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Cellular Partitioning of Nanoparticulate versus Dissolved Metals in
Marine Phytoplankton
Gretchen K. Bielmyer-Fraser,*,† Tayler A. Jarvis,† Hunter S. Lenihan,‡ and Robert J. Miller§
†
Valdosta State University, 1500 North Patterson Street, Valdosta, Georgia 31698, United States
Bren School of Environmental Science and Management, University of California, Santa Barbara, California 93106, United States
§
Marine Science Institute, University of California, Santa Barbara, California 93106, United States
‡
ABSTRACT: Discharges of metal oxide nanoparticles into aquatic environments are increasing with their use in society, thereby increasing exposure risk
for aquatic organisms. Separating the impacts of nanoparticle from dissolved
metal pollution is critical for assessing the environmental risks of the rapidly
growing nanomaterial industry, especially in terms of ecosystem effects. Metal
oxides negatively affect several species of marine phytoplankton, which are
responsible for most marine primary production. Whether such toxicity is
generally due to nanoparticles or exposure to dissolved metals liberated from
particles is uncertain. The type and severity of toxicity depends in part on
whether phytoplankton cells take up and accumulate primarily nanoparticles or
dissolved metal ions. We compared the responses of the marine diatom,
Thalassiosira weissf logii, exposed to ZnO, AgO, and CuO nanoparticles with the
responses of T. weissf logii cells exposed to the dissolved metals ZnCl2, AgNO3,
and CuCl2 for 7 d. Cellular metal accumulation, metal distribution, and algal population growth were measured to elucidate
differences in exposure to the different forms of metal. Concentration-dependent metal accumulation and reduced population
growth were observed in T. weissf logii exposed to nanometal oxides, as well as dissolved metals. Significant effects on population
growth were observed at the lowest concentrations tested for all metals, with similar toxicity for both dissolved and
nanoparticulate metals. Cellular metal distribution, however, markedly differed between T. weissf logii exposed to nanometal
oxides versus those exposed to dissolved metals. Metal concentrations were highest in the algal cell wall when cells were exposed
to metal oxide nanoparticles, whereas algae exposed to dissolved metals had higher proportions of metal in the organelle and
endoplasmic reticulum fractions. These results have implications for marine plankton communities as well as higher trophic
levels, since metal may be transferred from phytoplankton through food webs vis à vis grazing by zooplankton or other pathways.
■
applications.10 Exposure of aquatic organisms to nanoparticles
is difficult to quantify; however, some modeling efforts have
reported the presence of metal oxides in aquatic systems at
levels that may cause toxicity.11
Phytoplankton require the essential metals zinc (Zn) and
copper (Cu) for enzyme functioning, but at elevated
concentrations, these metals may exert toxicity.12−14 Most
ecotoxicity work on phytoplankton has been done using
dissolved metals, with a few recent studies on nanoparticles. For
example, ZnO nanoparticles are known to reduce population
growth rates in the diatoms Skeletonema marinoi, Thalassiosira
pseudonana, and Thalassiosira weissf logii.13,14 Metal oxide
nanoparticles dissolve in seawater to varying degrees, and
release free metal ions,15 which may be the predominant cause
of toxicity to aquatic organisms. However, the dynamics of
dissolution and release of metal ions, and how these processes
vary with concentration and material type is poorly understood.
INTRODUCTION
Diatoms are the dominant primary producers in the ocean, and
because they are small and have a high surface-to-volume ratio,
they can take up and accumulate substantial amounts of
contaminants.1−4 This uptake may cause multiple impacts to
marine food webs: reductions in population growth rate and
possibly nutritional content of phytoplankton cells could
reduce resources available for consumers,5 and accumulation
of contaminants in phytoplankton can lead to trophic transfer
and resulting toxic effects on consumers.6 Metals are an
important class of such contaminants, and now are being
discharged into coastal ecosystems as nanomaterials in addition
to traditional bulk and dissolved forms. Nanomaterials are now
widely utilized for their enhanced mechanical and optical
properties, as well as their efficient electrical conductivity,
relative to larger forms of similar materials.7 Because of their
growing application in an array of fields including electronics,
chemical, cosmetics, and biomedicine,7,8 nanomaterials are
emerging as a new class of contaminants, with unknown
environmental consequences.9 Metal oxide nanomaterials in
particular are commonly used because they are synthesized
relatively easily and have myriad industrial and consumer
© 2014 American Chemical Society
Received:
Revised:
Accepted:
Published:
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March 25, 2014
October 13, 2014
October 22, 2014
October 22, 2014
dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450
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prior to inoculation. Synthetic seawater was made by mixing
Instant Ocean salt with 18 mΩ Milli-Q water and aerating at
least 24 h before use.32 The sterilized media was inoculated
with 2.5 × 105 algal cells and the algae were cultured for 7 d
with continuous aeration. Algae were incubated under cool
white fluorescent lights (12 h light: 12 h dark) at a light
intensity of 36 μmol photons m−2 s−1 and a temperature of 20
°C. Algal densities were measured using a hemocytometer
(Hausser Scientific, Horsham, PA) and a compound microscope.
Nanoparticles. ZnO nanoparticles were obtained from
Meliorum Technologies (Rochester, NY, USA) and characterized for size, morphology and chemical composition.33,34
ZnO nanoparticles were spheroid, 100% zincite, and 20−30 nm
in diameter. CuO nanoparticles were obtained from SigmaAldrich (St. Louis, MO, USA) and described as <50 nm and
99.8% pure; characterization with ICP-OES and TEM showed
that they were 84.8 ± 2.7% pure (impurities included Na, Ca,
Si, and Mg) and 20−100 nm in diameter.35 AgO nanoparticles
were obtained from QuantumSphere Inc. (Santa Ana, CA) and
described as 20−30 nm in diameter. TEM characterization
showed that the AgO particles were 20−70 nm in diameter, and
no detectable impurities were found.
Experimental Design. T. weissf logii was cultured as
described above, but using F/2 nutrients without trace metals
and without ethylenediaminetetraacetic acid (EDTA). T.
weissf logii was exposed to ZnO, AgO, and CuO nanoparticles,
as well as ZnCl2, AgNO3, and CuCl2 for 7 d. Nanomaterial
stock suspensions were prepared by adding 10 mg of metal
oxide nanoparticles to 10 mL of ultra pure 18 mΩ Milli-Q
water, vortexing for 30 s, sonicating for 30 min, dilution (1:10)
with 30 ppt synthetic seawater containing 10 mg L−1 alginate
(previously made in a 1 mg L−1 stock in 18 mΩ Milli-Q water),
and vortexing again for 30 s. Dissolved metal solutions were
prepared by adding a 10 mg L−1 stock solution of each metal
(Zn as ZnCl2, Ag as AgNO3, and Cu as CuCl2) to synthetic
seawater (30 ppt salinity). Different volumes of each stock
solution were added separately to the 1 L Erlenmeyer flasks to
achieve the following nominal concentrations: 0.1, 0.5, and 1.0
mg L−1 of ZnO and ZnCl2, 1.0 and 10 mg/L of AgO and
AgNO3, and 0.25 and 5.0 mg L−1 of CuO and CuCl2. A metalfree control treatment was also included in each experiment.
Metal concentrations were chosen from past studies reporting
growth effects in phytoplankton.6,12,14 Before inoculation with
T. weissf logii and at 1, 3, 5, and 7 d during the experiment water
samples were collected in 15 mL polypropylene centrifuge
tubes from each flask, filtered (0.45 μm), and acidified with
trace metal grade nitric acid (Fisher Scientific, Pittsburgh, PA)
for later metal analysis. Measured dissolved metal concentrations are presented in Table 1. For the nanoparticle
exposures, we took the difference between nominal and
dissolved concentrations to represent the portion of the
metal in nanoparticle form; thus the nanoparticle exposures
represented a combination of dissolved and nanoparticulate
metal. There were three replicate flasks per treatment.
Approximately 5 × 108 cells L−1 were added to each flask at
the start of the experiments. The cultures were continuously
aerated with sterile 1 mL pipettes and stirred once a day by
swirling. Algal cell density was measured at 0, 3, 5, and 7 d. The
algae were exposed to the nanoparticles and dissolved metals
throughout log phase growth. At the end of the exposure
period, the algae were concentrated by centrifugation in 50 mL
polypropylene centrifuge tubes at 3500 rpm for 15 min.
Greater understanding of these dynamics is needed to elucidate
differences in metal adherence, uptake, and accumulation
patterns in phytoplankton exposed to nanoparticulate metals
and dissolved metals.
Metal ions typically traverse algal cell membranes through
ion/voltage-gated channels16 and once inside the cell, exert
toxicity by binding to metabolic sites normally occupied by
other essential ions.17,18 Nanoparticles can directly diffuse
across the membrane in some cases (i.e., small in size; positive
ions on the surface of nanoparticles), pass through ion channels
and transporter proteins, or enter the cell through endocytosis.19,20 Once inside the cell, nanoparticles can interact with
oxidative organelles such as mitochondria, and cause oxidative
stress by disrupting the balance between oxidant and
antioxidant processes and generation of reactive oxygen
species.21−23
Depressed growth of phytoplankton populations can cascade
to reduce production of primary consumers, especially
zooplankton and larval fish. In addition, accumulated metal in
phytoplankton may be a significant source of dietary metals to
primary consumers.12 However, pathways and patterns of metal
accumulation and transfer to consumers are difficult to predict
and thus not well understood. Several studies have shown that
the chemical form of the metal within the prey can influence
the assimilation of metals by consumers,24−29 and different
subcellular fractions of accumulated trace metals vary in their
trophic availability to consumers, perhaps reflecting ligand
binding.24,25 Metals bound to different ligands, such as proteins
or other organic molecules, could correspondingly differ in
bioavailability.24,30 For example, Cheung and Wang29 demonstrated differences in the assimilation efficiency and transfer of
cadmium (Cd), Zn, and silver (Ag) to the gastropod Thais
clavigera depending on the subcellular compartmentalization of
the metals in the prey (barnacles, oysters, mussels, snails, and
limpets). Some researchers have suggested that the most
bioreactive metal fraction in organisms is that which is bound
to metallothioneins and possibly small peptides like glutathione.24,30
In a prior study, we found significantly reduced survival and
reproduction of the copepod Acartia tonsa fed a diet of
nanoparticulate ZnO-exposed diatoms.6 The dietary zinc
concentrations causing effects to the copepod consumers
(10−20 ug g−1 dw) were similar to those reported to cause
effects in copepods fed diets (3−14 ug g−1 dw) previously
exposed to dissolved zinc.6,12 However, the effects of the diets
on copepods were different: nanoparticulate ZnO-exposed diets
decreased copepod survival most severely, whereas dissolved
zinc-exposed diets affected reproduction to the greatest
degree.6,12 We hypothesized that this difference was due to
differential partitioning of the two metal forms in the diatom
cells. In this study, we examined the accumulation of
nanoparticulate metal oxides (nZnO, nAgO, and nCuO) and
corresponding dissolved metals (ZnCl2, AgCl, and CuCl2) in
the widespread coastal marine diatom, Thalassiosira weissf logii,
and compared the effects of the different materials on diatom
population growth rates.
■
METHODS
Algal Culturing. Thalassiosira weissf logii was obtained from
AlgaGen LLC (Vero Beach, FL) and cultured in autoclaved 1 L
Erlenmeyer flasks following APHA guidelines.31 The algal
medium consisted of 300 mL of synthetic saltwater (30 ppt
salinity) and F/2 nutrients, and was autoclaved and cooled
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Table 1. Mean ± Standard Deviations of Measured
Dissolved Metal Concentrations in the Thalassiosira
weissf logii Media during Exposure to Controls, ZnO, Zn as
ZnCl2, AgO, Ag, as AgNO3, CuO, and Cu as CuCl2 for 7 d
Methyldiaminetetraacetic acid (EDTA), to remove loosely
bound metal from the cell wall, and then algal cells were
centrifuged at 3500 rpm for 15 min. The supernatant fractions
(representing loosely bound metals washed from the algal
surface) were preserved in 15 mL centrifuge tubes. The
remaining algal pellets were resuspended in 1 mL of purified 18
mΩ Milli-Q water and sonicated for approximately 1 min to
lyse the algal cells, which was verified using a light microscope.
The sample was then differentially centrifuged to separate the
cells into cell wall, organelle, endoplasmic reticulum, and
cytosol fractions, using previously established methods.12
Briefly, the lysed cells were centrifuged at 4000 rpm for 15
min to obtain the cell wall fraction. The organelle fraction was
then obtained by centrifuging the remaining supernatant for 30
min at 10 000 rpm. The supernatant was then centrifuged at
100,000 rpm for 1 h to obtain the total membrane fraction
(endoplasmic reticulum). The remaining supernatant was the
cytosol fraction. Each fraction was digested by addition of 1 mL
of trace metal grade nitric acid (Fisher Scientific, Pittsburgh,
PA) and 1 mL of purified 18 mΩ Milli-Q water and then
analyzed for metal concentration as described below. Data are
presented as accumulated metal per gram algal dry weight
(Table 2). This measurement includes metal in both dissolved
and particulate form.
Metal Analysis. Water samples and digested algae fractions
were diluted with 18 mΩ Milli-Q water, and analyzed for metal
concentration using graphite furnace atomic absorption
spectrophotometry (GFAAS; PerkinElmer, AAnalyst 800).
Certified 1 g/mL metal standards dissolved in 2% HCl (Fisher
Chemical, Fairlawn, NJ) were used for GFAAS analysis of each
metal. Detection limits for each metal were 1 μg L−1, based on
these protocols. Standards were analyzed at the beginning and
end of each analysis.
Data Analysis. Statistical differences between treatments (p
< 0.05; n = 3) were calculated using Analysis of Variance
(ANOVA) followed by a posthoc Tukey’s test to determine
measured metal (μg L−1)
nominal metal concentration
control Zn
10 μg L−1 ZnO
10 μg L−1 Zn
100 μg L−1 ZnO
100 μg L−1 Zn
500 μg L−1 ZnO
500 μg L−1 Zn
1000 μg L−1 ZnO
1000 μg L−1 Zn
control Ag
1000 μg L−1 AgO
1000 μg L−1 Ag
10000 μg L−1 AgO
10000 μg L−1 Ag
control Cu
250 μg L−1 CuO
250 μg/L Cu
5000 μg L−1 CuO
5000 μg L−1 Cu
3.92
9.41
8.40
94.2
94.4
203
461
301
920
1.80
321
828
1275
8152
2.97
78.0
173
769
2506
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.80
0.70
0.99
4.32
9.60
20.2
26.1
22.5
42.5
0.40
49.0
91.0
38.6
479
0.86
0.95
33.0
54.0
857
Subsamples of the concentrated algae were collected for the
following additional procedures. First, the algae was filtered and
washed with 0.5 M ammonium formate (to reduce the salt from
the algae while maintaining the osmotic gradient for calculation
of algal dry weight), and then dry weights were determined by
drying multiple aliquots of algae from each treatment in
preweighed aluminum weigh boats in an oven at 80 °C for 1 d.
Second, Zn, Ag, and Cu distribution within the algal cells was
determined.
Metal Distribution in Algal cells. The intact algal cells
from each treatment were washed with a metal chelator, 0.01
Table 2. Cellular Metal Distribution (Mean ± Standard Deviation × 10−9; μg Dissolved Metal/g Dry Weight) in Thalassiosira
weissf logii after Exposure to Controls, Metal Oxide Nanoparticles (ZnO, AgO, and CuO), or Dissolved Metals (Zn as ZnCl2, Ag
as AgNO3, and Cu as CuCl2) for 7 d (n = 3)
nominal metal concentration
control Zn
0.01 mg L−1 ZnO
0.01 mg L−1 Zn
0.1 mg L−1 ZnO
0.1 mg L−1 Zn
0.5 mg L−1 ZnO
0.5 mg L−1 Zn
1.0 mg L−1 ZnO
1.0 mg L−1 Zn
control Ag
1.0 mg L−1 AgO
1.0 mg L−1 Ag
10 mg L−1 AgO
10 mg L−1 Ag
control Cu
0.25 mg L−1 CuO
0.25 mg L−1 Cu
5.0 mg L−1 CuO
5.0 mg L−1 Cu
a
EDTA wash
0.58
17.2
5.49
19.4
16.0
26.7
58.7
50.9
120
1.15
6.08
4.27
14.6
16.1
1.02
5.78
6.79
14.9
17.3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.1
14
1.6
18
2.8
8.4
15
0.3
19
0.4
1.2
0.7
4.0
3.7
0.2
2.2
1.9
0.2
2.4
cell wall
0.77
48.0
1.93
105
7.80
227
18.4
204
31.0
0.98
120
34.6
329
111
1.35
108
53.0
626
112
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.2
14
0.6
54
0.9
22
9.2
12
3.5
0.0
33
8.0
90
20
0.3
18
14
40
23
organelles
0.83
13.4
2.13
33.0
9.12
39.5
35.3
50.9
72.5
1.04
12.3
72.1
51.6
116
2.86
12.1
68.5
40.1
143
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.3
1.6
0.5
1.3
1.6
2.1
10
0.0
3.1
0.3
2.7
12
17
19
0.3
3.7
20
14
29
endoplasmic reticulum
0.68
10.6
2.50
19.0
6.78
16.7
13.8
26.4
24.1
0.93
5.99
19.8
10.1
47.4
0.88
4.46
46.6
26.0
85.6
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.2
3.1
0.3
2.2
1.0
0.5
6.3
0.8
3.0
0.1
1.5
2.6
2.1
8.4
0.3
1.5
14
8.3
16
cytosol
totala
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
4.09
100
14.1
223
45.7
342
142
369
175
5.06
160
145
410
313
7.19
141
190
738
400
1.23
11.3
2.10
34.5
5.60
35.2
15.4
37.2
30.2
0.95
15.6
13.8
4.74
22.7
1.08
11.0
14.7
30.6
42.0
0.1
3.8
0.8
2.8
0.7
3.6
8.3
12
3.2
0.2
2.5
3.0
1.3
3.1
0.1
6.5
3.3
7.1
11
Indicates the calculated total.
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differences between treatments. Data were first assessed for
normality and homogeneity of variances using the Shapiro−
Wilk’s test and Bartlett’s test, respectively.
■
RESULTS
Metal Concentrations. Concentrations of Zn, Ag, and Cu
in the experimental media spiked with ZnCl2, AgNO3, and
CuCl2 were similar to nominal concentrations, with values
averaging 85% of nominal values (Table 1). In contrast,
concentrations of dissolved Zn, Ag, and Cu in the experimental
media spiked with nZnO, nAgO, and nCuO were considerably
lower than nominal values, particularly at higher concentrations
(Table 1). For nZnO, Zn concentrations were similar to
nominal values at 0.1 mg L−1; however, at 0.5 and 1.0 mg L−1,
values were two and 3-fold lower, respectively, than those
values for ZnCl2 at the same nominal concentrations (Table 1).
For 1 and 10 mg L−1 nAgO, Ag concentrations were
approximately 3- and 7-fold lower than those observed for
AgNO3. Likewise, for 0.25 and 5 mg L−1 nCuO, Cu
concentrations were approximately 2- and 3-fold lower than
those observed for CuCl2 (Table 1).
Metal Toxicity to T. weissf logii. Exposure of T. weissf logii
to increasing metal concentrations (both as nanoparticles and
dissolved metals) resulted in significantly reduced population
growth (Figures 1−3). Algal growth curves were similar
between nAgO and AgNO3 exposures and between nCuO
Figure 2. Thalassiosira weissf logii growth over 7 d after exposure to
various concentrations of (A) AgO nanoparticles and (B) Ag as
AgNO3. Asterisks indicate significant differences from control values
(p < 0.05; n = 3).
and CuCl2 exposures but some differences were observed for
Zn (Figures 1−3). T. weissf logii exposed to ZnCl2 had a
significantly greater growth reduction than algae exposed to
nZnO by 7 d (Figure 1). Lowest observable effect
concentrations (LOECs; dissolved metal concentrations only)
were 94.2 and 94.4 μg L−1 for nZnO and ZnCl2 exposures, 321
and 828 μg L−1 for nAgO and AgNO3 exposures, and 78.0 and
173 μg L−1 for nCuO and CuCl2 exposures, respectively.
Metal Accumulation and Distribution in T. weissf logii.
Metals accumulated in a concentration-dependent manner in T.
weissf logii after exposure to both metal oxide nanoparticles and
dissolved metals. All cell fractions of metal-exposed algae were
significantly elevated above the control cell fractions. Additionally, metal accumulation patterns were significantly different
depending on the form of metal exposed (Figures 4−6; Table
2).
In general, T. weissf logii exposed to metal oxide nanoparticles
had higher proportions of metals in the cell wall fractions
compared with organelle fractions. By contrast, higher
proportions of metals were observed in the organelle fraction
after exposure to dissolved metals (Figures 4−6; Table 2).
Significantly higher Zn concentrations (p < 0.05; n = 3) were
observed in nZnO-exposed cell fractions compared with the
corresponding fractions from cells exposed to ZnCl2 (e.g., cell
wall from ZnO-exposed algae compared to cell wall from
ZnCl2-exposed algae), with only one exception (1 mg L−1,
EDTA fraction). Significant differences (p < 0.05; n = 3) were
also observed between all nAgO-exposed cell fractions and
Figure 1. Thalassiosira weissflogii growth over 7 d after exposure to
various concentrations of (A) ZnO nanoparticles (as seen in Jarvis et
al. 2013) and (B) Zn as ZnCl2. Asterisks indicate significant differences
from control values (p < 0.05; n = 3).
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Figure 5. Cellular metal distribution (mean ± standard deviation; μg
metal/cell) in Thalassiosira weissf logii after exposure to AgO
nanoparticles and Ag as AgNO3 for 7 d (n = 3). Asterisks indicate
significant differences between a cellular fraction at a specific
concentration of either metal oxide nanoparticles or dissolved metals
(p < 0.05; n = 3). ER = endoplasmic reticulum. All metal-exposed
fractions were significantly higher than controls.
Figure 3. Thalassiosira weissflogii growth over 7 d after exposure to
various concentrations of (A) CuO nanoparticles and (B) Cu as
CuCl2. Asterisks indicate significant differences from control values (p
< 0.05; n = 3).
Figure 6. Cellular metal distribution (mean ± standard deviation; μg
metal/cell) in Thalassiosira weissf logii after exposure to CuO
nanoparticles and Cu as CuCl2 for 7 d (n = 3). Asterisks indicate
significant differences between a cellular fraction at a specific
concentration of either metal oxide nanoparticles or dissolved metals
(p < 0.05; n = 3). ER = endoplasmic reticulum. All metal-exposed
fractions were significantly higher than controls.
weissf logii to AgNO3 and CuCl2 as compared with nAgO and
nCuO (Figures 5 and 6; Table 2). The proportion of metal
loosely bound to the cell wall (liberated from the EDTA wash)
after exposure to nanoparticle or dissolved metals were similar
or slightly lower when exposed to dissolved metals (Figures
4−6; Table 2). The total cellular concentrations were similar
between nanoparticulate and dissolved Ag and Cu, yet
significantly more Zn accumulated in algae exposed to nZnO
compared with ZnCl2 (Figures 4−6; Table 2). The ratio of total
Zn accumulated after exposure to ZnO as compared with
exposure of ZnCl2 approximated 5, 2.5, and 2, for the 0.1, 0.5,
and 1.0 mg L−1 treatments, and largely corresponded to Zn
accumulation in the cell wall fractions (cell wall and EDTA
washed; Table 2).
Figure 4. Cellular metal distribution (mean ± standard deviation; μg
metal/cell) in Thalassiosira weissf logii after exposure to ZnO
nanoparticles and Zn as ZnCl2 for 7 d (n = 3). Asterisks indicate
significant differences between a cellular fraction at a specific
concentration of either metal oxide nanoparticles or dissolved metals
(p < 0.05; n = 3). ER = endoplasmic reticulum. All metal-exposed
fractions were significantly higher than controls.
AgNO3-exposed cellular fractions except one (10 mg L−1,
EDTA fraction) and between all nCuO-exposed cell fractions
and CuCl2-exposed cellular fractions. For Ag and Cu,
proportionally higher metal concentrations were observed in
the endoplasmic reticulum fraction after exposure of T.
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Article
DISCUSSION
The differences between dissolved and nominal metal
concentrations demonstrated in Table 1 reflect the different
exposure scenarios, with nanoparticulate ZnO, AgO, and CuO
solutions containing nanoparticle and dissolved metals, and
ZnCl2, CuCl2, and AgNO3 solutions containing solely dissolved
metal. The dissolution of ZnO, AgO, and CuO nanoparticles
was probably higher at the lower exposure concentration, a
process well characterized in our previous study.6 Cytoxicity
can result from both metal oxide nanoparticles and dissolved
metal exposure.12,13,20 In the present study, cytotoxicity
resulted in decreased population growth of T. weissf logii after
exposure to both metal oxide nanoparticles and dissolved
metals, similar to previous results.13 Miller et al.14 reported
decreased population growth rates in the marine phytoplankton
species Skeletonema marinoi, Isochrysis galbana, and Dunaliella
tertiolecta after exposure to 1000 μg L−1 ZnO nanoparticles and
in Thalassiosira pseudonana after exposure to 500 μg L−1 ZnO
nanoparticles. Similarly, population growth of the algal species
T. pseudonana, Chaetoceros gracilis, and Phaeodactylum tricornutum was reduced after exposure to 10 mg L−1 ZnO
nanoparticles for 100 h.27 Franklin et al.13 reported toxicity
values (72-h IC50−60 μg L−1 Zn) for both ZnO nanoparticles
and ZnCl2 to the freshwater alga, Pseudokirchneriella subcapitata, and attributed toxicity solely to dissolved Zn. Likewise,
Aruoja et al.36 reported an IC50 value of 0.71 mg Cu L−1
(similar to the results in this study) for growth of the algae
Pseduokirchneriella subcapitata after exposure to CuO nanoparticles, where much of the toxicity was attributed to liberation
of soluble Cu ions. At lower metal concentrations, metal
nanoparticles may more quickly dissolve in solution, complicating results for short-term assays.6 Nevertheless, we found that
toxicity levels were similar for nanoparticulate and dissolved
forms of Zn, Cu, and Ag at the concentrations tested. This
contradicts results of Shi37 who reported greater toxicity (i.e.,
decreased chlorophyll concentration) of nCuO than ionic Cu in
duckweed, a freshwater plant, thus illustrating that results may
be taxon- and media-dependent. More studies should be
performed at lower exposure levels in the future to better
elucidate differences in toxicity between the different forms of
metals.
Compared with controls, significantly higher metal concentrations were observed in T. weissf logii cells after exposure to
metals in nanoparticulate or dissolved form. The Cu and Zn
concentrations in the control algae were similar to past studies
and likely reflect their essentiality.12 Silver is a nonessential
metal, yet the control algae contained low concentrations,
perhaps resulting from low Ag concentrations (∼2 ppb) in the
dilution water and values near the detection limit of the
GFAAS. These values were negligible and at least 2 orders of
magnitude below the concentrations measured in the Agexposed algae. Metal accumulation in T. weissf logii cells were
well within the range of values reported in the literature.6,12,38
DeSchamphelaere et al.37 reported a Zn concentration of 490
μg g−1 dw in the alga, Pseudokirchneriella subcapitata, after
exposure to 60 μg L−1 and Guan and Wang39 reported Zn
concentrations ranging from 27.2 to 280 μg g−1 dw in the alga,
Chlamydomonas reinhardtii, after exposure to 2 to 200 μg L−1
Zn.
The distribution of metal within the treated algal cells
differed, depending on the form of metal in the exposure media.
Exposure of T. weissf logii to metal oxide nanoparticles resulted
in a relatively higher proportion of metal in the cell wall
fraction, whereas exposure to dissolved metals resulted in a
relatively higher proportion of metal in the organelle, and in
some instances, endoplasmic reticulum fractions. Peng et al.40
reported Zn bioaccumulation in three algal species, Thalassiosira pseudonana, Chaetoceros gracilis, and Phaeodactylum
tricornutum after exposure to 10 mg L−1 ZnO nanoparticles
for 100 h. The ZnO-exposed algae exhibited apparent cell wall
damage and internal degradation, characterized by a lack of
recognizable organelles that was attributed to release of
dissolved Zn upon partial dissolution. Histology was not
assessed in the current study but it is plausible given the metal
accumulation pattern that metal oxide nanoparticles may cause
more damage to the cell wall than dissolved metals, and
absorption of both forms of metals may cause damage to the
organelle fraction. The greater proportion of metals in the cell
wall fraction, as compared to the internal fractions, after
nanoparticulate metal exposure may be due to size exclusion of
the nanoparticles; dissolved metals may move relatively quickly
as ions through channels into the cell.16 This may be
exacerbated by aggregation of nanoparticles, which was not
evaluated in this study but is expected to occur at levels that
vary with the organic matter content of the media and other
factors.15
The internal distribution of metals in the algal cells also
varied to some degree, depending on the form of metal used in
the exposure media. The control T. weissf logii and the algae
exposed to ZnO nanoparticles had a higher proportion of Zn in
the cytosol than in the endoplasmic reticulum. After exposure
of T. weissf logii to dissolved Zn, the proportion of Zn in the
endoplasmic reticulum increased relative to the cytosol. A
similar pattern of metal accumulation was observed with
dissolved Cu and to some extent dissolved Ag exposure.
Consistent with the findings of this study, Xu et al.41 also
reported a decline in the relative distribution of Zn in the
cytoplasm of T. weissf logii after exposure to dissolved Zn. In
mammalian cells, Zn concentrations in the cytosol are much
higher than in the endoplasmic reticulum, and when in excess,
Zn is generally sequestered by the endoplasmic reticulum.42
This may also be the situation for Zn, Cu, and Ag in algal cells.
Differences in metal distribution in T. weissf logii after exposure
to nanoparticles or dissolved metals likely resulted from
differences in rates and/or modes of uptake. Additionally,
depending on exposure concentration, various degrees of
dissolution and release of metal ions from metal oxide
nanoparticles apparently occurred over time,12,13,35 resulting
in exposure of both dissolved and nanoparticulate metals, thus
influencing the metal uptake and distribution in the algae.
The patterns of metal distribution that we report have broad
implications for food chain transfer and effects of metals in
consumers. Bielmyer et al.12 previously reported a significant
decrease in copepod reproduction after they consumed
Thalassiosira pseudonana that were exposed to dissolved metals,
resulting in accumulation values of 120 μg g−1 dw Ag, 3.05 μg
g−1 dw Zn, or 31.9 μg g−1 dw Cu within the algal cells. These
concentrations were lower than those shown to cause a
decrease in copepod survival. Jarvis et al.6 demonstrated that
feeding on nZnO-exposed T. weissf logii resulted in significantly
reduced survival and reproduction of the copepod Acartia tonsa.
The effect levels observed from copepods fed nanoparticleexposed diets were similar to those reported for dissolvedmetal-exposed diets.6,12 Contrary to past dietary studies with
dissolved metals12,38,43,44 survival was a more sensitive end
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(5) Karl, D. M.; Bidigare, R. R.; Letelier, R. M. Long-term changes in
plankton community structure and productivity in the North Pacific
Subtropical Gyre: The domain shift hypothesis. Deep Sea Res., Part II
2001, 48.8, 1449−1470.
(6) Jarvis, T. A.; Miller, R. C.; Lenihan, H. S.; Bielmyer, G. K.
Toxicity of ZnO nanoparticles to the copepod, Acartia tonsa, exposed
via a phytoplankton diet. Environ. Toxicol. Chem. 2013, 32, 1264−
1269.
(7) Klaine, S. J.; Alvarex, P. J. J.; Batley, G. E.; Fernandes, T. F.;
Handy, R. D.; Lyon, D. Y.; Mahendra, S.; McLaughlin, M. J.; Lead, J.
R. Nanomaterials in the environment: Behavior, fate, bioavailability,
and effects. Environ. Toxicol. Chem. 2008, 27, 1825−1851.
(8) Blaise, C.; Gagne, F.; Ferard, J. F.; Eullaffroy, P. Ecotoxicity of
selected nanomaterials to aquatic organisms. Environ. Toxicol. 2008,
23, 591−598.
(9) Masciangioli, T.; Zhang, W. X. Environmental technologies at the
nanoscale. Environ. Sci. Technol. 2003, 37, 102A−108A.
(10) Gu, H.; Soucek, M. D. Preparation and characterization of
monodisperse cerium oxide nanoparticles in hydrocarbon solvents.
Chem. Mater. 2007, 19, 1103.
(11) Gottschalk, F.; Sonderer, T.; Scholz, R. W.; Nowack, B.
Modeled environmental concentrations of engineered nanomaterials
(TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci.
Technol. 2009, 43, 9216−9222.
(12) Bielmyer, G. K.; Grosell, M.; Brix, K. Toxicity of silver, zinc,
copper, and nickel to the copepod Acartia tonsa exposed via a
phytoplankton diet. Environ. Sci. Technol. 2006, 40, 2063−2068.
(13) Franklin, N. M.; Rogers, N. J.; Apte, S. C.; Batley, G. E.; Gadd,
G. E.; Casey, P. S. Comparative toxicity of nanoparticulate ZnO, bulk
ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella
subcapitata): The importance of particle solubility. Environ. Sci.
Technol. 2007, 41, 8484−8490.
(14) Miller, R. J.; Lenihan, H. S.; Muller, E. B.; Tseng, N.; Hanna, S.
K.; Keller, A. A. Impacts of metal oxide nanoparticles on marine
phytoplankton. Environ. Sci. Technol. 2010, 44, 7329−7334.
(15) Keller, A. A.; Wang, H.; Zhou, D.; Lenihan, H. S.; Cherr, G.;
Cardinale, B. J.; Miller, R. J.; Ji, Z. Stability and aggregation of metal
oxide nanoparticles in natural aqueous matrices. Environ. Sci. Technol.
2010, 44, 1962−1967.
(16) Campbell, P. G. C.; Errécalde, O.; Fortin, C.; Hiriart-Baer, V. P.;
Vigneault, B. Metal bioavailability to phytoplankton-applicability of the
biotic ligand model. Comp. Biochem. Physiol., Part C: Toxicol.
Pharmacol. 2002, 133, 189−206.
(17) Sunda, W. G. Trace metal interactions with marine
phytoplankton. Biol. Oceanogr. 1988, 6, 411−442.
(18) Morel, F. M. M.; Hudson, R. J. M.; Price, N. M. Limitation of
productivity by trace metals in the sea. Limnol. Oceanogr. 1991, 36,
1742−1755.
(19) Verma, A.; Uzun, O.; Hu, Y.; Hu, Y.; Han, H. S.; Watson, N.;
Chen, S.; Irvine, D. J.; Stellacci, F. Surface-structureregulated cellmembrane penetration by monolayer-protected nanoparticles. Nat.
Mater. 2008, 7, 588−595.
(20) Chang, Y. N.; Zhang, M.; Xia, L.; Zhang, J.; Xing, G. The toxic
effects and mechanisms of CuO and ZnO nanoparticles. Materials
2012, 5, 2850−2871.
(21) Zhang, D. X.; Gutterman, D. D. Mitochondrial reactive oxygen
species-mediated signaling in endothelial cells. Am. J. Public Health
2007, 292, H2023−H2031.
(22) Yang, H.; Liu, C.; Yang, D. F.; Zhang, H. S.; Xi, Z. Comparative
study of cytotoxicity, oxidative stress and genotoxicity induced by four
typical nanomaterials: The role of particle size, shape and composition.
J. Appl. Toxicol. 2009, 29, 69−78.
(23) Toduka, Y.; Toyooka, T.; Ibuki, Y. Flow cytometric evaluation
of nanoparticles using side-scattered light and reactive oxygen
speciesMediated fluorescenceCorrelation with genotoxicity.
Environ. Sci. Technol. 2012, 46, 7629−7636.
(24) Bielmyer, G. K.; Grosell, M. Emerging issues in marine metal
toxicity. In Essential Reviews in Experimental Biology, Bury, N., Handy,
R., Eds.; Kings College: London, UK, 2011, Vol. 2, pp 129−158.
point than reproduction after exposure to nanoparticle-exposed
diets.6 The differences in metal accumulation and distribution
in phytoplankton observed in the present study could
contribute to the unique effects of ingestion of phytoplankton
exposed to nanomaterials, compared with dissolved metals.
Metal oxide nanoparticles bound to the cell walls or frustules of
phytoplankton may facilitate rapid uptake of large amounts of
metals by consumers, resulting in more acute toxicity effects.
Metal ions that readily adsorb to algal surfaces may be released
under changing pH within the digestive tract of consumers.45
Accumulation of metal oxide nanomaterials on phytoplankton
cell walls may facilitate trophic transfer of these contaminants
and cause reverberating effects on oceanic food webs.
It is important to note that most of the metal concentrations
in this study were higher than those presently found in most
marine environments. These concentrations nevertheless
enabled us to clearly measure decreases in T. weissf logii
populations and significant differences in metal accumulation
and distribution. We implicitly assume that patterns of metal
distribution across cellular fractions do not depend heavily on
concentration. More research is needed to examine accumulation and effects of metals at lower concentrations because
ecosystem productivity can be affected by even small decreases
in phytoplankton growth.46 Additionally, metal accumulation
and distribution in this lower trophic level may have important
implications up the food chain, decreasing fitness of consumers.
Adult mortality rates of primary consumers such as
zooplankton can modulate interannual variability of pelagic
food webs and modify elemental cycling in the ocean;47
widespread contamination by metals, whether nanoparticulate
or dissolved, may thus have complex and unpredictable effects
on oceanic ecosystems.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: (229) 333-5766 E-mail: [email protected].
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
Funding for the present study was provided in part by the
University of California Center for Environmental Implications
of Nanotechnology (UC CEIN) supported by the National
Science Foundation and the Environmental Protection Agency
under Cooperative Agreement Number DBI-0830117, and by
the Valdosta State University Graduate School (Valdosta, GA,
USA). A VSU equipment grant provided the funding for the
atomic absorption spectrophotometer.
■
REFERENCES
(1) Trequer, P.; Nelson, D. M.; Van Bennekom, A. J.; DeMaster, D.
J.; Leynaert, A.; Queguiner, B. The silica balance in the world ocean: A
reestimate. Science 1995, 268, 375−379.
(2) Field, C. B.; Behrenfeld, M. J.; Randerson, J. T.; Falkowski, P.
Primary production of the biosphere, integrating terrestrial and
oceanic components. Science 1988, 281, 237−240.
(3) Lewis, M. A. Use of fresh-water plants for phytotoxicity testing
A review. Environ. Pollut. 1995, 87, 310−336.
(4) Luoma, S. N.; Hogstrand, C.; Bell, R. A.; Bielmyer, G. K.; Galvez,
F.; LeBlanc, G. A.; Lee, B. G.; Purcell, T. W.; Santore, R. C.; Santschi,
P. H.; Shaw, J. R. In Silver in the Environment: Transport, Fate, and
Effect; Andren, A. W., Bober, T. W., Eds; University of Wisconsin Sea
Grant, Madison, Wisconsin, USA, 1999; pp 65−97.
13449
dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450
Environmental Science & Technology
Article
Daphnia magna strauss. Environ. Toxicol. Chem. 1998, 17 (3), 412−
419.
(46) Cloern, J. E.; Grenz, C.; Vidergar-Lucas, L. An empirical model
of the phytoplankton:carbon ratioThe conversion factor between
productivity and growth rate. Limnol. Oceanogr. 1995, 40, 1313−1321.
(47) Ohman, M. D.; Hirche, H. J. Density-dependent mortality in an
oceanic copepod population. Nature 2001, 412 (6847), 638−641.
(25) Rainbow, P. S.; Luoma, S. N.; Wang, W. X. Trophically available
metalA variable feast. Environ. Pollut. 2011, 159, 2347−2349.
(26) Reinfelder, J. R.; Fisher, N. S. The assimilation of elements
ingested by marine copepods. Science 1991, 251, 794−796.
(27) Hutchins, D. A.; Wang, W. X.; Fisher, N. S. Copepod grazing
and the biogeochemical fate of diatom iron. Limnol. Oceanogr. 1995,
40, 989−994.
(28) Wallace, W. G.; Lee, B. G.; Samuel, L. N. Subcellular
compartmentalization of Cd and Zn in two bivalves. I. Significance
of metal-sensitive fractions (MSF) and biologically detoxified metal
BDM. Mar. Ecol. 2003, 249, 183−197.
(29) Cheung, M. S.; Wang, W. X. Influence of subcellular metal
compartmentalization in different prey on the transfer of metals to a
predatory gastropod. Mar. Ecol.: Prog. Ser. 2005, 286, 155−166.
(30) Bielmyer, G. K.; Bell, R. A.; Klaine, S. J. The effects of ligandbound silver to Ceriodaphnia dubia. Environ. Toxicol. Chem. 2002, 21,
2204−2208.
(31) APHA. Standard Methods for the Examination of Water and
Wastewater, 15th ed; American Public Health Association: Washington, DC, 1981.
(32) Bielmyer, G. K.; Arnold, R.; Tomasso, J.; Klaine, S. J. Influence
of salt source on synthetic saltwater quality. Chemosphere 2004, 57,
1707−1711.
(33) Godwin, H. A.; Chopra, K.; Bradley, K. A.; Cohen, Y.; Harthorn,
B. H.; Hoek, E. M. V.; Holden, P.; Keller, A. A.; Lenihan, H. S.; Nisbet,
R. M.; Nel, A. E. The University of California Center for the
Environmental Implications of Nanotechnology. Environ. Sci. Technol.
2009, 43, 6453−6457.
(34) Keller, A. A.; Hongtao, W.; Dongxu, Z.; Lenihan, H. S.; Cherr,
G.; Cardinale, B. J.; Miller, R. J.; Zhaoxia, J. Stability and aggregation of
metal oxide nanoparticles in natural aqueous matrices. Environ. Sci.
Technol. 2010, 44, 1962−1967.
(35) Hanna, S. K.; Miller, R. J.; Zhou, D.; Keller, A. A.; Lenihan, H. S.
Accumulation and toxicity of metal oxide nanoparticles in a softsediment estuarine amphipod. Aquat. Toxicol. 2013, 142−143, 441−
446.
(36) Aruoja, V.; Dubourguier, H. C.; Kasemets, K.; Kahru, A.
Toxicity of nanoparticles CuO, ZnO, and TiO2 to microalgae
Pseudokirchneriella subcapitata. Sci. Total Environ. 2009, 407, 1461−
1468.
(37) Shi, J.; Abid, A. D.; Kennedy, I. M.; Hristova, K. R.; Silk, W. K.
To duckweeds (Landoltia punctata), nanoparticulate copper oxide is
more inhibitory than the soluble copper in the bulk solution. Environ.
Pollut. 2011, 159, 1277−1282.
(38) DeSchamphelaere, K. A. C.; Canli, M.; Lierde, V. V.; Forrez, I.;
Vanhaecke, F.; Janssen, C. R. Reproductive toxicity of dietary zinc to
Daphnia magna. Aquat. Toxicol. 2004, 70, 233−244.
(39) Guan, R.; Wang, W. Dietary assimilation and elimination of Cd,
Se, and Zn by Daphnia magna at different metal concentrations.
Environ. Toxicol. Chem. 2004, 23, 2689−2698.
(40) Peng, X.; Palma, S.; Fisher, N. S.; Wong, S. S. Effect of
morphology of ZnO nanostructures on their toxicity to marine algae.
Aquat. Toxicol. 2011, 102, 186−196.
(41) Xu, Y.; Wang, W. X.; Dennis, P. H. Influences of metal
concentration in phytoplankton and seawater on metal assimilation
and elimination in marine copepods. Environ. Toxicol. Chem. 2001, 20,
1067−1077.
(42) Qin, Y.; Dittmer, P. J.; Park, G.; Jansen, K. B.; Palmer, A.
Measuring steady-state and dynamic endoplasmic reticulum and Golgi
Zn2b with genetically encoded sensors. P. Natl. Acad. Sci. U.S.A. 2011,
108, 7351−7356.
(43) Hook, S. E.; Fisher, N. S. Sublethal effects of silver in
zooplankton: Importance of exposure pathways and implications for
toxicity testing. Environ. Toxicol. Chem. 2001, 20, 568−574.
(44) Hook, S. E.; Fisher, N. S. Relating the reproductive toxicity of
five ingested metals in calanoid copepods with sulfur affinity. Mar.
Environ. Research 2002, 53, 161−174.
(45) Taylor, G.; Baird, D. J.; Soares, A. M. V. M. Surface binding of
contaminants by algae: Consequences for lethal toxicity and feeding to
13450
dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450