Inuences of ambient carbon nanotubes on

toxic metals accumulation in Daphnia magna

Zhi-Guo Yu, Wen-Xiong Wang*
Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay,
Kowloon, Hong Kong
a r t i c l e i n f o
Article history:
Received 29 March 2012
Received in revised form
30 August 2012
Accepted 28 January 2013
Available online 28 March 2013
Keywords:
Daphnia
Metals
Carbon nanotubes
Biokinetics
Bioaccumulation
a b s t r a c t
With the rapid increase of carbon nanotube (CNT) applications, there are considerable
concerns of their inevitable releases into the aquatic environments. CNTs may interact
with and further inuence the fate and transport of other pollutants such as toxic metals.
In the present study, non-covalent and nontoxic dispersant polyvinyl pyrrolidone (PVP)
was used to provide a relatively stable test solution for CNTs. The dissolved uptake rate
constant (k
u
) and the dietary assimilation efciency (AE) of cadmium (Cd) and zinc (Zn)
were then quantied in a freshwater zooplankton Daphnia magna in the presence of
different CNTs (without functionalized e single-walled nanotubes-SWNTs, multi-walled
nanotubes-MWNTs, and with functionalized e F-SWNTs, F-MWNTs, containing oxygen
functional groups at the defect sites of CNTs) concentrations. We demonstrated that
different CNTs exposures led to distinctive metal accumulation patterns. Non-
functionalized CNTs signicantly decreased the metal uptake rate from the dissolved
phase, possibly because of their effects on the physiological activity of animals. In contrast,
the F-CNTs (F-SWNTs and F-MWNTs) adsorbed the metals and increased the metal accu-
mulation in daphnids in a concentration-dependent manner, due to the ingestion of F-
CNTs associated metals. The AEs of metals in D. magna were elevated by CNTs physical
blocking of the animal guts. Our present study showed that CNTs could serve as a new
pathway for metal accumulation. This raised a new environmental problem of CNTs since
they may induce the accumulation of toxic metals from the dietary exposure.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Ideal carbon nanotubes (CNTs) are pure carbon macromole-
cules consisting of one or more concentric layers of rolled
graphene sheets with various lengths and diameters or
different functional groups. The release of manufactured
nanoparticles into the environment is likely to rise because of
the increasing application of these particles in our daily life. It
is inevitable that they will enter into ecologically sensitive
environments such as waters and soils (Petersen et al., 2008).
The toxicological effects of carbon nanotubes (CNT) on
different organisms have been rapidly documented
(Templeton et al., 2006; Cheng et al., 2007; Smith et al., 2007;
Kang et al., 2008; Mueller and Nowack, 2008; Kim et al., 2010),
and one of the most signicant risks is their potential accu-
mulation by organisms and transfer along the food chains
(Baun et al., 2008; Ghafari et al., 2008).
In aquatic environments, CNTs will interact with and
affect the behavior of other pollutants such as toxic metals
(Rao et al., 2007). Kim et al. (2009, 2010) investigated the
* Corresponding author. Tel.: 852 23587346; fax: 852 23581559.
E-mail address: wwang@ust.hk (W.-X. Wang).
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http://dx.doi.org/10.1016/j.watres.2013.01.059
interactions of lysophosphatidylcholine (LPC)-single-walled
nanotubes (SWNTs) and multi-walled nanotubes (MWNTs,
dispersed in natural organic matter, NOM) with soluble cop-
per, and quantied the bioavailability and toxicity of Cu to a
freshwater cladoceran Daphnia magna. They demonstrated
that both MWNTs and SWNTs enhanced the Cu toxicity to
animals, but the mechanisms of such interaction were
different for these two CNTs. Specically, the interaction of
MWNTs and NOM may reduce the adsorption of Cu ion by
NOM, whereas SWNTs may enhance the Cu accumulation in
the animals. However, detailed biokinetic mechanisms of
interaction between Cu or any other toxic metals and CNTs
remain essentially unknown at present.
Although the nanoparticles are expected to eventually
reach the aquatic systems (Petersen et al., 2008), the hydro-
phobic interactions of as-produced CNTs usually lead to a
strong tendency to aggregate (Lin et al., 2010). Therefore,
obtaining a long-term stable suspension is one of the most
important challenges in studying CNTs in aquatic systems. To
provide efcient and facile dispersion of CNTs in aqueous
solutions, many studies used the non-covalent dispersants
which can minimize the modication of the electronic or
mechanical properties of CNTs. In previous studies, Roberts
et al. (2007) and Kim et al. (2010) both employed LPC as the
dispersant for SWNTs in their study of D. magna. However,
these authors found that the stability of LPC coated SWNTs
was destroyed during the bioassay because of the apparent
ingestion of LPC by the animals. Alpatova et al. (2010) instead
selected polyvinyl pyrrolidone (PVP) as dispersant, which was
considered nontoxic since its 50% lethal oral dosage in rats
was 100,000 mg per kg body weight (Burnette, 1960). PVP can
successfully provide a stable SWNTs-suspension in a variety
of aqueous systems including natural waters, but there is no
report on the stability of PVP-CNTs suspensionin the presence
of aquatic animals. Maintaining a stable suspension of CNTs is
critical for the biokinetic measurements of metals in the
animals.
In this study, we specically quantied the biokinetics of
Cd and Zn in a freshwater cladocean D. magna in their
interaction with CNTs. D. magna is a lter-feeder and has
been a model organism in ecotoxicological study. Cd and Zn
are the common toxic metals found in freshwater ecosys-
tems (Guan and Wang, 2004; Tan and Wang, 2008). We
contrasted the inuences of both functionalized CNTs (F-
CNTs) and non-functionalized CNTs (NF-CNTs), as well as
single-walled CNTs (SWNTs) and multi-walled CNTs
(MWNTs), on two biokinetic processes including metal up-
take from the dissolved phase and metal assimilation from
the dietary phase. Aquatic animals accumulated metals
through multiple pathways such as the water or dietary
pathway. It is expected that food particles and CNTs may
interact with each other, but whether such interaction can
affect the metal uptake is unknown. Petersen et al. (2009)
reported that CNTs accumulation in the guts of D. magna
may inhibit the digestion of algal food. We hypothesized that
CNTs may serve as a new pathway for aquatic animals to
accumulate the toxic metals. This study can therefore pro-
vide important information on the biological fate and
transport of toxic metals as affected by different CNTs in the
aquatic environments.
2. Materials and methods
2.1. Carbon nanotubes (CNTs)
Non-functionalized-SWNTs (NF-SWNTs), non-functionalized-
MWNTs (NF-MWNTs), functionalized-SWNTs (F-SWNTs), and
functionalized-MWNTs (F-MWNTs, containing oxygen func-
tional groups at defect sites of CNTs) were purchased from
Chengdu Nanotechnology Co. with purity of 95%, 95%, 90%,
and 90%, respectively. All the CNTs were produced by catalytic
chemical vapor deposition, and were used as received. NF-
SWNTs and F-SWNTs had an inside diameter of 0.8e1.6 nm,
an outer diameter of 1e2 nm, and a length of 5e30 mm. For NF-
MWNTs and F-MWNTs, the inside diameter and length were
approximately 8e10 nm and 10e20 mm, respectively. Carboxyl
group content for F-SWNTs and F-MWNTs was 2.73% and
3.86% of weight, respectively.
2.2. Dispersion of carbon nanotubes and stability of
CNTs in suspension
The non-covalent dispersant PVP (approximately 29 kDa, from
SigmaeAldrich) was used to disperse the CNTs. In a pre-
liminary experiment, the 48-h acute toxicity test was con-
ducted using 7-d-old D. magna. The test consisted of a control
and 4 PVP concentrations (0.005, 0.05, 1, and 5 g/L), each with
four replicates of ten individuals. In each beaker, 100 mL of
test medium was allocated to the 10 daphnids. No food was
provided during the exposure period. After 48 h of exposure,
the daphnids without resuming swimming upon gentle
agitation were considered as immobilized. In this test, PVP
was not toxic to daphnids, i.e., there was no toxic symptom at
a PVP concentration as high as 5 g/L. The PVP-CNTs suspen-
sion was prepared according to the methods described by
Alpatova et al. (2010). Briey, CNTs (4.0e4.5 mg) were added
into 40 ml of 1 g/L-PVP solution (PVP dissolved in MilliQ water)
in a 40-ml glass tube. The suspension was then sonicated for
15 min using a probe sonicator (Branson) at an intensity of
60 W, and was then left to settle for 24 h. The supernatant was
collected and used as the stock solution. The concentration of
CNTs in the supernatant was 80e90 mg/L, depending on the
stability of the suspension of different CNTs.
In order to quantitatively assess the long-term stability of
CNT suspension, a combination of different characterization
methods was employed. UVeVIS spectrophotometry (8500 II)
was used to determine the concentration of CNTs in sus-
pensions at 500 nm (Bahr et al., 2001; Li et al., 2006; Lee et al.,
2007). Standard curve was constructed by recording the
different dilutions of 20 mg/L CNTs-PVP suspensions (0, 1,
2.5, 5 and 10 mg/L), which were prepared using the same
procedures as described above. The coefcient was deter-
mined and used to estimate the concentration of exfoliated
CNTs in suspensions in the following experiments. It was
difcult to reach a full extent of exfoliation, thus CNTs con-
centrations calculated from the calibration curves were the
estimated values. Meanwhile, the effective hydrodynamic
diameter and z-potential of suspended CNTs were deter-
mined after 24, 48, and 72 h of settling. The size and charge
were measured by dynamic light scattering (DLS, Brookhaven
wa t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 1 7 9 e4 1 8 7 4180
Instruments) and phase analysis light scattering techniques,
respectively.
2.3. Organisms, medium and radioisotopes
The test organisms, D. magna, were raised in ltered (GF/C
Whatman, Maidstone, UK) pond water collected from Hong
Kong University of Science and Technology campus at a
density of one individual per 10 ml of water. Before the
experiment, green algae Chlamydomonas reinhardtii cultured
previously in WC medium (Tan and Wang, 2008) were fed to
daphnids at 5 10
4
cells/mL (neonates 3 d old) or 10
5
cells/
mL (adults >3 days old) daily. The growth conditions for both
green algae and daphnids were 23.5

C with a 14: 10 h light:
dark cycle. Individual culture systems were maintained to
obtain healthy neonates and then 7-day old organisms were
used for all the tests. In all experiments, modied SM7 with a
low Ca concentration (20 mg/L) (Tan and Wang, 2008; Lin and
Xing, 2008) was used in order to prevent the potential in-
uences of high Ca concentration on the stability of CNTs
suspension. The pH of solutions was maintained at 8.0 0.2
by adding 0.01 M HCl or NaOH.
Radioactive isotopes,
109
CdCl
2
(Boston, MA, USA) and
65
ZnCl
2
(Roskilde, Denmark), were used as tracers in the pre-
sent study. Radioisotopes were diluted in 0.1 N HCl as stock
solution. The radioactivity was detected by Wallac 1480
NaI(T1) gamma detector at 88 keV for
109
Cd and 1115 keV for
65
Zn (Turku, Finland). Counting propagated errors were
generally <5%.
2.4. Daphnia effects on PVP-CNTs stability
Roberts et al. (2007) found that D. magna was able to reduce the
stability of CNTs. The effects of the presence of daphnids on
the stability of CNT suspension of different concentrations
were assessed by monitoring the concentration and zeta po-
tential of CNTs over 8e12 h. Specically, 200-ml breakers were
lled with 100 mL of test solutions (CNTs in modied SM7) at
different concentrations of CNTs (0.5, 1, 2, and 4 mg/L). Each
concentration treatment had 6 beakers. Ten 7 days old D.
magna were added to each of the three replicates of each
concentration, while no D. magna was exposed in the
remaining three replicates (serving as the control). The CNTs
concentrations (l 500 nm) was measured every 2 h over a
period of 8 h. Test solutions were not renewed and organisms
were not fed during this period of time. In a different experi-
ment, we also tested the stability of NF-SWNTs at different
concentrations (0.5, 1, 2 and 4 mg/L) without the presence of
daphnids.
2.5. Metal adsorption onto CNTs
Adsorption experiments quantied the sorption of Cd(II) and
Zn(II) onto the CNTs. The concentration of CNTs was 4 mg/L,
and
109
Cd and
65
Zn as well as Cd(II) (as CdCl
2
) and Zn(II) (as
ZnCl
2
) were added to the SM7 medium to achieve a concen-
tration of0.5 mg/L and 5 mg/L, respectively. The pH of the so-
lutions was maintained at 8.0 0.2 by adjusting with 0.01 M
HCl or NaOH. The sorption of
109
Cd and
65
Zn onto CNTs was
then measured over a period of 24 h at room temperature. At
different time intervals, a subsample was rst collected to
measure the radioactivity (representing the total radioactivity
in the aqueous phase and in the CNTs). Another sample was
then added to the ultralter (1 kDa disk membrane lter from
EMD Millipore) and ultracentrifuged (Wang et al., 2005), and
the radioactivity of the ltrates was measured. The amounts
of metals adsorbed on CNTs were calculated as the difference
between the total radioactivity in the medium and the radio-
activity in the ltrate. The % of metals adsorbed on CNTs was
then calculated as the ratio of radioactivity adsorbed to the
total radioactivity in the medium. There were triplicated
measurements at each time point.
2.6. Dissolved uptake of Cd(II) and Zn(II) inuenced by
CNTs
For NF-SWNTs and NF-MWNTs, there were 5e6 treatments
including SM7, PVP (with addition of PVP to SM7 to achieve
1 mg/ml), and 0.5, 1, 2, 4 mg (CNTs)/L in the PVP-SM7 medium.
For F-SWNTs and F-MWNTs, there were seven experimental
treatments, including SM7, PVP, 1 and 4 mg F-CNTs/L, EDTA,
EDTA1 or 4 mg F-CNTs/L. In this experiment, EDTA (0.1 mM,
sufcient to chelate all metals if they were not adsorbed) was
added into the experimental system to specically complex
with any free Cd(II) and Zn(II) (unbound with CNTs).
Before the exposure, selected daphnids of uniform size
were depurated for 2 h to clear their guts. Thirty daphnids for
each treatment were equally divided into three replicates and
placed into beakers containing 100 ml medium for 8 h. The
metal concentrations in the medium were 0.5 mg/L for Cd and
5 mg/L for Zn, respectively. Radioisotopes
109
Cd and
65
Zn were
also spiked as the radiotracers to followthe uptake behavior of
Cd and Zn by the animals. No food was added during the
experimental period in order to avoid the uptake of metals
associated with food.
At 2, 4, 6, and 8 h, daphnids were collected and rinsed with
SM7 for 1 min to remove the weakly adsorbed metals and
CNTs. Radioactivity in the animals was counted, after which
they were returned to the medium immediately. The inux
rate (I, mg/g/h) was calculated as the slope of the linear
regression between the accumulated Cd and Zn in daphnids
and the exposure time (Tan and Wang, 2008). At the end of
dissolved uptake experiment, the animals were dried at 80

C
overnight and their dry weights were recorded. The uptake
rate constant from the water (k
u
, L/g/h) was calculated as the
slope of the linear regression between the inux rate and the
Cd or Zn concentration in the medium through the origin of
the natural coordinate.
2.7. Assimilation of Cd and Zn inuenced by CNTs
We specically quantied the assimilation of metals radio-
labeled onto F-CNTs, as well as the assimilation of metals
radiolabeled onto algae as inuenced by the presence of NF-
CNTs. To radiolabel the F-CNTs,
109
Cd (1 mg/L, plus stable Cd)
and
65
Zn (5 mg/L, plus stable Zn) were rstly added to F-CNTs
suspension of different concentrations, and equilibrated for
12 h. EDTA (0.1 mM) was then added to the suspension to bind
the free metal ions. The algae C. reinhardtii were radiolabeled
by
109
Cd and
65
Zn using the methods described in Tan and
wa t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 1 7 9 e4 1 8 7 4181
Wang (2008). Briey, C. reinhardtii at the early log phase were
harvested and resuspended in a modied WC medium
(without ethylenediaminetetraacetic acid, Zn, and Cu) at an
initial cell density of 2 10
5
cells/ml. The addition of radio-
isotopes was 148 kBq/L of
109
Cd and 296 kBq/L of
65
Zn. After 3 d
growth, the algae were centrifuged and resuspended in M7
medium immediately before the experiment. The algal cell
density was determined using a hemocytometer.
For F-SWNTs and F-MWNTs, there were six treatments,
including radiolabeled algae added to SM7, radiolabeled algae
added to SM7 media amended with PVP, radiolabeled F-CNTs
(SWNTs and MWNTs) at concentrations of 2 and 10 mg/L. The
daphnids were pulse fed with radiolabeled particles (algae or
F-CNTs) for 15 min, and then depurated in SM7 medium
containing 10
5
cells/ml of nonradioactive food for 24 h. The
daphnids were assayed for radioactivity every 3 h during the
rst 12 h and every 12 h during the subsequent 12 h. The water
and food were renewed after each radioactivity measurement.
The dietary AE was calculated as the percentage of radioac-
tivity retained in the daphnids after 12 h of depuration.
Another experiment specically quantied the AEs of
metals from the radiolabeled algae in the presence of NF-
SWNTs and NF-MWNTs. Radiolabeled algae were added to
different exposure medium, including SM7, PVP, CNTs in SM7-
PVP mediumat1, 2, 4 mg/L CNTs, respectively. Each treatment
had triplicated beakers containing 10 individual daphnids.
Daphnids were then added and pulse fed the radiolabeled
algae for 15 min. The ingested radioactivity was immediately
measured. After that, 10
5
cells/ml of nonradioactive food was
added to the new corresponding medium and daphnids were
depurated for 24 h. Radioactivity in the daphnids was moni-
tored regularly and the AE was quantied as described above.
One-way analysis of variance (ANOVA) was performed to
compare metal adsorption by CNTs, dissolved uptake and
assimilation efciency of Cd and Zn.
3. Results and discussion
3.1. Stability of CNTs in suspension with and without
daphnids
The stability of PVP-facilitated CNTs suspensions in SM7 with
or without daphnids is shown in Fig. 1. The UVevis absorption
A. no daphinds
Time (h)
z
e
t
a
-
p
o
t
e
n
t
i
a
l

(
m
V
)
-60
-50
-40
-30
-20
-10
0
B. no daphnids. NF-SWNTs
Time (h)
C
N
T
s

(
m
g
/
L
)
0
1
2
3
4
5
C. with daphnids. NF-SWNTs
%

C
N
T
s

r
e
m
a
i
n
i
n
g

i
n

s
u
s
p
e
n
s
i
o
n
0
20
40
60
80
100
0.5 mg/L
1.0 mg/L
2.0 mg/L
4.0 mg/L
40
60
80
100
0.5 mg/L
1.0 mg/L
2.0 mg/L
4.0 mg/L
Time (h)
40
60
80
100
1 mg/L
2 mg/L
4 mg/L
Time (h)
0 20 40 60 80 100 0 2 8 10 12
8
4 6
0 2 4 6 0 2 4 6 8
40
60
80
100
1 mg/L
2 mg/L
4 mg/L
D. with daphnids. NF-MWNTs
E. with daphnids. F-SWNTs
F. with daphnids. F-MWNTs
Fig. 1 e (A). The z-potentials of different CNTs in suspension without the presence of daphnids. (C): NF-SWNTs; (O): NF-
MWNTs; (
;
): F-SWNTs; (D): F-MWNTs. (B). Concentrations of NF-SWNTs remaining in suspension without daphnids. (C):
0.5 mg/L; (O): 1 mg/L; (
;
): 2 mg/L; (D): 4 mg/L (C-F): % CNTs remaining in suspension with the presence of daphnids. C: NF-
SWNTs; D: NF-MWNTs; E: F-SWNTs, F: F-MWNTs. All the values are the means of 3 replicates, and the standard deviations
are generally smaller than the dot sizes.
wa t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 1 7 9 e4 1 8 7 4182
and zeta potential measurements were both employed to
measure the content and stability of CNTs as a function of
settling time. The calibration curves for the intensity of
absorbance as a function of CNTs concentration were per-
formed for all suspensions. The calibration curves were linear
(r
2
0.994) (data not shown). The zeta potentials of different
CNTs were in the order of NF-SWNTs > NF-MWNTs > F-
SWNTs > F-MWNTs, indicating that F-CNTs was better
dispersed than the NF-CNTs (Fig. 1A). F-CNTs were rich in
carboxyl and resulted in higher electrostatic repulsion due to
more surface charge. This observation was conrmed by the
time-dependent z-potential test. The concentrations of NF-
SWNTs-PVP were nearly constant throughout the exposure
period (Fig. 1B).
We also investigated the stability of CNTs-PVP suspensions
in the presence of daphnids (Fig. 1CeF). The CNT concentra-
tions measured by the optical method at 500 nm were recor-
ded for all exposure medium. All CNTs mediumwere partially
destabilized by the presence of daphnids during the 8-h
exposure. After 8 h, the concentrations of CNTs (NF-SWNTs,
NF-MWNTs, F-SWNTs, F-MWNTs) dispersed in the test me-
dium were 60e80%, 90e92%, 91e94%, 92e98% of the initial
concentrations, respectively, and for SWNTs the nanotube
aggregation was found at the bottomof test solutions. Overall,
the stability of the suspension of different CNT-based mate-
rials was in the order of F-MWNTs > F-SWNTs NF-
MWNTs > NF-SWNTs.
D. magna had an effect on the stability of PVP-CNTs, espe-
cially for NF-SWNTs, and soluble SWNTs decreased as a result
of aggregation at the bottom of test beaker. However, such
decrease in the concentration of different CNTs appeared to
be small for the F-CNTs and NF-MWNTs, and should not
signicantly affect the bioassay results within the time frames
of our biokinetic studies (<8 h). Roberts et al. (2007) employed
LPC as dispersants in their toxicity testing, and hypothesized
that LPC-CNTs may be ingested by D. magna as a result of
lter-feeding. The animals then excreted SWNTs alone to the
test media following the metabolism of LPC.
3.2. Adsorption of Cd and Zn by CNTs
CNTs with oxygen containing functional groups had great
capacity to sorb metals from water. Tian et al. (2010) reported
that metal catalyst residues in the production of commercial
CNTs could absorb the trace metals. To understand the
change in metal uptake from the dissolved phase and the
interaction of metals and CNTs, adsorption was investigated.
Fig. 2 shows the retained percentage of metals absorbed by the
CNTs. There was nearly no absorption of Cd(II) and Zn(II) onto
NF-CNTs, possibly due to the distinctive purity of different
commercial CNTs (Fig. 2A, B). The concentrations of NF-CNTs
employed in our study were much lower than those used by
Tian et al. (2010). For F-CNTs, the functional groups improved
the ion-exchange capabilities of CNTs, thus the capacity of
adsorption for metals increased correspondingly. Within the
rst 30 min, about 60% and 25% of Cd(II) was adsorbed onto F-
SWNTs and F-MWNTs, respectively, and about 50%and 20%of
Zn(II) was adsorbed onto F-SWNTs and F-MWNTs. F-CNTs had
more intense interaction with metals than the pristine CNTs,
while the distinctive adsorption capacity between F-SWNTs
and F-MWNTs was caused by the different functional group
densities.
3.3. Inuences of CNTs on metal uptake from the
dissolved phase
In all treatments, uptake of Cd and Zn from the dissolved
phase followed a linear pattern during the 8-h exposure at
different NF-SWNTs concentrations ranging from 0.5 to 4 mg/
L (not shown). The calculated dissolved uptake rates of Cd and
A. NF-SWNTs
%

m
e
t
a
l
s

r
e
m
a
i
n
i
n
g

i
n

s
o
l
u
t
i
o
n
60
70
80
90
100
Cd
Zn
B. NF-MWNTs
60
70
80
90
100
Cd
Zn
C. F-SWNTs
Time (min)
%

m
e
t
a
l
s

a
d
s
o
r
b
e
d
0
20
40
60
80
100
Cd
Zn
D. F-MWNTs
Time (min)
1 10 100 1000 1 10 100 1000
0
20
40
60
80
100
Cd
Zn
Fig. 2 e Percentages of Cd and Zn not adsorbed onto NF-SWNTs (A) and NF-MWNTs (B), and percentages of Cd and Zn
adsorbed onto F-SWNTs (C) and F-MWNTs (D). Values are mean SD (n [ 3).
wa t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 1 7 9 e4 1 8 7 4183
Zn were signicantly inuenced by different NF-SWNTs con-
centrations (Table 1). Generally, metal accumulation and k
u
decreased with increasing NF-SWNTs concentrations.
Compared to the controls, the k
u
of Cd and Zn was much
lower, and at 4 mg/L NF-SWNTs the inux rates of Cd and Zn
were only 26% of the PVP treatment. For NF-MWNTs exposure
(Table 1), in contrast to SWNTs treatments, the newly accu-
mulation patterns of Cd and Zn varied. For example, the
calculated k
u
of Cd in all NF-MWNTs treatments were signif-
icantly lower than the controls. For Zn, its accumulation in
PVP treatments was nearly 2-fold higher than that measured
at 4 mg/L NF-MWNTs after 8 h of exposure. It appeared that in
all treatments, the dissolved uptake rates of Cd and Zn were
similar betweenSWNTs and MWNTs treatments, and the k
u
of
Cd and Zn was elevated in the PVP medium.
In our study, both NF-SWNTs and NF-MWNTs signicantly
reduced the uptake of Cd and Zn with increasing NF-CNTs
concentration. Since neither NF-SWNTs nor NF-MWNTs
could adsorb metals at these exposed concentrations, such
reduced metal uptake was rather tricky. Roberts et al. (2007)
found the accumulation of SWNTs on the external surfaces
of animals. We also observed the surface adsorption of NF-
CNT in our study, and such decreased accumulation of
metals may be caused by surface physical blocking, which led
to a decreased ability of swimming in the medium (Roberts
et al., 2007).
The biokinetics of metals at different F-CNT concentra-
tions was also quantied (Fig. 3). Without the presence of
EDTA, Cd and Zn accumulation increased linearly with
exposure time at a much faster rate for all F-CNTs concen-
trations treatments. Accumulation of metals was closely
related to the F-CNTs concentration. However, the calculated
k
u
did not show statistically signicant difference among the
different treatments (data not shown).
We hypothesized that metal accumulation by daphnids
under F-CNTs exposure may result from direct ingestion. To
test this hypothesis, we also added EDTA to complex the
metal ions in the medium (i.e., any metal complexed by EDTA
was not available for biological uptake). As a result, no sig-
nicant increase of metal accumulation over the 8-h exposure
time in all EDTAtreatments was found and the slope was near
zero. However, the y-intercept increased consistently with
increasing F-CNTs concentration. Such intercept was opera-
tionally dened as the initial ingestion of labeled F-CNTs, and
strongly suggested that the higher accumulation of Cd and Zn
may be due to the ingestion of these F-CNTs by the daphnids.
The F-CNTs apparently had a differential effect on the
dissolved uptake as compared to NF-CNTs. The F-CNTs were
rich in carboxyl groups, which were capable of adsorbing
metal ions (acid adsorption), especially for the divalent metal
ions. Such sorption may be responsible for the elevated
accumulation of Zn and Cd. In order to well explain this
observation, we added EDTA to control the uptake of metals
from the aqueous phase. The data further supported our hy-
pothesis, and suggested that metals may be accumulated
through the ingestion of F-CNTs by the animals. Previously, it
was hypothesized that F-CNTs may behave similarly as the
natural organic matters (NOM), which could decrease the
dissolved uptake of metals by binding with the free ions (Guo
et al., 2001; Zhong and Wang, 2009; Tian et al., 2010). Clearly,
metals bound with F-CNTs had the potential of being taken up
by D. magna through direct ingestion of these nanomaterials.
Although the physical properties of NF-CNTs and F-CNTs
were similar, our study showed that their inuences on metal
accumulation were clearly different. F-CNTs signicantly
increased the metal accumulation, while NF-CNTs typically
reduced the metal uptake. Thus, the chemical properties
played an important role in the biokinetics of metals, and the
differential toxic effects of F-CNTs and NF-CNTs should
certainly be considered.
3.4. Inuences of CNTs on metal assimilation
Depuration of ingested metals associated with radiolabeled
algae in shown in Fig. 4. During the 24 h depuration, the per-
centage of ingested Cd and Zn retained in daphnids decreased
sharply within the initial 3 h, and then decreased at a slower
rate. However, for all CNTs treatments, the retentionof metals
leveled off more slowly than the controls. With an increase of
NF-SWNTs concentration, the metal retention also increased.
Daphnids exposed in SM7 and PVP medium had signicantly
lower AEs of Cd and Zn than those exposed at different con-
centrations of NF-SWNTs (Table 2). At the highest NF-SWNTs
concentration, the Cd AE (51.6%) was also the highest. In
contrast, signicantly lower Cd AE was observed in the SM7,
PVP and 1 mg/L NF-SWNTs treatments (28%). Similar results
were also found for Zn, with the highest AE being 3-folds of
the controls.
The dietary assimilation of Cd and Zn inuenced by NF-
MWNTs showed a similar pattern (Fig. 4). With elevating NF-
MWNTs concentration, the AEs of Cd and Zn increased at
the same time. The highest AE was 51.6%and 51.5%for Cd and
Zn, respectively, for NF-MWNTs 4 mg/L treatment (Table 2),
and the lowest AEs were found in the control treatments.
There was no signicant difference between the controls and
the low (1 mg/L) NF-MWNTs exposure.
Our study clearly suggested that the dietary assimilation of
metals from algae was inuenced by NF-CNTs. Because the
addition of NF-CNTs signicantly increased the AEs, CNTs and
radiolabeled algae were probably ingested by the animals
simultaneously, and the interactions between algae and
Table 1 e The quantied Cd and Zn k
u
(L/g/h) of Daphnia
magna in the presence of NF-SWNTs or NF-MWNTs.
Values are mean SD (n [ 3). Different letters indicates
signicant difference between the two treatments
( p <0.05).
Treatments Cd Zn
SM7 (control) 0.22 0.015
a
0.21 0.003
a
PVP (control) 0.34 0.034
b
0.31 0.018
b
NF-SWNTs (0.5 mg/L) 0.26 0.028
a
0.20 0.034
a
NF-SWNTs (1 mg/L) 0.18 0.019
c
0.15 0.018
c
NF-SWNTs (2 mg/L) 0.12 0.004
d
0.11 0.009
d
NF-SWNTs (4 mg/L) 0.09 0.009
d
0.08 0.004
d
SM7 (control 0.191 0.021
a
0.195 0.011
a
PVP (control) 0.210 0.075
a
0.218 0.025
a
NF-MWNTs (1 mg/L) 0.118 0.006
b
0.144 0.001
b
NF-MWNTs (2 mg/L) 0.082 0.004
c
0.117 0.002
c
NF-MWNTs (4 mg/L) 0.064 0.011
c
0.105 0.011
c
wa t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 1 7 9 e4 1 8 7 4184
nanotubes may prolong the gut residence time of algae. Two
possibilities may explain the elevated AEs. First, it was dif-
cult for CNTs to be assimilated by the daphnids, and these
CNTs in the guts may slow down the digestion and assimila-
tion of ingested algae. Second, the internal gut surface sorp-
tion of CNTs may interrupt their physiological activities.
Petersen et al. (2008) directly employed
14
C-labeled MWNTs
as food, and conrmed that daphnids could not fully excrete
MWNTs out of their guts after 24 h of depuration. In another
experiment (Petersen et al., 2009), they fed algae during the
same time and showed that a considerable fraction of inges-
ted CNTs was excreted (50e85%) within the rst few hours,
but little was released afterwards. In our study, the high
retention of the radioactivity of Cd and Zn in the animals may
similarly be explained by the addition of CNTs during the
whole depuration period.
A. F-SWNTs
N
e
w
l
y

a
c
c
u
m
u
l
a
t
e
d

C
d
(

g
/
g

d
r
y

w
t
)
0.0
0.5
1.0
1.5
B. F-MWNTs
0
1
2
3
D. F-MWNTs
Time (h)
0 2 4 6 8 10
0
10
20
30
40
C. F-SWNTs
Time (h)
0 2 4 6 8 10
N
e
w
l
y

a
c
c
u
m
u
l
a
t
e
d

Z
n
(

g
/
g

d
r
y

w
t
)
0
4
8
12
Fig. 3 e Newly accumulation of Cd (A and B) and Zn (C and D) by Daphnia magna during 8-h exposure in the presence of F-
CNTs. (C): SM7 as control; (O): PVP as control; (;): 1 mg CNTs/L; (D): 4 mg CNTs/L; (-): EDTA; (,) EDTAD1 mg CNTs/L; (A):
EDTAD4 mg CNTs/L. Values are mean SD (n [ 3).
A. NF-SWNTs (Cd)
0
20
40
60
80
100
C. NF-SWNTs (Zn)
Time (h)
%

C
d

r
e
t
a
i
n
e
d

i
n

d
a
p
h
n
i
d
s
0
20
40
60
80
100
B. NF-MWTs (Cd)
0
20
40
60
80
100
D. NF-MWNTs (Zn)
Time (h)
0 5 10 15 20 25 0 5 10 15 20 25
0
20
40
60
80
100
%

Z
n

r
e
t
a
i
n
e
d

i
n

d
a
p
h
n
i
d
s
Fig. 4 e Retention of Cd (A and B) and Zn (C and D) in D. magna in the presence of different concentrations of CNTs during the
24-h depuration. The daphnids were previously pulse fed with radiolabeled algae for 15-min. (C): SM7; (O): PVP; (
;
): 1 mg
CNTs/L; (D): 2 mg CNTs/L; (-): 4 mg CNTs/L. Values are mean SD (n [ 3).
wa t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 1 7 9 e4 1 8 7 4185
Radiolabeled F-CNTs were also employed to measure the
AEs of metals. Algae were then added during the depuration
time. Fig. 5 shows the retention of metals bound directly with
F-CNTs. For Cd, both F-SWNTs and F-MWNTs treatments did
not show signicant difference from the control (SM7 and PVP
treatments, radiolabeled algae). The AEs for F-SWNTs and F-
MWNTs were dependent on their concentrations (Table 3).
Similar results were found for Zn, but the AEs of F-SWNTs and
F-MWNTs (29.6e34.6%) were signicantly higher than those of
the controls (12e14%). Overall, metals bound with F-CNTs
could be ingested by D. magna, and were difcult to be
removed by daphnids after 24 h of depuration. The slow
depuration of metals bound with F-CNTs was similar to the
depuration of
14
C-labeled MWNTs examined by Petersen et al.
(2009). The difference of AEs between Cd and Zn from radio-
labeled F-CNTs may be explained by the sorption capacity F-
CNTs (Zn(II) >Cu(II) >Cd(II) (Rao et al., 2007). Cd may be easier
to desorb from CNTs than Zn, and was thus less inuenced by
the CNTs concentration.
In order to address the mechanisms of NF-CNT and F-CNT
interaction with daphnids, further understanding of the
ingestion process is necessary. The ltering apparatus of D.
magna had a ne mesh size from 240 nm to 640 nm (Gophen
and Geller, 1984; Lewinski et al., 2010). Food particles are
captured by setae, and then transported to food groove. CNTs
in the present study ranged from 30 nm to 40 nm (data not
shown), and could be captured by daphnids during their
feeding activity. Generally, given the relatively small particle
sizes of CNTs, D. magna may have low ingestion rate of these
nanoparticles, particularly for the better dispersed F-CNTs.
Thus, carbon nanotubes associated with algae were probably
more efciently ingested by the animals than the only func-
tionalized CNTs.
4. Conclusion
A better understanding of the interaction of CNTs and metals
can help predict the fate and transport of contaminants in
aquatic ecosystem. In the present study, inuences of carbon
nanotubes on metal uptake from the dissolved phase and
assimilation from the dietary source were investigated. The
aqueous uptake of Cd(II) and Zn(II) by D. magna was greatly
reduced by NF-CNTs. In contrast, higher metal uptake was
documented in the presence of F-CNTs. For the dietary accu-
mulation of Cd and Zn, NF-CNTs signicantly facilitated the
retention of these metals in the animals. Metals bound with F-
CNTs were also efciently retained by the animals. This study
for the rst time demonstrated that metal accumulation in D.
magna was affected by different carbon nanotubes, and
demonstrated that CNTs may either reduce or enhance metal
accumulation.
Table 2 e The ingestion rate (g/g/day) of D. magna and the
dietary assimilation efciencies of Cd and Zn in the
presence of NF-SWNTs or NF-MWNTs. Values are
mean SD (n [ 3). Different letters indicates signicant
difference between the two treatments ( p <0.05).
Treatments Ingestion rate AE (%)
Cd Zn
SM7 (control) 1.24 0.13
a
27.6 3.5
a
17.0 0.9
a
PVP (control) 1.17 0.03
a
27.8 2.1
a
13.0 0.2
a
NF-SWNTs (1 mg/L) 1.27 0.03
a
28.7 6.8
a
22.1 4.3
a
NF-SWNTs (2 mg/L) 0.42 0.10
b
49.6 9.3
b
43.1 3.1
b
NF-SWNTs (4 mg/L) 0.15 0.01
c
51.6 4.9
c
50.9 10.2
b
SM7 (Control) 1.20 0.05
a
26.3 3.2
a
26.9 1.1
a
PVP (control) 1.16 0.02
a
27.7 2.3
a
21.8 0.6
a
NF-MWNTs (1 mg/L) 1.15 0.08
a
29.7 2.8
a
28.7 3.8
a
NF-MWNTs (2 mg/L) 0.45 0.06
b
39.6 2.5
b
43.6 4.3
b
NF-MWNTs (4 mg/L) 0.17 0.00
c
51.6 5.0
c
51.5 5.0
b
Cd
%

C
d

r
e
t
a
i
n
e
d

i
n

d
a
p
h
n
i
d
s
0
20
40
60
80
100
Zn
Time (h)
0 5 10 15 20 25
%

Z
n

r
e
t
a
i
n
e
d

i
n

d
a
p
h
n
i
d
s
0
20
40
60
80
100
Fig. 5 e Retention of Cd and Zn in D. magna during the 24-h
depuration following the 15-min pulse exposure to
radiolabeled F-CNTs or algae. (C): radiolabeled algae in
SM7; (O): radiolabeled algae in PVP; (D): radiolabeled F-
SWNTs at 2 mg/L; (
;
): radiolabeled F-MWNTs at 2 mg/L;
(,): radiolabeled F-SWNTs at 10 mg/L; (-): radiolabeled F-
MWNTs at 10 mg/L. Values are mean SD (n [ 3).
Table 3 e The assimilation efciencies of Cd and Zn
sorbed with F-SWNTs or F-MWNTs in D. magna. Values
are mean SD (n [3). Different letters indicates
signicant difference between the two treatments
( p <0.05).
Treatments AE (%)
Cd Zn
SM7 (control) 27.5 3.5
a
17.0 0.9
a
PVP (control) 27.7 2.1
a
13.0 0.3
a
F-SWNTs (2 mg/L) 24.3 2.6
a
35.9 5.3
b
F-SWNTs (10 mg/L) 31.6 2.3
b
32.0 3.8
b
F-MWNTs (2 mg/L) 26.8 1.6
a
37.3 4.7
b
F-MWNTs (10 mg/L) 33.2 2.8
b
27.3 2.5
b
wa t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 1 7 9 e4 1 8 7 4186
Acknowledgments
We thank the two anonymous reviewers for their comments.
This study was supported by a General Research Fund
from the Hong Kong Research Grants Council (663011) to
W.-X. Wang.
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