Effects of ph on cd and zn uptake by plant

Aquatic Toxicology 49 (2000) 145–157
Effects of pH on cadmium and zinc uptake by the midge
larvae Chironomus riparius
L. Bervoets *, R. Blust
Department of Biology, Uni6ersity of Antwerp
(
RUCA
)
, Groenenborgerlaan
171
,
2020
Antwerp, 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, R
2
values 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
www.elsevier.com/locate/aquatox
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.: +32-3-2180349; fax: +32-3-
2180497.
E-mail address
:
(L. Bervoets)
0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S0166-445X(99)00066-1
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
146
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 biological 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 cm
2

) 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 Toxicology
49 (2000) 145 – 157
147
2
.
2
. Experimental procedures
In the culture, and in all acclimation and exper-
imental conditions the medium was artificial
River Water (RW). The composition of1lofthis
chemically defined freshwater was 0.096 g
NaHCO
3
, 0.004 g KCl, 0.123 g MgSO
4
.7H
2
O and
0.06 g CaSO
4
.2H
2
O, 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
radioisotopes
109
Cd and
65
Zn (Amersham Interna-
tional, UK) were used as tracers, 46.2 MBq/lof
each tracer being added to the metal stock solu-
tions. In all experimental exposure solutions the
resulting metal concentrations were 0.1 mMCd
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. pH
accl
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 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 was
not possible to maintain a pH of 10.0. For sim-
plification purposes, pH
accl
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 acclimation 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 after6hof
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:
M
uptake
2+
=
ACT
midge
60.CE.W

midge.t.SA
in which M
2+
uptake
is the cadmium or zinc uptake,
ACT
midge
is the
65
Zn or
109
Cd 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), W
midge
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
65
Zn/nmol total Zn and 462 Bq
109
Cd/nmol Cd). The counted larvae were dried
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
148
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, pK
a
=
6.1) was used to control the pH at 5.5 and 6.0;
MOPS (3-(N-Morpholino)propanesulfonic acid
pK
a
=7.2) was used to control the pH at 7.0;
EPPS (N-(2-Hydroxyethyl)piperazine-N%-(3-pro-
panesulfonic acid), pK
a
=8.0) was used to control
the pH at 8.0, and CHES (2-(N-cyclohexy-
lamino)ethane-sulfonic acid, pK
a
=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
−1

of 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
wereB 0.1 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 QLog KSpecies
CdOH
+
3.91 3.74
Cd(OH)
2
0

7.64 7.38
Cd(OH)
3
−
8.68 8.42
CdCl
+
1.97 1.80
2.59 2.34CdCl
2
0
CdCl
3
−
2.40 2.14
1.331.47CdCl
4
2−
2.11CdSO
4
0
2.45
3.103.44Cd(SO
4
)
2
2−
4.35CdCO
3
0

4.01
4.824.99ZnOH
+
10.20Zn(OH)
2
0
9.94
13.6513.90Zn(OH)
3
−
15.3415.50Zn(OH)
4
2−
0.360.53ZnCl
+
0.69 0.43ZnCl
2
0
ZnCl
3
−
0.450.70
0.32 0.15ZnCl
4
2−
1.982.32ZnSO
4
0
3.26Zn(SO
4

)
2
2−
2.92
Zn(SO
4
)
3
4−
2.032.03
4.76ZnCO
3
0
5.10
ZnHCO
3
+
11.03 10.69
a
K=thermodynamic stability constant; Q=conditional
stability constant, valid at the calculated freshwater ionic
strength of 0.009 M.
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
149
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 (9 0.01) mMCd
and 1.07 (9 0.03) mM Zn. No significant differ-
ences were measured between filtered and
unfiltered samples, indicating that precipitation of
certain metal species (e.g. CdCO
3
0
, ZnCO

3
0
) 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
(pH
accl
) which were exposed to six different pH
values (pH
exp
). 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 acclimation group cad-
mium uptake increases with increasing pH of
exposure with the exception of pH
exp
10.0 in the
acclimation groups pH
accl
5.5, 8.0 and 9.5. In the
acclimation groups pH
accl

5.5 and pH
accl
8.0 no
significant difference between uptake at pH
exp
9.0
and 10.0 was observed (pH
accl
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:
* P5 0.05; ** P5 0.01; *** P5 0.001. Statistical
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
150
Fig. 2. Uptake of cadmium by midge larvae in function of
exposure pH for the different pH acclimation groups
(Cd
total

=0.1 mmol l
−1
, temp 1591°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 (Zn
total
=1.0 mmol
l
−1
, temp 159 1°C). Means with standard deviation are given.
9.0 to 1.5 nmol g
−1
at pH
1
10.0 was observed
(t= 3.06, df =9, PB 0.05). The highest increase
in cadmium uptake was measured in the pH
accl
8.0
group, where the mean uptake increased from 1.4
nmol g
−1
atapH
exp
of 5.5 to 4.3 nmol g
−1
at a
pH
exp

of 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 pH
accl
8.0. 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 pH
accl
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; pH
accl
8 t= 0.72, df 16, P =0.50) and at
acclimation group pH
accl
9.5, a significant de-
crease in Cd uptake from 3.2 nmol g
−1
at 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)
F
s
Mean of squaresSource of variation df
(a)Cadmium uptake
Exposure pH 45.613 33.81*
Acclimation pH 5 18.22 13.51*
Interaction 15 1.73 1.29
a
(b) Zinc uptake
Exposure pH 3 20257 27.65*
9.74*71335Acclimation pH
Interaction 2.85*208815
a
ns, not significant;
* P50.001.
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
151
with increasing pH of exposure, with a decrease in
uptake at pH
exp
10.0 for the acclimation groups
pH
accl
8.0 and 9.5. In the latter cases a significant
decrease was measured (pH

accl
8 t= 3.39, df=17,
PB 0.005; pH
accl
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/gatan
exposure of 5.5 –19.0 nmol/g at an exposure pH
exp
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 pH
accl
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 (M
act
), pH of exposure (pH
exp
) and
pH of acclimation (pH
accl
) on metal uptake. A
coefficient of proportionality (C
f
) 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:

Me
upt
=C
f
*(Me
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):
Me
2+
+X =XMe;K
MeX
=[XMe]/[Me
2+
][X] (1)
XH= H
+
+X;K
a
=[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]BB[X]
T
), gives
[XMe]= (K
MeX
K
HX
[X]
T

/H
+
+K
HX
) [Me
2+
] (4)
If it is assumed that metals taken up by C.
riparius is proportional to [  XMe], that is
Me
upt
[ XMe], combining this relation with Eq.
(4) gives:
Me
upt
=F([Me
2+
]/(H
+
+K
a
)) (5)
Where F(=kKK
a
[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 (pH
exp
), 26% of the variation was de-
scribed. Adding the term which accounts for the
effect of the pH of acclimation (pH
accl
) 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.
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
152
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 0.6659 0.116 and
2.819 0.66 nmol/g, for cadmium and zinc, respec-
tively and K
a
values were 4.4891.94 10
−5
and
2.389 1.19 10
−5
m for both cadmium and zinc
uptake (means9 S.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: F
3,17
=23.7,
PB 0.001). With a Duncan post hoc test it was
shown that all groups differed significantly from
each other (PB 0.001) 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: F
3,17
=15.1, PB 0.001). 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 (P B 0.001).
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
BSEL1Variable L2
(1) Cd
upt
=C
f
*(Cd
act

k
)(R
2
=0.06**, n=154)
0.5350.1790.1780.357*Coefficient
−0.088***k-exponent −0.1140.026 −0.062
(2) Cd
upt
=C
f
*(Cd
act
k
pH
exp
l
)(R
2
=0.26***, n=154)
Coefficient 0.011
c
0.128*** 0.045k-exponent 0.083 0.173
2.889 4.305l-exponent 3.597*** 0.708
(3) Cd
upt
=C
f
*(Cd
act
k

pH
exp
l
pH
acll
m
)(R
2
=0.38***, n=154)
Coefficient 0.001
b
k-exponent 0.1850.1050.0400.145***
2.9430.609 4.1613.552***l-exponent
1.165m-exponent 1.8551.510*** 0.345
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
Variable L
2

L
1
SEB
(1) Zn
upt
=C
f
*(Zn
act
k
)(R
2
B0.01ns, n=154)
Coefficient 30.02* 15.04 14.98 45.06
−0.008
ns
k-exponent
(2) Zn
upt
=C
f
*(Zn
act
k
pH
exp
l
)(R
2
=0.24***, n=154)

Coefficient 0.135
ns
0.222*** 0.048k-exponent 0.174 0.270
4.480*** 0.889 3.591l-exponent 5.369
(3) Zn
upt
=C
f
*(Zn
act
k
pH
exp
l
pH
acll
m
)(R
2
=0.36***, n=154)
0.517
ns
Coefficient
0.228*** 0.042k-exponent 0.186 0.270
l-exponent 4.476*** 0.774 3.702 5.250
m-exponent 1.609*** 0.391 1.218 2.000
a
B: partial regression coefficients; SE: standard error for
partial regression coefficients; L
1

,L
2
: confidence limits for
partial regression coefficients. Zinc uptake in midge larvae in
nmol/g.
ns
not significant; * P50.05; *** P50.001
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
153
Fig. 4. Metal uptake rate by C. riparius at the pH of acclima-
tion (Cd
total
=0.1 mmol l
−1
, temp 1591°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. Cd
2+
and Zn
2+
) 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 2.79 mM
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.
L. Ber6oets, R. Blust
/
Aquatic Toxicology

49 (2000) 145 – 157
154
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 uptake 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 pH
accl
5.5 and
8.0 and decreases at pH

accl
of 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
L. Ber6oets, R. Blust
/
Aquatic Toxicology
49 (2000) 145 – 157
155
probably due to the fact that the model does not
discriminate between effects of pH in acclimated
and non-acclimated exposure groups. This be-
came clear when only the data of uptake at the
pH of acclimation were included in the model. As
with 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 complex
than a simple competition between metal ions
and protons as implied in the biological ligand
model.
Although the described variation increases for
both models when only the acclimated larvae are
considered, still more than 20 and 30% of the
variation in uptake of respectively cadmium and
zinc remains undescribed. As stated before, this is
mainly due to the high natural variation in metal
uptake, especially apparent in short term uptake
studies and at low environmental metal levels.
Evidence for this was found by calculating, using
Analysis of Variance, the relative magnitudes of
the variance components (Sokal and Rohlf,
1981). For the data where pH
accl
=pH
exp
we cal-
culated that 19.2 and 28.9% of the variation for
respectively cadmium and zinc uptake can be
ascribed to the ‘whitin groups’ variation.
Acknowledgements
We thank A. Vermeulen of the KBIN-Brussels
for supply of the egg ropes of the midge larvae.
This work was sponsored by the Fund for Joint
Basic Research of Belgium (project 2.0127.94).
LB is a research fellow and RB a research associ-
ates of the National Fund for Scientific Research

Flanders.
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