed to pH higher than 9.0 in combination with metals (Belanger and Cherry, 1990) In that study impaired

reproduc-tion and mortality of Ceriodaphnia dubia was

observed below pH 6 and above pH 9 when daphnids were exposed to pH only However

cadmium and zinc by larvae of the midge C.

riparius was examined using chemically defined

solutions At least three factors are important in

determining the effect of pH on cadmium and

zinc uptake by midge larvae The effect of pH on

metal uptake is the combined result of (1)

changes in the free metal ion activity: this

deter-mines the fraction of the metal in solution which

is available for uptake, (2) changes in pH of

exposure and (3) changes in pH of acclimation

These two latter factors influence the permeability

of the exchange surfaces for metal ions and other

physiological processes

when the organisms were exposed to zinc and

copper at pH 6, 8 and 9 an inverse relationship

between pH and effect was observed, regardless of

acclimation conditions

The decreased uptake at pH 10.0 in our study

probably is the result of the decrease in metal ion

activity of both cadmium and zinc Although

metal ion activities were very low at pH 10.0 (0.11

and 0.19 nM, respectively for cadmium and zinc)

uptake is still relatively high An explanation for

this relative high metal uptake at pH 10.0 might

be that an increase in pH alters the metal uptake

process by decreasing the protonation of the

bind-ing sites That is, the biological availability of the

free metal ion increases with increasing pH

Another possible explanation could be that one

or more of the inorganic metal species, which are

dominant at the highest pH, are available to the

midge larvae However, adding these species in

the uptake model, could not increase the

de-scribed variation in metal uptake Moreover it is

unlikely that the carbonate species are available to

aquatic organisms (Blust et al., 1991; Campbell,

1995)

4.2 Effect of acclimation

The effect of pH on the uptake of metals by the

midge larvae is not only determined by the effect

on chemical speciation but also by physiological

effects At all exposure conditions acclimation

had a remarkable but inconsistent effect on

up-take of both metals The marked effect of

accli-mation on cadmium and zinc uptake by the midge

larvae is a strong indication that pH has not only

an effect on the speciation of the metals or

proto-nation of the binding sites but also alters the

physiological condition of an organism and thus

indirectly 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 zinc

uptake (Bervoets et al., 1996b) A possible

hy-pothesis for the acclimation effect is a pH

depen-dent behaviour of the larval C riparius Wildi et

al (1994) found an increase in larval mucus

secre-tion at lower pH, which could result in a retarded

diffusion of the metals along the concentration

gradient Another possibility is an effect of pH on respiration Alibone and Fair (1981) observed an

increase of respiration rate in Daphnia magna with

increasing pH No behavioural or physiological data in literature were found on the effect of pH higher that 9.0

4.3 Modelling metal uptake

With the empirical non-linear model for the pooled data no more than 26% and 24% of the variation in cadmium and zinc respectively uptake could be described An increase of described vari-ation up to 38% and 36% respectively was ob-served when the factor that accounts for pH of acclimation was added The high proportion of undescribed variation is largely due to the natural variation in metal uptake by the midge larvae Also in other studies on cadmium uptake by midge larvae, a high variation in metal uptake within a treatment was observed (Seidman et al., 1986; Timmermans et al., 1992; Bervoets et al.,

1995, 1996a) Moreover, when the non-linear up-take models were fitted to the mean upup-take values

up to 63% of the cadmium uptake and 54% of the zinc uptake could be described

Another possible explanation for the high pro-portion of undescribed variation is that the pH of acclimation has an inconsistent effect on metal uptake From the modelling of the metal uptake it was obvious that pH of acclimation had a positive effect on the metal uptake (Table 3, Table 4) with

a coefficient of 1.51 for cadmium and 1.61 for zinc However, metal uptake increases with in-creasing pH of acclimation between pHaccl5.5 and 8.0 and decreases at pHacclof 10.0 in all exposure groups and for both metals When using only the data of larvae acclimated to the exposure condi-tions it was possible to describe a relatively high proportion of the variation in metal uptake (78 and 64% of the variation, respectively for cad-mium and zinc uptake)

With the empirical non-linear model it was not possible to take into account the non-consistent effect of metal uptake in function of pH However with the model of Hare and Tessier (1996) it was not possible to describe any of the observed varia-tion in metal uptake using the pooled data This is