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Limnol. Oceanogr., 56(1), 2011, 257–267
2011, by the American Society of Limnology and Oceanography, Inc.
doi:10.4319/lo.2011.56.1.0257
E
Contrasting patterns of cadmium bioaccumulation in freshwater cladocerans
Qiao-Guo Tan and Wen-Xiong Wang*
Section of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology
(HKUST), Clear Water Bay, Kowloon, Hong Kong
Abstract
We investigated the patterns of cadmium (Cd) bioaccumulation in three freshwater cladocerans with
contrasting calcium (Ca) contents and Ca uptake and loss kinetics. Ca content and dissolved Cd uptake rate were
significantly correlated. The large interspecies differences in sensitivity to aqueous Cd exposure were explained by
the differences in the Cd uptake rate. The high-Ca species, Daphnia galeata and Ceriodaphnia dubia, had higher
dissolved Cd uptake rates and were more sensitive to aqueous Cd exposure than the low-Ca species, Moina
macrocopa. Food was the dominant Cd source for all of the cladocerans. Feeding on the same Cd-containing
food, M. macrocopa attained the highest Cd body concentration due to its higher assimilation efficiency and
ingestion rate, which led to its susceptibility to dietary Cd despite its relatively high intrinsic tolerance. C. dubia
suffered most from the dietary Cd exposure, probably due to its higher intrinsic sensitivity. The efflux of Cd from
the cladocerans was well described by a two-compartment model. Although the efflux rate constants from both
compartments were comparable among the species, D. galeata had a much higher proportion of the internalized
Cd distributed into the fast compartment, and thus eliminated Cd at a much faster rate. The diversity of Cd
bioaccumulation patterns in cladocerans indicates that no simple generalization can be made from data on only a
few species. Because cladocerans are often the major zooplankton in lakes, their contrasting Cd accumulation and
loss suggest that the biogeochemistry of Cd would be different in waters dominated by different cladocerans.
The interspecies variation in metal concentration among
aquatic organisms is remarkable (Luoma and Rainbow
2005). Metal concentration in different invertebrates
collected from the same water body can have differences
by up to two orders of magnitude (Phillips and Rainbow
1988; Rainbow et al. 2004). Accordingly, the bioaccumulation-related physiological traits (e.g., accumulation rate,
loss rate) can also vary substantially among species, even
for those closely related (Buchwalter et al. 2008; Pan and
Wang 2009). Comparative studies of the metal accumulation and elimination processes in the framework of a
biodynamic model can provide insights and quantitative
explanations for the observed interspecies variation in
metal bioaccumulation (Luoma and Rainbow 2005). For
example, the Cd concentration in different phantom midges
feeding on the same prey varied by eight times, a finding
that has been explained by their distinct assimilation
efficiencies (Croteau et al. 2001). Pan and Wang (2009)
explained the 65-fold difference in Cu concentration in five
marine bivalves collected from the field using laboratory
quantified biodynamic parameters (i.e., assimilation efficiency and efflux rate constant). Because bioaccumulation
is the link between exposure and toxicity, the biodynamics
can also provide insights into the risks associated with
metal exposure. For instance, the high Cd uptake rate
coupled with low elimination rate in several aquatic insects
agreed well with their higher sensitivity to metal stress as
observed in the field (Buchwalter et al. 2007, 2008).
Nevertheless, the relationship between metal accumulation
(and subsequently body concentration) and toxicity is
usually complicated due to the existence of detoxification
(e.g., binding to metallothioneins and formation of
insoluble granules), especially in the case of chronic
exposure (Rainbow 2002).
Cladocerans play important roles in freshwater food
webs because they are often the major grazers of algae and
the prey of planktivorous fish. Among the cladocerans,
Daphnia spp. have received more attention than (any) other
cladocerans in metal bioaccumulation and ecotoxicological
studies, partly due to their relatively higher sensitivity to
metals (Mark and Solbe´ 1998; Sarma and Nandini 2006).
For example, Daphnia is ranked the sixth highest in the
sensitivity to acute Cd exposure out of 55 freshwater
genera, whereas Ceriodaphnia, Moina, and Alona are
ranked thirteenth, seventeenth, and twenty-sixth, respectively (USEPA 2001). The large interspecies difference in
sensitivity to metals impedes the extrapolation of the results
obtained from one cladoceran to another, even if they are
closely related species, and therefore necessitates an
understanding of the underlying mechanisms governing
the sensitivity of cladocerans.
Among cladoceran species, Daphnia spp. have higher Ca
content than Ceriodaphnia spp. and non-Daphnia species
(Wærva˚gen et al. 2002; Jeziorski and Yan 2006; Tan and
Wang 2010). Water is the dominant Ca source for
cladocerans, and a positive correlation has been found
between the body Ca content and the Ca influx rate from
water (Tan and Wang 2010). Cd2+, as a mimic of Ca2+, can
enter cells through the Ca uptake pathways, including the
Ca channel and Na+–Ca2+ exchangers (Craig et al. 1999;
Ahearn et al. 2001; Burke et al. 2003). Significant coupling
between Cd and Ca uptake in fish has been observed
(Zhang and Wang 2007). It is thus intriguing to examine
whether there are corresponding differences in Cd bioaccumulation among cladocerans with contrasting Ca contents and Ca biokinetics, and whether the bioaccumulation
* Corresponding author: [email protected]
257
258
Tan and Wang
can be used to explain the differences in their sensitivity to
Cd.
In the present study, we quantified the Cd biodynamics
in three cladoceran species, Ceriodaphnia dubia, Daphnia
galeata, and Moina macrocopa. These species are closely
related (Olesen 2000), and nevertheless they have contrasting Ca contents and Ca biodynamics (Tan and Wang 2010).
The responses of these cladocerans to both aqueous and
dietary Cd exposure were investigated in parallel. The
results obtained from this study were compared with those
previously documented for Daphnia magna (Tan and Wang
2009; Q.-G. Tan and W.-X. Wang unpubl.). Our objectives
were to test the relationship between the Cd and Ca influx
rates among different cladocerans, and to examine the
interspecies differences in Cd bioaccumulation as well as
their differential responses to Cd exposure.
Methods
Culturing of organisms—Ceriodaphnia dubia and Moina
macrocopa were obtained from C. K. Wong at Chinese
University of Hong Kong, and Daphnia galeata was
obtained from P. B. Han at Jinan University (Guangzhou,
China). The animals were routinely cultured in filtered
(GF/C, Whatman) creek water, and the green alga
Chlamydomonas reinhardtii (60 pg per cell) was offered as
food. The creek water was collected from an unpolluted
creek on the campus of The Hong Kong University of
Science & Technology (22u20911.30N, 114u15959.40E, Hong
Kong). An aliquot of at least 10 mL of water was allocated
to each individual, and the water was refreshed every 2 to
3 d. The algae (C. reinhardtii) were batch cultured in
Woods Hole modified CHU 10 (WC) medium (Guillard
and Lorenzen 1972) with an inoculation cell density of
approximately 104 cells mL21. After 5 d of growth with
aeration, the cell density increased to approximately 106
cells mL21. The algal culture was then centrifuged, and the
pellet was resuspended into filtered creek water and offered
to the animals at the daily dose of 5 3 105 to 106 cells (13.7–
27.3 mg carbon) per individual, depending on the age of the
animals. A temperature of 23.5uC and a light–dark
photoperiod of 14 h : 10 h were used to culture the
organisms for all experiments.
Also for all experiments, cladocerans in the early adult
stage (i.e., C. dubia: 5 d, 0.9 mm body length; D. galeata: 5 d,
1.6 mm; and M. macrocopa: 3 d, 1.3 mm) were used, except
where noted. Newly born (, 24 h) cladocerans were
separated from the routine cultures and raised in an Elendt
M7 medium (20 mg Ca L21; Samel et al. 1999) until early
adulthood following the same protocol as described for the
routine culture. A simplified Elendt M7 (SM7, 20 mg Ca L21)
medium was used in cases where the complexation of
ethylenediaminetetraacetic acid (EDTA) with Cd had to be
avoided. The SM7 medium contains only CaCl2, MgSO4,
K2HPO4, KH2PO4, NaNO3, NaHCO3, Na2SiO3, H3BO3,
and KCl. The pH values of M7 and SM7 media were adjusted
to 8.00–8.20 before use by adding HCl or NaOH as necessary.
Influx of Cd from solution and sensitivity to aqueous
Cd exposure—The influx rates of Cd from solution were
quantified in the three cladoceran species using the
radiotracer technique across the Cd concentration range
of 1 to 200 mg L21. For each species, three replicates, each
containing 20 individuals in 100 mL of exposure medium,
were used for each Cd concentration. The exposure media
were prepared by adding an appropriate volume of CdCl2
stock solution (1000 mg Cd mL21) into the SM7 medium
spiked with 109Cd (as CdCl2, half life: 163.5 d) at the
concentration of 3 to 6 mCi L21 (or 1.3–2.6 mg L21), and the
media were equilibrated overnight. The total Cd concentration was thus the sum of the stable and the radioactive
Cd. The cladocerans were exposed in the media for 8 h,
during which the animals were picked out for radioactivity
measurement every 2 h. Before each measurement, the
animals were collected in a mesh and allowed to swim for
approximately 1 min in a series of three beakers each
containing 200 mL of SM7 medium to remove the weakly
adsorbed 109Cd. At the end of exposure, the animals were
filtered onto a polycarbonate membrane, dried at 60uC for
2 d, and weighed to the nearest 10 mg. At the start and end
of exposure, an aliquot of 0.5 mL of exposure medium was
also sampled for radioactivity measurement, and the
average of the two values was considered as the radioactivity in the exposure medium and was used in calculating
influx rates. The decreases in radioactivity in the medium
due to uptake by cladocerans were negligible in all of the
experiments. The newly accumulated Cd (C, mg g21) was
calculated with the following equation:
C~A|Cw =Aw
ð1Þ
where A is the radioactivity in the animals (counts per min
g21, CPM g21), Aw is the average radioactivity in the
exposure solution (CPM L 21 ), and C w is the Cd
concentration in the exposure solution (mg L21). The
increase in radioactivity in animals formed a linear pattern.
A linear regression (not through the origin) was thus
conducted between C and the duration of exposure (h), and
the slope was considered as the influx rate (Jw, mg g21 h21),
which was expressed on a dry weight (dry wt) basis.
The sensitivity of cladocerans (early adult stage) to
aqueous Cd exposure was quantified by conducting 48-h
acute toxicity tests in SM7 medium. Each test consisted of a
control and five Cd concentrations. Four replicates, each
containing 10 individuals in 100 mL of test medium, were
used for each treatment. After 48 h of exposure, the
cladocerans that did not resume swimming upon gentle
agitation were considered as immobilized and counted. The
48-h median effective concentration (EC50) and 95%
confidence intervals were calculated according to the
trimmed Spearman-Karber method based on the nominal
Cd concentrations (Hamilton et al. 1977).
Dietary assimilation of Cd and sensitivity to dietary
Cd exposure—The assimilation efficiencies (AE) of Cd
from algae (C. reinhardtii) ingested by the three cladoceran
species were quantified under five food concentrations,
ranging from 2 3 103 to 105 cells mL21. The algae (C.
reinhardtii) used in the pulse feeding were radiolabeled in
modified WC medium (with Zn, Cu, and EDTA eliminated
Cladoceran cadmium bioaccumulation
from the standard recipe). Specifically, algae at the
exponential phase were centrifuged and resuspended into
the modified WC medium spiked with 109 Cd (20–
35 mCi L21) with the initial cell density at around 2 3
105 cells mL21. After 3 d of growth, the cell density reached
approximately 106 cells mL21. The radiolabeled algae were
then centrifuged and resuspended in M7 medium. The
centrifugation and resuspension process was repeated once
to remove the weakly bound 109Cd. After being counted for
cell density using a hemocytometer, the algae were
immediately used in the AE experiments.
Three replicate beakers were employed for each species
3 food concentration treatment. Each beaker contained 20
to 40 individuals (exact number recorded) in 200 mL of M7
medium. The 109Cd-labeled algae were added into each
beaker at the corresponding concentration, and the animals
were allowed to feed in the dark for 20 min. After the pulse
feeding, the animals were gently rinsed with M7 medium
and measured for radioactivity. Then, the animals were
transferred to a new M7 medium (10 mL per individual)
with the addition of nonlabeled algae at the corresponding
concentration for the 24-h to 48-h depuration, during
which the animals were periodically measured for radioactivity. After each measurement, the medium and food were
refreshed in order to reduce the recycling of egested 109Cd
and maintain the food concentration.
We quantified the ingestion rates of the animals after
dietary Cd exposure. Three treatments were used, including
a control, a low-Cd, and a high-Cd treatment. Algae were
cultured in a WC medium spiked with 32 mg L21 of Cd
when used as a low-Cd food or 108 mg L21 of Cd when used
as a high-Cd food. The free Cd2+ concentrations for
culturing the low-Cd and high-Cd food were 1.0 and
10.0 mg L21, respectively, as calculated by MINEQL+
(version 4.50, Environmental Research Software, Hallowell, Maine). The animals were fed the algae daily at the
dose of 5 3 105 to 106 cells per individual from birth (i.e., ,
24 h) for 5 d. The 5-d duration was chosen due to the short
life span of M. macrocopa (Tan and Wang 2010). The
concentration of EDTA contained in the Elendt M7
medium (1.68 mmol L21) ensured that the uptake of Cd
from water by cladocerans was negligible. At the end of the
dietary exposure, three subsamples of each cohort were
collected, rinsed with clean medium, and used for Cd body
burden measurement. The remaining individuals were used
for ingestion rate measurement.
Three replicates were used for each treatment, and each
replicate contained 20 individuals in 100 mL of M7 medium
with the addition of 104 cells mL21 of 109Cd-labeled algae (C.
reinhardtii). The animals were allowed to feed in the dark for
20 min and then were picked out, rinsed, and measured for
radioactivity. Afterward, the animals were collected and
dried at 60uC for 2 d and weighed to the nearest 10 mg. The
radioactivity in the algal cells was measured by first filtering
1 mL of the algal suspension with known cell density onto a
1-mm polycarbonate membrane, and then rinsing and
measuring it for radioactivity. The weight-specific ingestion
rate (IR, g g21 d21) was calculated based on the radioactivity
in the animals after the 20-min feeding and the radioactivity
in an algal cell.
259
Efflux of Cd—The efflux of Cd from cladocerans was
also quantified using the radiotracer technique. Briefly, the
animals were radiolabeled with 109Cd and then depurated
in a clean environment, and the elimination rate of 109Cd
was monitored. The animals were labeled by culturing them
in SM7 medium spiked with 109Cd (3 mCi L21) for 3 d,
during which food (C. reinhardtii) was added daily at the
dose of 5 3 105 to 106 cells per individual. After the 3-d
exposure, the age of C. dubia, D. galeata, and M.
macrocopa individuals was 5, 5, and 3 d, respectively. For
each species, the animals were divided into three replicates,
each containing 40 to 50 individuals, and depurated in M7
medium for 6 d. Ten milliliters of medium were allocated to
each individual, and 106 algal cells were offered to each
individual daily. The radioactivity in the animals was
measured every half day during the first 4 d and every day
during the last 2 d. After each measurement, the medium
was refreshed, and new food was added. The efflux rate
constant (ke) of Cd was estimated according to the decrease
in radioactivity in animals over time.
Chemical analysis and statistics—The cladocerans for Cd
content measurement were collected by filtering them onto
a polycarbonate membrane, after which they were rinsed
with clean medium, dried at 60uC for 2 d, and weighed to
the nearest 10 mg. The algae for Cd content measurement
were centrifuged and resuspended into M7 medium. The
centrifugation and resuspension process was repeated twice
in order to eliminate the weakly adsorbed Cd. Two
aliquots, 1 mL each of the concentrated algal suspension,
were filtered onto a preweighed GF/F (Whatman) filter and
dried (60uC, 2 d) for weight measurement. Another two
aliquots of the algae were digested in HNO3 for Cd
measurement.
Both the cladoceran and algal samples were digested in
1 mL of 40% HNO3 at 80uC for 2 d and diluted
appropriately for Cd measurement. The Cd concentrations
in the biological and water samples were measured using
furnace atomic absorption spectrometry (AAnalyst 800,
PerkinElmer). The concentration range of the standard
curve was 0.25 to 2 mg L21. All the measured Cd
concentrations of the media for the influx rate experiment
and toxicity tests were within 10% deviation of the nominal
concentration (59% within 5% deviation), and thus the
nominal concentrations were used for all calculations. The
radioactivity of 109Cd was measured using a Wallac 1480
NaI(T1) gamma counter (Turku).
Groups of data (i.e., AE, ke, IR) were compared with an
analysis of variance (ANOVA) followed by Tukey’s
multiple comparison tests. The percentages (i.e., AE,
relative IR) were arcsine-transformed to meet the mathematical assumption of normal distribution before the
ANOVA. Significant difference was accepted at p , 0.05.
All statistical analyses were conducted in Statistical
Package for the Social Sciences (SPSS) 16.0.
Results
Aqueous Cd uptake and sensitivity of cladocerans—The
influx rates of Cd in the three cladoceran species were
260
Tan and Wang
Fig. 1. (A) The relationship between influx rate of Cd from solution (Jw) and Cd concentration in medium (Cw) described by linear
regression on the log–log plot. Ceriodaphnia dubia: Jw 5 0.042 3 Cw0.711; Daphnia galeata: Jw 5 0.056 3 Cw0.833; Moina macrocopa: Jw 5
0.0035 3 Cw1.050; Daphnia magna: Jw 5 0.129 3 Cw0.855. (B) The percentage of individuals immobilized after a 48-h exposure to medium
containing different concentrations of Cd. The concentration–response curves were four-parameter logistic regressions generated with
SigmaPlot 10. Data for D. magna were from Q.-G. Tan and W.-X. Wang (unpubl.). The error bars represent standard deviations (n 5 3 in
A, n 5 4 in B).
quantified across the Cd concentration range of 1 to
200 mg L21. No obvious saturation of influx was observed
within this concentration range. The relationship between
the influx rate and Cd concentration could be satisfactorily
described by a linear regression on the log–log plot
(Fig. 1A). Due to the low specific activity of the radiotracer
(109Cd, , 2.3 mCi mg21), the lowest Cd concentration we
used (1.3 mg L21) was relatively high and can only be found
in contaminated waters (USEPA 2001; Croteau et al. 2002).
However, the Cd uptake kinetics we obtained can be
extrapolated to lower Cd concentrations considering that
no saturation occurred, and the extrapolation has been
shown to be valid in D. magna (Yu and Wang 2002a; also
see D. magna in Fig. 1A). Daphnia galeata consistently had
the highest influx rates, while M. macrocopa had the lowest.
The magnitude of interspecies difference in influx rate was
dependent on the aqueous Cd concentration, and the influx
rate in D. galeata was approximately one order of
magnitude higher than that in M. macrocopa.
Moina macrocopa was much more tolerant to aqueous
Cd than the other two species (Fig. 1B). The 48-h EC50 of
M. macrocopa was 737 mg Cd L21, which was 5.3 and 7.2
times higher than that of C. dubia and D. galeata,
respectively (Table 1).
Table 1. The 48-h median effective concentration (EC50) of
Cd and the calculated median effective influx rate (EJ50) of Cd in
four cladoceran species. Values in parentheses are 95% confidence
intervals. The EJ50 was calculated as the influx rate of Cd at the
Cd concentration of EC50 (see Fig. 1 for the equations).
Species
C. dubia
D. galeata
M. macrocopa
D. magna*
EC50 (mg L21)
138
102
737
17.3
(134, 144)
(91.8, 114)
(637, 852)
(16.1, 18.5)
EJ50 (mg g21 h21)
1.40
2.65
3.59
1.56
* Data from Q.-G. Tan and W.-X. Wang (unpubl.).
(1.36,
(2.42,
(3.08,
(1.46,
1.44)
2.90)
4.18)
1.66)
Dietary Cd assimilation and sensitivity of cladocerans—
The AE of Cd can be estimated from the percentage of the
pulse-ingested 109Cd retained in animals at the point when
the depuration curve begins to level off (Fig. 2). This is
because the physiological loss of assimilated Cd is a much
slower process than the egestion of unassimilated Cd. The
AE values of Cd in C. dubia, D. galeata, and M. macrocopa
were therefore calculated as the percentage of 109Cd
retained in the animal after 24, 32, and 24 h of depuration,
respectively. The results are listed in Table 2. The Cd AE
decreased significantly with increased food concentration
(2-way ANOVA, F4,30 5 75.1, p , 0.001) and was
significantly different among species (2-way ANOVA,
F2,30 5 100.4, p , 0.001). Moina macrocopa had the
highest AE (51.1–76.0%), while there was no significant
difference between C. dubia (24.2–74.5%) and D. galeata
(26.5–53.6%). The decrease in Cd AE with increasing food
concentration was most conspicuous in C. dubia (3-fold),
while the AE in M. macrocopa was least affected by food
concentration (1.5-fold).
For the dietary exposure experiment, the measured
specific Cd content of algae used in the control, low-Cd,
and high-Cd treatments was 0.038 6 0.003 mg g21, 84.2 6
0.6 mg g21, and 286 6 27 mg g21 dry weight (n 5 2),
respectively. After 5 d of exposure to Cd-loaded algae, the
Cd concentration in cladocerans was elevated in a dosedependent manner, but it was lower than the Cd
concentration in the algae on which they were fed. Moina
macrocopa consistently had the highest Cd concentration
(Fig. 3A); however, C. dubia showed the highest sensitivity
to dietary exposure when measured in terms of weightspecific ingestion rate (Fig. 3B). The weight-specific IR of
C. dubia, D. galeata, and M. macrocopa in the control
treatment were 0.40 6 0.02 g g21 d21, 0.35 6 0.01 g g21 d21,
and 0.44 6 0.04 g g21 d21, respectively. The IR of C. dubia
feeding on high-Cd algae was reduced to 51% of the
control. Feeding on high-Cd algae also caused a significant
Cladoceran cadmium bioaccumulation
261
slower rate (Fig. 4). The depuration data were well fitted by
a two-compartmental elimination model, which assumed
no exchange between compartments (Fig. 4). A linear
regression was conducted between the natural log (i.e., ln)
of the percentage of 109Cd retained in cladocerans and the
time of depuration between day 1.5 and 6 for each curve
(Fig. 4, dotted lines). The absolute value of the slope was
considered as the efflux rate constant of the slow
compartment (ke2), and e(y2intercept) was the percentage of
109Cd in the slow compartment at the start of depuration.
The efflux rate constant of the fast compartment (ke1) was
estimated from the depuration data between day 0 and 1.5
after the slow compartment was subtracted (Newman and
Clements 2008).
There was no significant interspecies difference in ke1
(2.26–2.70 d21, F2,6 5 0.479, p 5 0.641). Although
significant differences in ke2 (0.073–0.105 d21) were
observed among the three species, the differences were ,
1.5-fold (Table 3). There were large interspecies differences
in the distribution of Cd in different compartments. At the
start of depuration, a much higher proportion (67.3%) of
Cd in D. galeata was in the fast compartment than in C.
dubia (28.2%) and M. macrocopa (39.9%; Fig. 4). Therefore, at the end of the 6-d depuration, D. galeata retained
the least percentage of Cd.
Discussion
Fig. 2. The percentage of 109Cd retained in cladocerans (i.e.,
Ceriodaphnia dubia, Daphnia galeata, and Moina macrocopa)
during the 24- to 48-h depuration under different food concentrations (i.e., 2 3 103 to 105 cells mL21 of Chlamydomonas
reinhardtii). The 109Cd was ingested during the 20-min pulse
feeding on 109Cd-labeled C. reinhardtii. The dashed lines indicate
the time point for calculating the assimilation efficiency.
reduction in IR in M. macrocopa (to 80%), but not in D.
galeata. No significant reduction in IR was observed in
animals that were fed low-Cd algae.
Efflux of Cd—During the 6-d depuration, Cd was
rapidly eliminated from the cladocerans initially (i.e.,
between 0 and 1.5 d) and was then eliminated at a much
The consistently lower Cd uptake rate in M. macrocopa
was expected based on our previous findings that this
species had a strikingly low Ca uptake rate (Tan and Wang
2010) and that Cd is taken up by aquatic organisms as a
mimic of Ca through Ca uptake pathways (Craig et al.
1999; Ahearn et al. 2001; Burke et al. 2003). In our previous
studies, we quantified the Ca influx rates and Ca content in
Daphnia magna and the three cladocerans investigated in
the present study (Tan and Wang 2009; Tan and Wang
2010). By putting these data together, a positive correlation
can be found between the Ca and Cd uptake rate in the
four cladoceran species (including Daphnia magna); however, the correlation is not significant (p 5 0.076, Fig. 5A).
As mentioned in Tan and Wang (2010), the Ca influx rate
quantified during a short period (i.e., 4 h) might not be
representative of the average influx rate due to the
inherently large fluctuation across the molt cycle, and it is
probably underestimated to a different extent in different
species. Therefore, we also conducted a correlation analysis
between the Cd influx rate and the specific Ca content (%
of dry wt) of cladocerans (Fig. 5B), considering that the Ca
content provides a time-integrated measure of Ca influx
rate. The correlation was strong (R2 5 0.965) and
significant (p 5 0.018), which means that high-Ca
cladoceran species had a higher Cd influx rate. Pan and
Wang (2009) found that the large interspecies differences in
the Cu uptake rate in five bivalves can be explained by the
differences in their filtration rate. However, M. macrocopa,
which has the highest filtration rate, actually had the lowest
Cd uptake rate, which suggests that the uptake of Cd in
cladocerans is not limited by metal diffusion to uptake
sites.
262
Tan and Wang
Table 2. The assimilation efficiency of Cd (%) in Ceriodaphnia dubia, Daphnia galeata, and
Moina macrocopa feeding on algae (Chlamydomonas reinhardtii) of different concentrations (105
cells mL21 5 2.73 mg C L21). Values are mean 6 standard deviation (n 5 3). The means in each
column that do not share a common superscript letter were significantly different.
Food concentration (cells mL21)
Species
23103
53103
104
23104
105
C. dubia
D. galeata
M. macrocopa
74.560.8a
53.665.6b
76.065.9a
60.965.3ab
54.767.1a
70.562.2b
47.860.6a
48.963.0a
73.667.4b
32.161.4a
41.865.5a
65.366.5b
24.262.6a
26.562.4a
51.163.3b
Among the four species investigated, D. magna was the
most sensitive to aqueous Cd exposure, followed by D.
galeata and C. dubia. Moina macrocopa was the most
tolerant species and had a strikingly high EC50 (i.e.,
737 mg L21), which is similar to the results of Garcia et
al. (2004), who reported a 24-h LC50 (median lethal
concentration, equivalent to EC50 of the present study) of
680 mg L21 in 24-h neonatal M. macrocopa. By comparing
the Cd influx rate and sensitivity to aqueous Cd exposure
among the four cladoceran species, it is clear that species
with higher influx rate also had higher sensitivity (Fig. 1).
Therefore, the interspecies differences in influx rate provide
Fig. 3. (A) The body Cd concentration in Ceriodaphnia dubia
(C.d.), Daphnia galeata (D.g.), and Moina macrocopa (M.m.) after
a 5-d feeding on algae (Chlamydomonas reinhardtii) contaminated
by Cd (Cd concentration in algae [mg g21]: control 0.038 6 0.003,
low 84.2 6 0.6, high 286 6 27). (B) The weight-specific IR of
cladocerans relative to the corresponding control treatment. The
means in each column that do not share a common superscript
letter were significantly different. The error bars represent
standard deviations (n 5 3).
a sound explanation for the large differences in sensitivity
to aqueous Cd exposure. It was possible that the majority
of metal incorporated during the acute exposure remained
in the metabolically available pool and thus exerted toxicity
instead of being detoxified in different species (Rainbow
2002). Alternatively, because the species with a higher Cd
uptake rate also had a higher Ca uptake rate, their higher
sensitivity might be a result of higher susceptibility of Ca
uptake to the disturbance by Cd. In accordance with this
speculation, Grosell and Brix (2009) reported that the
freshwater snail Lymnaea stagnalis, which has a high Ca
uptake rate (0.32 mg g21 wet weight h21), were hypersensitive to Pb (EC20 , 4 mg L21) due to the reduction of Ca
uptake by Pb exposure.
By substituting EC50 values into the uptake kinetics
equations (Fig. 1A), we obtained the Cd influx rate
corresponding to 50% immobilization (or median effective
influx rate, EJ50; Table 1). While EC50 reflects the tolerance
of a species to aqueous Cd exposure, EJ50, which excludes
the confounding effect of interspecies differences in
bioaccumulation capability, reflects the intrinsic tolerance
of the species to internalized Cd. Compared to the large
variation in EC50 (42.6-fold) among species, the EJ50 values
were remarkably stable, with only a 2.6-fold variation
(Table 1), indicating that the four cladocerans actually
have quite comparable intrinsic sensitivity to Cd.
Taken together, we suggest that the four parameters,
including Ca content, Ca influx rate, Cd influx rate, and
sensitivity to aqueous Cd exposure, are intercorrelated in
cladocerans. Therefore, assuming similar detoxification
rates, if any, we expect cladocerans with higher Ca content
to be more sensitive to Cd exposure. Among the freshwater
zooplankton, Daphnia spp. have higher Ca content than
Ceriodaphnia spp. or non-daphnid cladocerans and copepods, and D. magna have the highest contents among the
cladocerans for which Ca content has been measured
(Wærva˚gen et al. 2002; Jeziorski and Yan 2006). In line
with our expectation, the sensitivity to acute Cd exposure in
freshwater zooplankton follows the same order: Daphnia
spp. . Ceriodaphnia spp. . non-daphnid cladocerans (M.
macrocopa and Alona affinis) and copepods (Cyclops
varicans), with D. magna being the most sensitive species
(USEPA 2001).
Moina macrocopa consistently had a higher AE of Cd
than C. dubia and D. galeata, especially at high food levels.
The Cd AE in M. macrocopa was quite similar to that
quantified in D. magna (50–80%) within the similar food
concentration range (Yu and Wang 2002b). The higher AE
Cladoceran cadmium bioaccumulation
Fig. 4. The percentage of 109Cd retained in cladocerans
during the 6-d depuration. The cladocerans were exposed in
109Cd-spiked medium (containing food) for 3 d before the
depuration. The dots are the measured values, and the error bars
represent standard deviations (n 5 3). The depuration data were
fitted with two-compartment first-order elimination model (i.e.,
the solid curves). Ceriodaphnia dubia: y 5 28.2e22.26x +
71.8e20.070x, Daphnia galeata: y 5 67.3e22.53x + 32.7e20.093x,
Moina macrocopa: y 5 39.9e22.70x + 60.1e20.073x. The dotted lines
represent the elimination from the slow compartment.
in M. macrocopa than in C. dubia and D. galeata agrees well
with the higher Cd concentration in the former species than
in the latter two species after feeding on the same Cdcontaminated food sources (Fig. 3A). The Cd concentration in M. macrocopa was 51–59% of the Cd concentration
in the algae on which they were fed, while that in C. dubia
and D. galeata was only 11–21% and 9–10% of that in the
algae, respectively. AE is an important parameter in
determining the trophic transfer of elements in aquatic
food webs (Wang 2002). M. macrocopa, which has a higher
Cd AE, theoretically has a greater potential to transfer Cd
from primary producers to predators at higher trophic
levels (e.g., predatory invertebrates, zooplanktivorous fish).
Although Cd is usually biodiminished along the planktonic
food chain (Wang 2002; Tsui and Wang 2007), predators at
higher trophic levels are still at the risk of suffering from
dietary Cd toxicity due to their longer life span and higher
sensitivity. For instance, feeding on M. macrocopa containing 56.6 mg g21 of Cd led to the Cd concentration in
catfish reaching the permissible limit of 0.20 mg g21 wet
weight and adversely affected the growth of that fish
(Ruangsomboon and Wongrat 2006). In the present study,
263
the Cd concentration in M. macrocopa fed on the algae
cultured under an environmentally realistic Cd concentration (Cd2+: 1.0 mg L21) reached 42.7 mg g21, which is near
the potentially hazardous level. This is noteworthy
considering the value of M. macrocopa in aquaculture as
feed for fish larvae (Evangelista et al. 2005).
It has been previously observed that a higher ambient Ca
concentration leads to a lower Cd AE in D. magna (Tan
and Wang 2008), and that elevating dietary Ca concentration reduces gastrointestinal Cd assimilation in rainbow
trout (Franklin et al. 2005). Both observations suggest that
Cd and Ca share common pathways for entering gut
epithelial cells. Therefore, we expected to see that the
species with a higher Ca AE would also have a higher Cd
AE, just like the coupling between Ca and Cd uptake from
water that we observed (Fig. 5). However, compared to Cd
AE, Ca AE was found to be significantly lower in M.
macrocopa (5.3%) than in C. dubia (10.6%) or D. galeata
(12.4%) (Tan and Wang 2010). One possible reason for the
decoupling between Cd and Ca assimilation could be that
other Cd uptake routes contributed substantially to the
assimilation of Cd, besides those shared with Ca.
The relative importance of food as a source of Cd (Sf)
can be calculated using the equation
Sf ~
Jf
|100%
Jf zJw
Jf ~AE|IR|Cf
ð2Þ
ð3Þ
where Jf and Jw are the influx rate of Cd from food and
water (mg g21 d21), respectively, IR is the weight-specific
ingestion rate (g g21 d21), and Cf is the Cd concentration in
food (mg g21). The Sf values in the three cladoceran species
were estimated for two food concentrations (i.e., 104 and 5
3 104 cell mL21), both of which are environmentally
realistic (DeMott et al. 2004). The AE values at the 5 3 104
cells mL21 food level (24.5%, 32.0%, and 57.4% in C.
dubia, D. galeata, and M. macrocopa, respectively) were
estimated by fitting an exponential decay model with food
concentration to the quantified Cd AE (Tan and Wang
2009). The IR values at 104 cells mL21 were measured in
the dietary toxicity experiment as described previously, and
the IR values at 5 3 104 cells mL21 were taken from Tan
and Wang 2010 (Table 4). In the dietary toxicity experiment, the Cd concentration in algae cultured in the medium
containing 1.0 mg Cd2+ L21 (equivalent to 1.2 mg Cd L21 in
SM7 medium as calculated using MINEQL+) was
Table 3. The efflux rate constant of Cd from the fast compartment (ke1) and slow
compartment (ke2), the estimated proportion of Cd influx into different compartments (f1, f2),
and the calculated ratio of steady-state Cd concentration in the two compartments (Css1 : Css2).
Values are mean 6 standard deviation (n 5 3). The means in each column that do not share a
common superscript letter were significantly different.
Species
ke1 (d21)
ke2 (d21)
f1 : f2
Css1 : Css2
C. dubia
D. galeata
M. macrocopa
2.2660.19a
0.07060.003a
2.5360.17a
2.7060.83a
0.09360.004b
0.07360.012a
0.70 : 0.30
0.93 : 0.07
0.81 : 0.19
1.0 : 3.8
1.0 : 0.7
1.0 : 2.2
264
Tan and Wang
Fig. 5. The correlation between the Cd influx rate from solution (JCd) and (A) the Ca influx rate from solution (JCa), (B) the specific
Ca content of four cladoceran species. C.d. 5 Ceriodaphnia dubia, D.g. 5 Daphnia galeata, D.m. 5 Daphnia magna, M.m. 5 Moina
macrocopa. The Ca influx rate and the Ca content of C.d., D.g., and M.m. are from Tan and Wang (2010); the Ca influx rate (at 20 mg Ca
L21) and the Ca content of D.m. are from Tan and Wang (2009); the Cd influx rate of D.m. is from Q.-G. Tan and W.-X. Wang (unpubl.).
84.2 mg g21. We thus used 1.2 mg Cd L21 to calculate Jw
and accordingly set Cf to be 84.2 mg g21. The calculated Sf
values are listed in Table 4. Food was the major (i.e., .
90%) Cd source for all three species, especially for M.
macrocopa, with a remarkably low Jw and a high AE. The
dominant role of dietary Cd found in the present study is in
agreement with the observations for D. magna (Guan and
Wang 2006; Tan and Wang 2008). We can probably
extrapolate this finding to other planktonic cladoceran
species because cladocerans have relatively high feeding
activity (e.g., . 0.5 g g21 d21) when provided with
abundant food, which is necessary for fueling their rapid
growth and frequent reproduction (Sarma et al. 2005; Tan
and Wang 2010).
As food was the major Cd source for the cladocerans, it
is important to assess the risk posed by dietary Cd
exposure. Moina macrocopa had the highest Cd concentration after the dietary exposure; however, this species was
less affected (in terms of ingestion rate) than C. dubia.
Daphnia galeata showed the highest tolerance to dietary
exposure. We can qualitatively explain the differential
responses of these species by using the body concentration
together with EJ50 (as an indicator of tolerance to
internalized Cd as described already; see Table 1). For
example, the highest sensitivity to dietary exposure in C.
dubia is the result of its highest intrinsic sensitivity (i.e.,
lowest EJ50) and intermediate body concentration. Although M. macrocopa had the highest accumulation, it is
less sensitive than C. dubia due to its highest intrinsic
tolerance (i.e., highest EJ50). However, we found poor
quantitative correlation between the sensitivity to dietaryassimilated Cd (i.e., reduction in ingestion rate) and the
ratio of body concentration to EJ50 (results not shown).
Altogether, the results suggest that although it is reasonable
to postulate that a species with higher tolerance to Cd
incorporated from water should have higher tolerance to
Cd assimilated from food, the correlation between the two
‘‘tolerances’’ is not linear.
The high sensitivity of C. dubia to dietary Cd was also
observed by Sofyan et al. (2007), although the sensitivity
they registered was much higher. They reported that C.
dubia feeding on algae (Pseudokirchneriella subcapitata)
containing 3.11 mg Cd g21 for 7 d reduced the feeding rate
to less than 40% of the control, while feeding on algae
containing 0.56 mg Cd g21 significantly inhibited reproduction. In comparison, D. magna showed lower sensitivity.
Feeding on algae (P. subcapitata) containing 62 mg Cd g21
for 21 d (16 h per day) reduced the number of neonates
Table 4. The relative importance of diet as the source of Cd (Sf) for different cladoceran
species at two food (Chlamydomonas reinhardtii) concentration levels (i.e., 104 and 5 3 104 cells
mL21). For the calculation, waterborne Cd concentration was assumed to be 1.2 mg L21 (or
1.0 mg L21 Cd2+ in SM7 medium), and the corresponding Cd concentration in our given diet was
84.2 mg g21. Jw, influx rate of Cd from solution. IR, weight-specific ingestion rate.
Food concentration (cells mL21)
Species
C. dubia
D. galeata
M. macrocopa
104
Influx rate of Cd
Jw (mg g21 d21) IR (g g21 d21)
1.15
1.56
0.10
* Data from Tan and Wang (2010).
0.40
0.35
0.44
53104
Sf (%)
IR*(g g21 d21)
Sf (%)
93.3
90.2
99.6
0.83
0.76
1.37
93.6
91.6
99.8
Cladoceran cadmium bioaccumulation
Fig. 6. The measured (see Fig. 4A) and predicted Cd
concentration in Ceriodaphnia dubia (C.d.), Daphnia galeata
(D.g.), and Moina macrocopa (M.m.) after feeding on Cdcontaminated algae (84.2 6 0.6 mg Cd g21) for 5 d. The prediction
was made based on the two-compartment model (see Discussion
for details). The assimilation efficiency and ingestion rate data
were from those listed (or used) in Table 4 at 5 3 104 cell mL21.
produced by approximately 30% but caused no mortality in
D. magna (Geffard et al. 2008). Moreover, Goulet et al.
(2007) observed no adverse effects of Cd-loaded algae (C.
reinhardtii, 70 mg g21) on the survival, feeding, growth, and
reproduction of D. magna during the 21-d culture.
The elimination of Cd from D. galeata was much faster
than from the other two species (Fig. 4). This is due to the
larger fraction of Cd distributed in the fast compartment
rather than to the slightly higher ke2 in D. galeata (Table 3).
After 3 d of exposure to both dietary and aqueous Cd,
28.2%, 71.8%, and 39.9% of Cd was distributed in the fast
compartment in C. dubia, D. galeata, and M. macrocopa,
respectively (Fig. 4). Based on these results, we estimated
the fraction of Cd influx into different compartments using
the two-compartment biodynamic model (Guan and Wang
2006; Newman and Clements 2008):
Cssi ~
fi |Jin
kei zgi
f1 zf2 ~1
Ct,i ~Cssi (1{e{(kei zgi )|t )
Ct ~Ct,1 zCt,2
ð4Þ
ð5Þ
ð6Þ
ð7Þ
where Cssi (mg g21) is the steady-state (i.e., the state when
influx equals efflux) Cd concentration in cladocerans
distributed in the fast (subscript 1) or slow (subscript 2)
compartment; Jin (mg g21 d21) is the total Cd influx from
both water and food; fi is the fraction of Jin distributed into
compartment i; gi (d21) is the growth rate constant of
compartment i; and Ct,i and Ct (mg g21) are the Cd
concentration in cladocerans at the exposure time of t (d).
The ratio of C3,1 : C3,2 was 0.393 (i.e., 28.2% : 71.8%), 2.06
(i.e., 67.3% : 32.7%), and 0.664 (i.e., 39.9% : 60.1%) in C.
dubia, D. galeata, and M. macrocopa, respectively (Fig. 4;
265
Eq. 6). By assuming that g1 5 g2 5 0.3 based on our
previous results on D. magna (Tan and Wang 2009), the
estimated f1 : f2 and Css1 : Css2 ratios are listed in Table 3.
The majority of the Cd (. 70%) was distributed into the
fast compartment upon internalization in all three species,
but especially in D. galeata (93%). However, after reaching
the steady state, the slow compartment contained the
majority of Cd in C. dubia (79%) and M. macrocopa (69%).
In contrast, D. galeata only had 41% of the total Cd in the
slow compartment. In addition, we validated the twocompartment model (Eqs. 3–6) by predicting the Cd
concentration in cladocerans after the 5-d feeding on
low-Cd–contaminated algae (see Fig. 4). The predicted
concentrations were 93% to 115% of the measured values
(Fig. 6).
The large interspecies variation in all of the investigated
bioaccumulation-related physiological traits indicates that
even the closely related freshwater cladocerans cannot be
treated as a homogeneous group in terms of metal
bioaccumulation. Therefore, in order to obtain a sound
generalization, any comparative study should not only be
based on the evolutionary phylogeny, but also on the
understanding of the physiological factors governing the
metal bioaccumulation (e.g., the Ca biokinetics in the
present study for Cd bioaccumulation). As zooplankton
play important roles in the biogeochemical cycle of trace
metals in lake ecosystems (Twiss et al. 1996), metal
biogeochemistry is thus expected to be different in lakes
dominated by different cladocerans.
In conclusion, there was a positive correlation between
the cladoceran Ca content and the Cd influx rate,
presumably due to the coupling of Ca and Cd during
their uptake. The species with a higher influx rate of Cd
from water was more susceptible to aqueous Cd stress.
The differential susceptibility to dietary Cd exposure
among cladocerans could also be explained by the
differences in their bioaccumulation capability and their
intrinsic sensitivity. Because food was the dominant Cd
source for cladocerans, the dietary toxicity of Cd should
be taken into account when assessing the risks of Cd
exposure. Ceriodaphnia dubia can serve as good bioindicator considering its high sensitivity to dietary Cd
exposure and its widespread distribution. Daphnia galeata,
with a relatively lower AE and a higher elimination rate,
acts as a recycler of Cd, whereas M. macrocopa, with just
the opposite characteristics, acts as an accumulator of Cd
and has higher potential to transfer Cd to higher trophic
levels.
Acknowledgments
We thank the two anonymous reviewers for their constructive
comments. This study was supported by a General Research Fund
from the Hong Kong Research Grants Council (663009) to W.X.W.
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Associate editor: Robert E. Hecky
Received: 06 July 2010
Accepted: 20 October 2010
Amended: 29 October 2010