ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 71 (2008) 765–773

www.elsevier.com/locate/ecoenv

Effects of interactions between algal densities and cadmium

concentrations on Ceriodaphnia dubia fecundity and survival
Suzelei Rodgher, Evaldo Luiz Gaeta Espı́ndola
São Carlos Engineering School, Water Resources and Applied Ecology Center, University of São Paulo,
Avenida Trabalhador São Carlense, 400, C.P 292, Cep 13.560-970 São Carlos, SP, Brazil
Received 8 November 2006; received in revised form 19 July 2007; accepted 24 August 2007
Available online 23 October 2007

Abstract

The influence of different densities of the algae Pseudokirchneriella subcapitata on the chronic toxicity of cadmium to Ceriodaphnia
dubia was investigated. The importance of algal cells as a source of metal to zooplankton was studied by exposing P. subcapitata cells to
free cadmium ions and supplying the algae as food to C. dubia. The results of a bifactorial analysis (metal versus food levels) showed
that metal toxicity to zooplankton was dependent on food level. Significant toxic effects on the fecundity and survival of C. dubia
were observed at low metal concentrations with high algal density. Algae contaminated with Cd2+ were less toxic to cladoceran than
was the Cd2+ in solution. Green algae retained cadmium and released low metal concentration in the test medium. We concluded that
algal cells are an important route of exposure to metal and a factor that has an appreciable influence on the expression of metal toxicity
to daphnids.
r 2007 Elsevier Inc. All rights reserved.

Keywords: Ceriodaphnia dubia; Sources of metal toxicity; Pseudokirchneriella subcapitata; Chronic toxicity

1. Introduction substances by improving their nutritional state (Chandini,

Until a few years ago, feeding activity had been largely
ignored in research published on accumulation of heavy
metals and their bioavailability to aquatic invertebrates,
presumably because feeding activity was not considered an
important source of contamination and uptake of metals in
previous studies. Recently, many studies have focused on
the effects of metals on survival, growth and reproduction
of filter-feeding organisms submitted to a variety of food
conditions, both in the laboratory and in the field
(Reinfelder et al., 1998).
The food, represented by the algae, can act as a channel
of metal contamination to cladocerans (Hook and Fisher,
2002) and can counteract metal toxicity, due to the algae’s
ability to exude organic compounds that complex metals
(Lombardi and Vieira, 2000). On the other hand, the algae
contribute to the resistance of the organisms to toxic
Corresponding author. Fax: +55 16 33738251.
E-mail addresses: surodgher@uol.com.br (S. Rodgher),
elgaeta@sc.usp.br (E. Luiz Gaeta Espı́ndola).
1989). Since aquatic organisms in their natural environ-
ment have food available during toxic exposure, attention
should be given to the role of food in toxicity tests (Lanno
et al., 1989).
A new approach in ecotoxicological studies has focused
on the influence of food on the effects of toxic agents on
zooplankton. Weltens et al. (2000) verified that particles
(sand, clay, algae) contaminated with cadmium are
potentially toxic to Daphnia, not only by acting as a source
of dissolved metal, but also because the particle-bound
fraction of Cd can become free and available within the
body of the filter-feeding organism. Barata et al. (2002)
confirmed that although water was the major pathway of
cadmium uptake for D. magna, a substantial amount of the
metal was obtained by the test organisms from the
contaminated algae.
Dietary exposure to metal of zooplankton may occur
because the algae can adsorb and take up dissolved metal
from the exposure solution before being ingested by the
organisms. However, algae may eliminate metal to the
solution during the daphnids’ exposure, which might result

0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2007.08.012
 ARTICLE IN PRESS
766 S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
in significant exposure of organisms to metal via water. The
elimination of metals from food in dietary toxicity studies
is recognized as an important factor that may confuse the
interpretation of the effects of dietary exposure (De
Schamphelaere et al., 2004). In this light, the aim of this
study was to evaluate the effects of varying the density of
the algae Pseudokirchneriella subcapitata on the chronic
toxicity of cadmium to the cladoceran Ceriodaphnia dubia,
when this organism was exposed to this metal in the water
and in contaminated food (cells of P. subcapitata). In these
experiments, total dissolved cadmium concentrations and
cadmium accumulated by algal cells were measured and
free metal ions were estimated in order to distinguish
between the effects of aqueous and dietary exposures as
sources of metal toxicity to these organisms. Feeding rates
of C. dubia were also measured in both treatments.
2. Materials and methods
2.1. Culture of test organisms
Cells of P. subcapitata and neonates of C. dubia were obtained from
cultures maintained at the Laboratory of Ecotoxicology and Ecophysiol-
ogy of Aquatic Organisms of the Water Resources and Applied Ecology
Center (CRHEA) of the University of São Paulo (São Paulo State, Brazil).
Cultures of C. dubia were maintained in a temperature-controlled
chamber at 2571 1C with a 12:12 h light/dark cycle, using reconstituted
water as the culture medium (pH 7.2–7.6, conductivity 160 mS/cm and
hardness between 42 and 48 mg/L of CaCO3). The water was changed
every other day. Reconstituted water consisted of four inorganic salts
dissolved in deionized water. Twenty millilitres of a solution (0.01 M of
CaSO4.2H2O) and 10 mL of another solution (0.002 M of KCL, 0.06 M of
NaHCO3 and 0.04 M of MgSO4.7H2O) were added to deionized water to a
final volume of 1 L (ABNT, 2005a).
The green algae P. subcapitata were cultivated in L.C. Oligo medium
(AFNOR, 1980), which was first autoclaved (121 1C) for 15 min in 2-L
Erlenmeyer flasks containing 1 L of the medium. The composition of the
culture medium is described in Table 1. This was inoculated with cells to a
concentration of around 1  104 cells/mL and the culture was exposed to
100 mE/m2/s2 in 12:12 h light/dark cycle, with constant aeration, at
2472 1C (ABNT, 2005b). Algal cells from exponentially growing
Table 1
Composition of the culture medium L.C. Oligo (AFNOR, 1980) used in the present study
Stock solution Compound
1 Ca(NO3)2  4H2O
2 KNO3
3 MgSO4  7H2O
4 K2HPO4
5 CuSO4  5H2O
(NH4)6Mo7O24  4H2O
ZnSO4  7H2O
CoCl2  6H2O
Mn(NO3)2  4H2O
C6H8O2  H2O
H3BO3
6 C6H5FeO7  5H2O
FeSO4  7H2O
FeCl3  6H2O
7 NaHCO3
P. subcapitata cultures were used as food for the zooplankton, C. dubia,
which was fed every other day with 1  105 cells/mL of the algae and a
suspension of yeast and commercial fish food (Vitormonios), in
accordance with the procedures set in the Brazilian guidelines ABNT
(2005a).
2.2. Toxicity tests
2.2.1. Exposure of algae to metal
Cells of P. subcapitata in the exponential growth phase were exposed
for 96 h to ascending levels of total dissolved cadmium of 0, 0.63, 1.51,
2.93, 6.85 and 12.60  107 M, corresponding to free cadmium ion
concentrations of 0, 0.60, 1.40, 2.70, 6.32 and 11.60  107 M, respectively.
Free cadmium ions were estimated using the MINEQL+ model (Schecher
and McAvoy, 1991). The test solutions used in this experiment were
prepared in volumetric flasks using volumetric pipettes. The stock solution
was 8.9 mM Cd(NO3)2.4H2O (solution standard of nitrate of cadmium for
atomic absorption, J.T. Backer). The dilution water used in the
preparation of the test was LC Oligo medium.
After the exposure period, the cells were centrifuged at 1500 rpm for
15 min (FANEM Excelsa centrifuge, model 206 PM), washed three times
with reconstituted water and resuspended in the zooplankton culture
water (reconstituted water). Finally, suspensions of the cells were stored in
polythene bottles in the dark at 4 1C during the chronic toxicity test with
zooplankton (De Schamphelaere et al., 2004).
Samples (2 mL) from each test flask were taken and fixed with Lugol’s
iodine solution to determine the cell density, by counting cells in an
Improved Neubauer Bright-Line hemocytometer under optical micro-
scope (Carl Zeiss), standard model 25. The mean number of cells produced
at each concentration, after this exposure period, was expressed as a
percentage growth reduction with respect to the control. These
percentages were used to calculate the IC50 (effective metal concentration
giving 50% inhibition of algal growth after 96 h exposure). The dry weight
of the algal cells was obtained by filtering a known volume of cells on a
pre-weighed filter. The filters with algal cells were dried for 24 h at 60 1C
and weighed to determine the cell mass per volume of culture (APHA,
1995).
2.2.2. Chronic toxicity test
The test solutions for the chronic toxicity tests with the cladocerans
were prepared as in the experiments with the algae and the dilution water
used in these preparations was reconstituted water. Acute toxicity tests
demonstrated a median effect concentration (EC50, 48 h) of 5.34  107 M
Cd to C. dubia (Rodgher, 2005). Based on the result of the acute toxicity
Concentration (M) Volume (mL) required from stock
solution to 1 L of medium
0.24 1.00
0.99 1.00
0.20 1.00
0.23 1.00
0.00016 0.50
0.00005
0.0002
0.0001
0.0002
0.0005
0.001
0.005 0.50
0.002
0.002
0.2 1.00
 ARTICLE IN PRESS
S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
tests, we prepared five nominal sublethal metal concentrations. Each
successive nominal concentration was about twice the previous one (0.11,
0.22, 0.44, 0.89, 1.78  107 M Cd). Test solution samples from each
nominal metal concentration were filtered, acidified and analyzed by
graphite-furnace atomic absorption spectrometry to determine the
dissolved cadmium concentrations. Considering that free metal ions
constitute a metal fraction of higher bioavailability to organisms, we used
the MINEQL+ program to calculate Cd2+. The free ion cadmium
concentrations were then predicted from the total dissolved cadmium
concentrations. The total dissolved cadmium concentrations used in the
chronic toxicity tests with zooplankton were 0, 0.11, 0.19, 0.44, 0.87,
1.78  107 M, corresponding to free cadmium ion concentrations of 0,
0.10, 0.17, 0.40, 0.86, 1.60  107 M, respectively.
In the chronic toxicity tests, 10 neonates of C. dubia (aged less than
24 h) were exposed, in 10 separate test vessels, to 15 mL of a certain metal
concentration and fed with one of three densities of P. subcapitata (low,
medium and high: 1  104 cells/mL, 1  105 cells/mL and 1  106 cells/mL,
respectively). The organisms were transferred to a freshly prepared test
solution every other day and fed with the respective algal concentration
daily. The test media were gently stirred using a fine Pasteur pipette twice a
day to ensure availability of algal cells to the organisms. A control test
without the added metal was also prepared. The toxicity tests lasted seven
to eight days, the period needed for production of the third brood, and the
survival of adults and number of live neonates per female were recorded
(ABNT, 2005a).
Two controls were used: a laboratory control (LC) that contained the
food concentration normally used in the maintenance of the test organism
(1  105 cells/mL plus the suspension of yeast and fish food) and another
control (C), containing only the algae at the density being tested. This was
a way of testing the physiological condition of the organisms being used in
the tests. The chronic toxicity tests were considered valid when a survival
of 80% was observed in adult organisms, corresponding to the laboratory
control, and when they produced at least 15 neonates per female (ABNT,
2005a).
In another experiment, cells of P. subcapitata exposed to free cadmium
for 96 h were offered as food to zooplankton. The concentrations of metal
used to contaminate P. subcapitata were 0, 0.60, 1.40, 2.70  107 M
Cd2+. Neonates of C. dubia were exposed to reconstituted water and fed
with three different densities of metal-contaminated P. subcapitata (low,
medium and high: 1  104, 1  105 and 1  106 cells/mL, respectively)
during seven days. The toxic effects of metal-contaminated algae on
fecundity and survival of C. dubia were verified. The feeding and water-
changing procedures for this test were similar to those used when this
organism was exposed to different metal concentrations.
2.2.3. Feeding rates of the c. dubia
The effects of free cadmium in water and of Cd+2 contaminated algal
diet on feeding rates of zooplankton at different algal levels were evaluated
using measured filtration and ingestion rates. The feeding experiments
with C. dubia were carried out in test vessels containing 50 mL of the
medium and 10 individuals under the same condition of the toxicity
chronic test. A control test, without the added metal, was also prepared.
Test organisms were exposed to two treatments (water and contaminated
food) for 24 h, after which the final algal concentration was measured
using a hemocytometer (Villarroel et al., 1999). The filtration rate (F) was
defined as the volume of medium swept clear per unit of time and
the ingestion rate (I) as the number of cells consumed by an animal
in a specific time interval. To calculate the average filtration (ml/ind/h)
and ingestion rates (cells/ind/h), the equations from Gauld (1951)
were used:
V ðln C 0  ln C t Þ
F¼  A,
n t
ln C 0  ln C 0t
A¼ ,
t in a total of 10 tests, was 1.65  107 M Cd2+, with lower
pffiffiffiffiffiffiffiffiffiffiffi
I ¼F C0Ct ,
767
where C0 and Ct are initial and final food concentrations (cell/mL), t is
time (duration of the experiment in hours), and n is the number of
daphnids in volume V (mL). A is a correction factor for changespin the
ffiffiffiffiffiffiffiffiffiffiffi
control with final concentration Ct0 after time t. The expression C 0 C t
represents the geometric mean of the food concentration during time t.
2.2.4. Metal analyses
At the end of the experiments with P. subcapitata, samples were taken
from each treatment to determine the metal accumulated by algal cells.
Aliquots of test solutions from experiments with algae and cladocerans
were filtered through a 0.45 mm membrane filter The filters with the
accumulated fraction were dried and submitted to acid digestion (HNO3
and H2O2) (APHA, 1995). The analysis of metal in the algal cells was
carried out without EDTA washing, in order not to remove the metals
adsorbed on the cell surface. The measured concentration of metal in algal
cells was taken as the total amount of metal accumulated by the cells (i.e.,
externally and internally bound metal), expressed as mg Cd/mg dry weight
of algae. At the end of the toxicity tests with zooplankton, samples also
were taken to determine the total dissolved metal and accumulated metal
by the cells. These measurements were taken at concentrations where toxic
effects to C. dubia could be identified. Filtered samples were preserved by
acidifying with concentrated nitric acid, for subsequent determination of
total dissolved metal. We used values of total dissolved metal together
with the chemical composition of the test medium to compute the
concentration of free metal ion using the MINEQL+ program. During the
experimental period the metal loss from the algae in the toxicity test with
contaminated food was quantified as the total dissolved concentration of
metal in the culture medium.
For each sample digested, three unused filters were digested and
analyzed as blanks (Van Loon, 1985). All the samples were analyzed by
graphite-furnace atomic absorption spectrometry (Varian AA 220). The
detection limit for Cd, calculated as described in Miller and Miller (1994),
was 4.45  1010 M.
2.2.5. Statistical analysis
The IC50 value of cadmium for the algae was determined by the
trimmed Spearman–Karber method (Hamilton et al., 1977). The algal cell
density data obtained from the experiment with the chlorophyte were
submitted to tests for normality (Shapiro–Wilk’s test) and homogeneity
(Bartlett’s test), and then, to Dunnett’s test (parametric test) to detect
significant differences between the controls and each metal treatment.
To test the significance of the effect of algal food densities, free metal
ion concentration and different densities of metal-contaminated alga on
the survival, fecundity and feeding rates of C. dubia, a two-way ANOVA
was employed on the chronic test data, using the BioEstat 2.0 program
(Ayres et al., 2000). The numbers of neonates produced by C. dubia
females and their feeding rates were submitted to tests for normality
(Shapiro–Wilk’s test) and homogeneity (Bartlett’s test) and, accordingly,
to Dunnett’s test (parametric) to detect significant differences between the
controls and each metal treatment. Tukey’s test (parametric) was used in
multiple comparisons to detect significant differences among the fecundity
obtained in treatments with different algal densities and metal concentra-
tions. Fisher’s exact test was used to distinguish significant differences in
the survival of zooplankton between the control and the various
treatments at the end of the chronic toxicity tests. The above statistical
tests were run using the Toxstat version 3.3 computer package (Gulley
et al., 1993).
3. Results
3.1. Exposure of algae to metal
The mean value for the IC50 of Cd2+ to P. subcapitata,
and upper limits of 0.70 and 2.60  107 M Cd2+,
respectively. Table 2 summarizes the effects of metal on
 ARTICLE IN PRESS
768 S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773

the algae and the results of metal analyses. A decrease in
the cell density of P. subcapitata was observed with
increasing concentrations of cadmium. Concentrations of
2.70  107 M Cd2+ reduced the algal cell density to less
than half that of the control (without added metal) and
dry weight fell similarly. Metal analyses demonstrated
that green algae were able to accumulate cadmium.
With increasing free cadmium ions in solution, the
metal accumulated by P. subcapitata also increased. For
example, when the concentration of Cd2+ increased
from 0.57 to 6.32  107 M, the metal concentration
accumulated by the green algae increased from 0.001 to
0.01 mmol/mg.
3.2. Chronic toxicity test with zooplankton
The fecundity and survival of C. dubia were significantly
affected by the food level, the metal ion concentrations,
and also by their interactions (Tables 3 and 4). The survival
of the cladocerans decreased with increasing Cd2+only at
high algal density. At 0.4, 0.86 and 1.60  107 M free
Cd2+, the number of neonates per female was higher at the
medium food level than at high and low levels. Treatment
with 1.60  107 M of free cadmium caused a decrease in
the number of neonates produced by C. dubia when
medium (105 cells/mL) and high (106 cells/mL) algal den-
sities were supplied as food (Fig. 1). On the other hand, the
toxic effects on zooplankton fecundity during exposure to
0.40 and 0.86  107 M of Cd2+ were observed only at high
food density. The mean numbers of neonates per female
when C. dubia was fed at high algal density and exposed to
0.40 and 1.60  107 M Cd2+ (2.75 and 1.65 neonates/
female, respectively) were significantly lower than those
obtained at medium algal density and at the same metal
concentrations (7.50 and 4.70 neonates/female, in 0.40 and
1.60  107 M Cd+2, respectively) (Fig. 1).
Fig. 2 demonstrates that the test organisms suffered a
significant reduction in mean neonate numbers when they
were fed with high and medium densities of algae exposed
to 1.40 and 2.70 of  107 M Cd2+. The survival of the
cladocerans was not significantly affected. The mean
number of neonates produced by C. dubia at the high
density of algae contaminated with 2.70  107 M Cd2+
(3.33 neonates/female) was significantly lower than that at
the medium density (7.29 neonates/female).
Considering both chronic toxicity tests, the number of
neonates produced per female and survival of C. dubia were
lower in the treatment with water contaminated with 0.4,
0.86 and 1.60  107 M Cd2+ at high algal density than
were fecundity and survival of C. dubia exposed to a high
food level contaminated with 0.6, 1.4 and 2.70  107 M
Cd2+ (Tukey’s test, Po0.05). At low food concentrations
(104 cells/mL), in both sets of chronic toxicity tests (water
and food contaminated with metal), the number of
neonates per female was not different to that found in
the control (Dunnett’s test, Po0.05). Before the metal
effect, there was the effect of low food reducing the
fecundity of test organisms, as this was also observed in the
control containing 1  104 cells/mL. At exposures of 0.4,
0.86 and 1.6  107 M of free cadmium and in the
experiment with food contaminated at 1.4 and
2.7  107 M Cd2+, the fecundity of C. dubia at low food
density was equal to that at high food level. The animals
might have been affected by metal when fed with high algal
densities in both treatments (water and contaminated
food), and limited by low food concentration.
3.3. Feeding rates
The filtration rates of C. dubia in control test conditions
(C) in both treatments (water and contaminated food) were
higher at low food concentration than at medium and high
food levels, whereas ingestion rates were higher at high
than at medium and low algal densities. These results were
generally observed during the metal exposures (Figs. 3
and 4). Metal in water did not affect the filtration and
ingestion rates of the zooplankton, and the feeding rates
only were affected by food levels. In experiments with algal
densities contaminated with Cd+2, filtration and ingestion
rates of C. dubia were only altered by food levels.
3.4. Metal analyses
Concentrations of total dissolved cadmium, free cad-
mium and cadmium accumulated by algal cells, at the end
of toxicity test with C. dubia, are given in Table 5. In the

Table 2
Values of dissolved total metal, free metal, metal accumulated by algal cells, cell density and dry weight in experiments with P. subcapitata algae with
cadmium

Dissolved total cadmium at start of exposure (107 M) C (0) 0.63 1.51 2.93 6.85 12.6
Free cadmium at start of exposure (107 M) 0 0.60 1.40 2.70 6.32 11.6
Cadmium bound to algal cells (mmol/mg) 0 0.001 0.001 0.004 0.01 0.05
(0.000) (0.000) (0.001) (0.002) (0.001)
Mean cell density after 96 h of exposure (106 cells/mL) 6.39 4.91a 3.94a 2.10a 0.77a 0.14a
(1.14) (1.34) (1.54) (0.65) (0.26) (0.14)
Dry weight (mg/L) 137.00 92.22a 89.91a 64.55a 24.92a 9.70a
(23.36) (27.34) (31.02) (19.91) (6.31) (2.73)

Mean values (SD). C, control.

a
Indicates statistically different from control (C) (Dunnett’s test, Po0.05).
 ARTICLE IN PRESS
S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773 769

Table 3 4. Discussion
Results of two-way analyses of variance applied to data on survival,
fecundity and feeding rates of C. dubia when exposed to cadmium at
Metal toxicity to zooplankton can vary with the density
several concentrations and fed with algae at various densities
of algae offered to the organisms. For example, at low
Parameter Source of variation F d.f. P metal concentrations, an increased algal level can enhance
the resistance of the species (Sarma et al., 2000). This effect
Survival Food 12.60 2.16 o0.001
was not observed in the present study, in which toxic effects
Metal 15.14 5.16 o0.001
Food  metal 2.09 10.16 o0.050 to C. dubia were demonstrated when exposed to low metal
concentrations (0.40 and 0.86  107 M Cd2+) while being
Fecundity Food 46.52 2.13 o0.001
fed with a high density of algae. In addition, the mean
Metal 11.97 5.13 o0.001
Food  metal 6.02 10.13 o0.001 numbers of neonates produced by C. dubia during exposure
to 0.86 and 1.60  107 M Cd+2 in the presence of high
Filtration rate Food 104.22 2.36 o0.001
algal density were significantly smaller than those obtained
Metal 2.08 4.30 NS
Food  metal 1.34 8.30 NS at medium algal density. The analysis of the combined
effects of food level and metal concentration showed that
Ingestion rate Food 197.39 2.30 o0.001
metal toxicity was dependent on food level. This result
Metal 2.79 4.30 NS
Food  metal 0.66 8.30 NS agrees with the independent analyses performed with each
food level.
d.f.: degree of freedom; P: probability; NS: not significant (P40.05). In general, cladocerans cannot tolerate an algal level
higher than 4  106 cells/mL, because such densities can
depress their feeding rate (Nandini and Sarma, 2000). In
Table 4 this study, the feeding rate of C. dubia was monitored. At
Results of two-way analyses of variance applied to data on survival, the highest food density used (106 cells/mL), C. dubia
fecundity and feeding rates of C. dubia when this organism was fed with filtered less volume of medium and ingested more algal
cadmium-contaminated algae at various densities
cells. On the other hand, organisms fed with a low algal
Parameter Source of variation F d.f. P density showed high filtration and low ingestion rates. The
feeding behavior of zooplankton could have consequences
Survival Food 3.00 2.13 NS
Metal 3.46 4.13 o0.05
for their life cycle (growth, reproduction, survival). Under
Food  metal 1.56 8.13 NS the control conditions, the fecundity of C. dubia was
observed to rise with increasing algal density supplied, and
Fecundity Food 43.78 2.90 o0.001
it was significantly reduced by density of 104 cells/mL of
Metal 32.01 4.90 o0.001
Food  metal 11.20 8.90 o0.001 P. subcapitata. Algal concentrations of 106 cells/mL prob-
ably occur in eutrophic environments, so that studies using
Filtration rate Food 25.02 2.18 o0.001
this algal density would help in the understanding of metal
Metal 3.92 2.18 NS
Food  metal 2.10 4.18 NS dynamics in polluted aquatic systems.
Algae have been reported to reduce contaminant toxicity
Ingestion rate Food 132.50 2.18 o0.001
to cladocerans. Antunes et al. (2004) observed a decrease in
Metal 7.14 2.18 NS
Food  metal 6.00 4.18 NS the chronic toxic effect of the pesticide lindane to D. magna
when this cladoceran was exposed to a high density of S.
d.f.: degree of freedom; P: probability; NS: not significant (P40.05). capricornutum (6  105 cells/mL). Hauri and Horne (2004)
showed that a large amount of algal food reduced the
availability of labile copper to C. dubia during chronic
treatments with 0.40, 0.86 and 1.60  107 M Cd2+, the toxicity tests, due to complexing of the metal by food
presence of 106 cells/mL of the green alga P. subcapitata particles. The authors reached a general conclusion that a
reduced the concentration of free cadmium ions to 0.10, high food level could supply additional energy for growth
0.26 and 0.63  107 M, respectively. In the treatment and reproduction, and at the same time, enhance specific
with 1.60  107 M Cd2+, the fraction of accumulated mechanisms of detoxification and of resistance to toxic
metal (0.020 mmol/mg) at high algal level was higher agents.
than that verified at the medium algal concentration The results of the present work are consistent with those
(0.012 mmol/mg). Our results demonstrated that metal- obtained by Klüttgen and Ratte (1994), who demonstrated
contaminated algae released low cadmium concentra- inhibited growth and reproduction of D. magna when this
tions back into the test medium during toxicity tests organism was exposed to concentrations from 1 to 5 mg/L
(Table 6). Algae contaminated with 1.40 and 2.70  107 M of Cd, together with a high food concentration of Chlorella
Cd2+ offered to zooplankton as food at high density vulgaris. According to the authors, the toxic effect of the
released low cadmium (0.09 and 0.18  107 M Cd, metal observed in these conditions resulted from the raised
respectively) back into the test medium at end of the test Cd uptake by the zooplankton, as a consequence of an
(Tables 5 and 6). increment in their metabolic rate, due to higher availability
 ARTICLE IN PRESS
770 S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773

high food medium food low food

20
a b

Mean number of neonates per female

100 a
a
16
80
c e
12 j
% survival

60 # gg
e o
m *
8 i l
40 d i l
f n n
h * *
20 4
*
# #
0 0
LC C 0.1 0.17 0.4 0.86 1.6 LC C 0.1 0.17 0.4 0.86 1.6
free cadmium concentration (10-7M) free cadmium concentration (10-7M)

Fig. 1. Percent of survival (A) and mean number of neonates (B) of C. dubia in chronic toxicity tests for cadmium, when fed with various algal densities of
P. subcapitata. LC (laboratory control) and C (control). Survival: # indicates statistically different from the control (C) (Fisher’s exact test, Po0.05).
Number of neonates per female: * indicates statistically different from the control (C) (Dunnett’s test, Po0.05) and means with different letters are
significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation. Each bar for L.C corresponds to one laboratory control of three
chronic toxicity tests performed with the three different algal densities.

high food medium food low food

a a
100 a b
20
Mean number of neonates per female

b
d
80 16
c h
g, h
j
% survival

60 12 *
e * i
f g i *
40 8
*
20 4

0 0
LC C 0.6 1.4 2.7 LC C 0.6 1.4 2.7
free cadmium concentration to which algae were free cadmium concentration to which algae were
exposed (10-7 M) exposed (10-7 M)

Fig. 2. Percent of survival (A) and mean number of neonates (B) of C. dubia when fed with various algal densities of P. subcapitata previously exposed to
cadmium. LC (laboratory control) and C (control). Survival: # indicates statistically different from the control (C) (Fisher’s exact test, Po0.05). Number
of neonates per female: * indicates statistically different from the control (C) (Dunnett’s test, Po0.05) and means with different letters are significantly
different (Tukey’s test, Po0.05). Error bars denote standard deviation. Each bar for LC corresponds to one laboratory control of three chronic toxicity
tests performed with the three different algal densities.

of food. Similarly, Smolders et al. (2005) also verified that the organisms were exposed to 0.4, 0.86 and 1.60  107 M
cadmium toxicity to D. magna increased with increasing of Cd2+ and fed with high algal density, metal analyses
food concentration. demonstrated that algal cells accumulated cadmium.
When the C. dubia females were exposed to free Cadmium associated with algae may form an alternative
cadmium ion at a high algal density, they filtered less source of metal contamination to zooplankton, besides the
volume of medium and ingested more algal cells with metal free metal ions from the water. For filter feeding
bound to the cells, until the toxic effect of the bound metal organisms, the digestive system is a considerable contam-
inhibited their feeding, reproduction and survival. When ination route, and a particle-bound toxic substance can
 ARTICLE IN PRESS
S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773 771

10

Ingestion rate ( x 104 cells/indl/h)

high food medium food low food
1000 a
c m
f h j

Filtration rate (µl/ind/h)

8
800 h
f
l d
j o 6
600
b
e g k,l n 4
400 i,j b h k n
d f
200 a g 2
e k m c i l o
i e g
0 0
C 0.1 0.17 0.4 0.86 1.60 C 0.1 0.17 0.4 0.86 1.60
free cadmium concentration (10-7 M) free cadmium concentration (10-7 M)

Fig. 3. Filtration and ingestion rates of algal cells by C. dubia when exposed to cadmium and fed with various algal densities of P. subcapitata. Means with
the different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation.

high food medium food low food

Ingestion rate ( x 104 cells/ind/h)

1000 d
10
c
Filtration rate (µl/ind/h)

a
800 e 8
g g
i
600 i 6
d
b
400 h b e j
f 4 h
200 f 2
a d h c f h j
0 0
C 0.6 1.4 2.7 C 0.6 1.4 2.7
free cadmium concentration to which free cadmium concentration to which
algae were exposed (10-7 M) algae were exposed (10-7 M)

Fig. 4. Filtration and ingestion rates of algal cells by C. dubia when fed with various algal densities of P. subcapitata previously exposed to cadmium.
Means with the different letters are significantly different (Tukey’s test, Po0.05). Error bars denote standard deviation.

Table 5
Total dissolved, free and accumulated metal at the end of chronic toxicity tests with C. dubia to cadmium

Control Water contaminated with metal (107 M Cd2+) Food contaminated with metal (107 M Cd2+)

0 0.40 0.86 1.60 0.60 1.40 2.70

Medium High Medium High Medium High Medium High Medium High Medium High Medium High
food food food food food food food food food food food food food food

Dissolved total 0.02 0.02 0.21 0.11 0.58 0.30 1.11 0.71 0.02 0.06 0.03 0.11 0.05 0.20
metal (107 M) (0.002) (0.002) (0.01) (0.02) (0.01) (0.01) (0.02) (0.01) (0.00) (0.01) (0.00) (0.01) (0.00) (0.00)
Free metal ions 0.02 0.02 0.19 0.10 0.52 0.26 1.10 0.63 0.02 0.05 0.03 0.09 0.04 0.18
(107 M) (0.002) (0.002) (0.01) (0.01) (0.03) (0.01) (0.02) (0.02) (0.01) (0.01) (0.01) (0.01) (0.00) (0.00)
Metal in algal cells ND ND 0.004 0.002 0.009 0.005 0.012 0.020 0.001 0.001 0.002 0.001 0.005 0.003
(m mol/mg) (0.001) (0.001) (0.001) (0.001) (0.002) (0.002) (0.00) (0.00) (0.00) (0.00) (0.001) (0.001)

Mean values (SD). ND: not detected.

readily be filtered from the water and released into their The importance of algal cells as contamination agents
digestive tract (Fliedner, 1997). At low algal density, for zooplankton was confirmed by the results obtained in
organisms filtered more water with metal and ingested toxicity tests with contaminated food. In this study, a
fewer algal cells than at a high food level. Low acquisition decrease in fecundity of C. dubia was observed when these
of food by cladocerans would result in less energy for organisms were fed on P. subcapitata cells exposed to
reproduction and for metal toxicity repair mechanisms Cd2+. As a result of adding medium and high algal
(Rose et al., 2002). densities contaminated with Cd2+, concentrations between
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772 S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773

Table 6
Dissolved cadmium concentration (107 M) in the culture medium during experiments with C. dubia fed with various algal densities of P. subcapitata
previously exposed to cadmium

Cadmium Day 2 Day 4 Day 8

concentration to
which algal cells
were exposed 104 cells/ 105 cells/ 106 cells/ 104 cells/ 105 cells/ 106 cells/ 104 cells/ 105 cells/ 106 cells/
(107 M) mL mL mL mL mL mL mL mL mL

0.60 ND 0.02 0.08 ND 0.03 0.05 ND 0.02 0.06

(0.00) (0.00) (0.00) (0.00) (0.01)
1.40 ND 0.05 0.12 ND 0.07 0.17 ND 0.03 0.11
(0.00) (0.01) (0.00) (0.02) (0.00) (0.01)
2.70 ND 0.10 0.21 ND 0.09 0.18 ND 0.05 0.20
(0.01) (0.01) (0.01) (0.01) (0.00) (0.00)

Mean values (SD). ND: not detected.

0.03 and 0.20  107 M Cd were released in the test
medium. This may occur either via desorption or elimina-
tion of metal from algal cells (De Schamphelaere et al.,
2004) and from elimination by the daphnids that have
incorporated dietary metal in their tissues (Guan and
Wang, 2004). Indeed, at such a low cadmium concentra-
tion, no toxic effects are expected. We observed effects on
the fecundity and survival of C. dubia up to 0.20  107 M
Cd2+. These observations suggest that toxic effects in
cladocerans given contaminated food can be attributed to
cadmium via dietary exposure. Our findings agree with
those of De Schamphelaere et al. (2004), who found
adverse effects on reproduction of D. magna after exposure
to dietary Zn. Sofyan et al. (2006) also demonstrated that
Cd in algal cells was available for trophic transfer and
capable of producing deleterious effects to C. dubia. In our
experiments involving feeding with Cd+2 contaminated
algae, C. dubia ingested more algal cells at 106 cells/mL
than at 105 and 104 cells/mL. According to Allen et al.
(1995) and Taylor et al. (1998), algal cells contaminated
with Cd can be collected and ingested normally by
daphnids, but the contaminant interferes with digestion,
resulting in cells passing through the gut without being
digested. This could result in profound changes at the
population levels, since the main parameters for population
growth and survival are dependent on the energy input
from feeding activity.
The toxic effects on C. dubia were less pronounced
when the zooplankton were exposed to food contaminated
with 0.60 and 1.40  107 M Cd2+ than those obtained
when the test organisms were exposed to 0.86 and
1.60  107 M Cd2+ in test solution at high algal density.
This result implies that free cadmium ions are more
bioavailable to cladocerans than metal in food, but a
substantial amount of the metal could be obtained from
their food. Consequently, it should be noted that metal
dietary exposure may be present in treatment with
contaminated water.
Food quality is at least as important as quantity for the
fecundity, population growth and survival of cladocerans
(Kilham et al., 1997). Nishikawa et al. (2003) found that
cells of the green algae Chlamydomonas acidophila suffered
a reduction in the level of polyphosphate after being
exposed to 10 and 20 mM of Cd. Metal could have affected
the nutritional value of the algal cells that were exposed to
it. Such changes could make the algae poor in nutritional
value and unviable as food for C. dubia. In the present
study, the cadmium concentrations used to contaminate
P. subcapitata were lower than those used by Nishikawa et
al. (2003). It is unlikely that the observed reduction in
fecundity of C. dubia might result from a dietary quality
effect. In general, negative effects may be related to the
direct toxic effects of metals on target cells or sensitive
tissues responsible for egg production (vitellogenesis)
(Hook and Fisher, 2002). Indirect effects of metal, due to
a diminution in the process of nutrient assimilation
(Munger et al., 1999) have negative repercussions on
growth and fecundity of zooplankton.
5. Conclusion
Our findings suggested that free cadmium ions were
more bioavailable to zooplankton than metal in food. The
addition of algae at high density in chronic toxicity tests in
treatments with water contaminated with cadmium might
have promoted binding sites for the metal and, conse-
quently, generated additional routes for exposure of test
organisms to metal. Food should be considered as an
additional source of metal exposure to cladocerans.
Ecotoxicological studies should consider the influence of
varying algal densities on the expression of metal toxicity
on daphinids. This would allow a better understanding of
the possible relationships between toxicants and aquatic
organisms.
Acknowledgments
We thank the National Research Council (CNPq:
Process 140156/2002-0) and the São Paulo State Research
Support Foundation (FAPESP: Process 10417/2002) for
 ARTICLE IN PRESS
S. Rodgher, E. Luiz Gaeta Espı´ndola / Ecotoxicology and Environmental Safety 71 (2008) 765–773
financial support. We also express our gratitude to
Alessandra Tonietto for assistance with the MINEQL+
program, to Dr. Liane Biehl Printes for valuable com-
ments, and to anonymous reviewers for improving the
manuscript.
Funding sources: National Research Council (CNPq:
Process 140156/2002-0) and the São Paulo State Research
Support Foundation (FAPESP: Process 10417/2002).
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