Environ. Sci. Technol.

2009, 43, 25892594

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Published on February 27, 2009 on http://pubs.acs.org | doi: 10.1021/es802806n

Effects of Soluble Cadmium Salts

Versus CdSe Quantum Dots on the
Growth of Planktonic
Pseudomonas aeruginosa
JOHN H. PRIESTER,
PETER K. STOIMENOV,
RANDALL E. MIELKE,
SAMUEL M. WEBB,|
CHRISTOPHER EHRHARDT,
JIN PING ZHANG,# GALEN D. STUCKY,
A N D P A T R I C I A A . H O L D E N * ,
Donald Bren School of Environmental Science &
Management, Department of Chemistry and Biochemistry,
Earth Sciences, and Materials Research Laboratory, University
of California, Santa Barbara, California 93106, Center for Life
Detection, Jet Propulsion Laboratory, California Institute of
Technology, Pasadena, California 91109, and Stanford
Synchrotron Radiation Laboratory, Stanford Linear
Accelerator Center, Menlo Park, California 94025

Received October 3, 2008. Revised manuscript received

January 15, 2009. Accepted January 22, 2009.

With their increased use, engineered nanomaterials (ENMs)

will enter the environment where they may be altered by bacteria
and affect bacterial processes. Metallic ENMs, such as
CdSe quantum dots (QDs), are toxic due to the release of
dissolved heavy metals, but the effects of cadmium ions versus
intact QDs are mostly unknown. Here, planktonic Pseudomonas
aeruginosa PG201 bacteria were cultured with similar total
cadmium concentrations as either fully dissolved cadmium acetate
(Cd(CH3COO)2) or ligand capped CdSe QDs, and cellular
morphology, growth parameters, intracellular reactive oxygen
species (ROS), along with the metal and metalloid fates
were measured. QDs dissolved partially in growth media, but
dissolution was less in biotic cultures compared to sterile controls.
Dose-dependent growth effects were similar for low
concentrations of either cadmium salts or QDs, but effects
differed above a concentration threshold of 50 mg/L (total cadmium
basis) where (1) the growth of QD-treated cells was more
impaired, (2) the membranes of QD-grown cells were damaged,
and (3) QD-grown cells contained QD-sized CdSe cytoplasmic
inclusions in addition to Se0 and dissolved cadmium. For
most concentrations, intracellular ROS were higher for QDversus cadmium salts-grown bacteria. Taken together, QDs were
* Corresponding author e-mail: holden@bren.ucsb.edu; tel: 805893-3195; fax: 805-893-7612.

Donald Bren School of Environmental Science & Management,

University of California, Santa Barbara.

Department of Chemistry and Biochemistry, University of

California, Santa Barbara.

Center for Life Detection, Jet Propulsion Laboratory, California

Institute of Technology.
|
Stanford Synchrotron Radiation Laboratory, Stanford Linear
Accelerator Center.

Earth Sciences, University of California, Santa Barbara.

#
Materials Research Laboratory, University of California, Santa
Barbara.
10.1021/es802806n CCC: $40.75

Published on Web 02/27/2009

2009 American Chemical Society

more toxic to this opportunistic pathogen than cadmium ions,

and were affected by cells through QD extracellular stabilization,
intracellular enrichment, and cell-associated decay.

Introduction
The rapid development of the engineered nanomaterials
(ENMs) industry has raised concerns about ENM releases to
the environment (1) where bacteria are abundant (2) and
can catalyze essential nutrient-recycling reactions during
growth (3) and influence ENM fates (4). Reported individual
ENM-bacterial biophysical interactions include biosorption,
ENM breakdown (5), and cellular uptake (6), with effects
including membrane damage and toxicity (7, 8). However,
such interactions are rarely evaluated in concert and over
growth-associated time scales. This leaves questions about
ENM fates and effects when bacteria are present, including
the quantitative importance to toxicity end points of intact
ENMs versus breakdown products.
For heavy metal-composed ENMs such as cadmium
selenide (CdSe) quantum dots (QDs), toxic metal ions may
cause cellular toxicity (9). CdSe QDs are fluorescent semiconductor ENMs of interest in photovoltaics (10) and in
diagnostics for stably labeling mammalian cells (11) and
bacteria (12). CdSe QDs are often capped with ZnS to enhance
fluorescence (13); core-shell configurations also stabilize
Cd(II) surface atoms against dissolution which increases
biocompatibility (9, 14). Still, there are concerns about cap
loss (9, 15) and the subsequent toxicity of bare QDs and
dissolved cadmium.
Cd(II) causes cellular toxicity by several mechanisms
including interfering with DNA repair (16) and metabolic
proteins (17), membrane lipid peroxidation (18), substitution
for physiological Zn(II), and reactive oxygen species (ROS)
formation (19). In gram-negative bacteria, Cd(II) readily
enters the cell through open gate Mg(II) transporters. Efflux
through cadmium-induced membrane proteins is the primary resistance mechanism (20). QDs also generate ROS,
which damage membranes (21); such damage may account
for QDs nonspecifically entering bacteria in the dark (22)
and in the light (12). Once in cells, QD toxicity may again be
from either intact nanoparticles (12) or from released Cd(II)
ions (23). However, still unknown are the relative toxicities
and fates of Cd(II) ions versus either absorbed or assimilated
intact QDs, especially when they co-occur in the bacterial
growth environment.
The objectives of this study were to quantify the effects
to bacteria of ligand capped CdSe QDs and Cd(II) ions, taking
into account that Cd(II) ions might be generated during
bacterial growth if QDs dissolve, and that QDs may also be
directly toxic. We asked: to what extent do QDs dissolve in
bacterial culture? Are QDs and/or Cd(II) ions assimilated by
cells? What is the toxicity of Cd(II) ions versus QDs? Assuming
that QDs would be toxic due to dissolved cadmium, we grew
a relatively cadmium-tolerant bacterial strain, Pseudomonas
aeruginosa PG201, with measurably high total cadmium that
was initially in the form of either Cd(II) ions or QDs. Citrate
capped, as opposed to core-shell (i.e., metal capped), QDs
were used to increase the likelihood that both Cd(II) and
QDs would coexist in culture, thus providing the opportunity
to differentiate ENM effects from heavy metal toxicity. It has
been previously reported (15, 21) that capped QDs with no
dissolved Cd(II) cause toxicity, but the relative effects of both
intact QDs and dissolved Cd(II) have not been evaluated
until now. Bacteria were also grown with soluble cadmium
salts to separately quantify the effects of Cd(II) ions. While
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CdSe QDs did dissolve in bacterial growth media, dissolution

was incomplete. After accounting for effects of Cd(II) ions,
the residual toxicity from intact QDs was relatively greater
than toxicity from cadmium salts alone. This work thus reveals
the separable effects of QDs and their released heavy metals,
and newly evaluates the effects of ENMs on an important
opportunistic pathogen.

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Experimental Section
Chemicals and Bacterial Culturing. Pseudomonas aeruginosa PG201, a well-studied environmental strain (24), was
cultured with either Cd(II) or CdSe QDs in Luria Bertrani
(LB) broth. All chemicals were reagent grade or better (Sigma
Chemical, St. Louis, MO; and Fisher Scientific, Hampton,
NH). See Supporting Information for QD synthesis and
culture details.
Cultures were amended with either cadmium acetate
(Cd(CH3COO)2 at 5, 10, 20, 37.5, 75, 115, and 150 mg/L as
cadmium) or CdSe QDs (10, 20, 37.5, 50, 75, 100 and 125
mg/L as cadmium). Controls included medium-only (no
cadmium) and uninoculated versions of each treatment. Five
independent replicates were prepared for each treatment
and control. Cultures were prepared in 200 L volumes in
96-well microplates (see Supporting Information for details).
At 6 h after inoculation, 3 replicates were subsampled by
aseptically removing 2 L for Cd(II) ion quantification (see
below). To test the effects of citrate on growth (at concentrations at and above those present with the QDs), an additional
treatment involved amending LB broth with 400, 800, or 1200
mg/L sodium citrate.
Growth experiments with Cd(CH3COO)2 and CdSe QDs
were repeated independently using larger volumes (7.5 mL)
and selected cadmium concentrations (with at least 3
replicates) for microscopy, analyses of intra- and extracellular
metals and macromolecules, and analysis of intracellular ROS.
The optical density (OD) was monitored (600 nm) to
determine if experimental scale-up created bias. Another
growth experiment was for assaying acidification by measuring the pH at 0 and 24 h for the control and the
Cd(CH3COO)2 and CdSe QD treatments amended with 75
mg/L total cadmium. Separate 7.5 mL cultures were also
grown to study selenium-only effects using sodium selenite
(Na2SeO3) over a concentration range up to 500 mg/L Se(IV).
Cell Harvesting. To determine the distribution of Cd(II)
ions versus intact QDs in bacterial culture, late exponential
phase (24 h) cultures of the 75 mg/L (total cadmium)
Cd(CH3COO)2 and CdSe QD treatments were harvested for
quantifying total cellular metal and metalloid contents by
inductively coupled plasma atomic emission spectroscopy
(ICP-AES), analyzing Se oxidation state by X-ray absorption
near edge spectra (XANES), electron microscopy, epifluorescence microscopy, and for determining the crystal structure of intracellular metal and metalloid by X-ray diffraction
(XRD). Intracellular biomacromolecules were also assayed
(see Supporting Information).
Intracellular ROS. Total ROS in abiotic CdSe and cadmium acetate solutions, and in midexponential phase P.
aeruginosa cultures, were quantified using the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay (25, 26) (see Supporting Information for assay details).
Total and Dissolved Cadmium Quantification. Dissolved
cadmium was quantified by the Measure-iT kit (Invitrogen,
Carlsbad, CA). Calibration standards were prepared using
Cd(CH3COO)2 (0, 25, 50, 75, and 100 mg/L as cadmium) in
all relevant aqueous conditions: nanopure H2O, sterile LB,
and filter-sterilized (0.2 m) late exponential phase (24 h)
culture supernatant. ICP-AES with a TJA High Resolution
IRIS instrument (Thermo Electron Corporation, Waltham,
MA) was used to quantify total selenium and cadmium.
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Instrument calibration was against commercial Cd and Se

standards (Sigma Chemical).
QD Integrity by XRD and XANES; and Abiotic Dissolution. Abiotic QD dissolution (see Supporting Information)
was measured. QD intracellular integrity was inferred in two
ways: using X-ray diffraction for cell-associated QD crystal
structure, and using XANES for cellular Se oxidation state.
Se-K-edge XANES spectra were collected at the Stanford
Synchrotron Radiation Laboratory (SSRL) beam line 11-2
under SPEAR3 to determine the oxidation state of Se, using
overnight-shipped (dry ice) triplicate cell pellets from a CdSe
QD (75 mg/L) treatment for 24 h cultures as described above.
Detailed descriptions of the XANES and XRD methods
are located in the Supporting Information.
Microscopy and Image Analysis. Phase-contrast microscopy (Nikon E800 at 1000 total magnification, with image
acquisition) was used for measuring (in micrographs using
Photoshop 5.5) the cell aspect ratios of ten cells per treatment
for culture aliquots dispensed directly onto microscope slides.
Total cell counts were determined by epifluorescence
microscopy of cells from separately grown late exponential
phase cultures (LB controls and 75 mg/L total cadmium as
either QDs or Cd(II) ions) that were SYBR gold-stained
(Invitrogen, Carlsbad, CA) and counted as before (27).
High-resolution microscopy was used to assess membrane
integrity and metal, metalloid, and QD localization in cells.
Scanning transmission electron microscopy (STEM) and
energy dispersive X-ray analysis (EDXA) were performed using
24 h cultures for the 75 mg/L (total cadmium) QD and Cd(II)
treatments plus controls. See the Supporting Information
for sample preparation and instrument details.
STEM micrographs were analyzed in Adobe Photoshop
for cell and nanoparticle dimensions, nanoparticle counts,
and membrane damage frequency. Cell dimensions were
measured from longitudinal and vertical cross sections (5
each), and cell surface areas and volumes were calculated
assuming cylindrical geometry.
Data Analysis. Cellular Se and Cd contents were normalized to cell counts as before (27). Statistical analyses were
performed with SPSS 12.0.1 (SPSS Inc., Chicago, IL) or
Microsoft Excel 2000 software. Means were compared by the
Student t test. Where applicable, standard errors were
propagated according to standard methods (28).

Results
Bacterial Growth and Relationship to QD Dissolution.
Growth of P. aeruginosa PG201 was inhibited by both
Cd(CH3COO)2 (Figure S1) and CdSe QDs (Figure S2) in that
increased total cadmium resulted in longer lag times, lower
specific growth rates, and lower yields (Tables S1, S2). Growth
parameters appeared related to total cadmium concentration
similarly for QD-treated and Cd(CH3COO)2-treated cultures
(Tables S1 and S2). The pH was constant during growth and
averaged 7.4 ( 0.1. Neither sodium citrate (Figure S3), nor
sodium selenite (data not shown) inhibited growth.
Because growth with either QDs or Cd(CH3COO)2 appeared similar (Figure S1 and S2; Tables S1 and S2), complete
QD dissolution was implied. Still, several tests were performed
to quantify QD dissolution: (1) a dialysis study of initial
dissolution kinetics in water (Supporting Information 2.2;
Figure S4); (2) dialysis studies to determine aqueous chemistry
effects on 24 h dissolution end points (Supporting Information 2.2; Figure S5); and (3) Cd(II) ion quantification in
cultures at 6 h, i.e., entry into exponential phase for most
cultures. Most relevant to growth, QDs were between 25 and
50% dissolved in cultures at 6 h (Figures S1 and S2 early
exponential phase), and the relationship between the total
and dissolved fraction was expectedly linear (Figure S6).
Relating specific growth rates to the concentration of
dissolved Cd(II) at 6 h (i.e., end of lag phase) revealed that

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FIGURE 1. Specific growth rate of P. aeruginosa versus

dissolved cadmium amended as either cadmium acetate
(squares) or CdSe QDs (diamonds). As shown in the graph, all
cadmium treatments result in reduced growth rates relative to
the no-cadmium control (dark circle). At QD concentrations
exceeding 50 mg/L (total cadmium basis), QDs remain relatively
toxic while cultures appear to resist Cd(II) ions.
QD- versus Cd(CH3COO)2-treated cultures were similarly
affected at lower total cadmium concentrations (Figure 1).
However, for dissolved cadmium concentrations exceeding
approximately 20 mg/L (i.e., total QD cadmium 50 mg/L)
growth rates of QD-treated cultures continued to decline
steeply as a function of Cd(II) dose while growth rates for
Cd(CH3COO)2-treated cultures were less sensitive to cadmium dose (Figure 1). When plotted against dissolved Cd(II),
the other growth curve metrics of lag time and yield
(maximum OD) also showed stronger effects with QDs in
comparison to Cd(CH3COO)2 (Figure S7).
Cellular Morphology and Estimated Intracellular Nanoparticles.Above50mg/Ltotalcadmium,QD-andCd(CH3COO)2grown cultures appeared to respond differently to the
dissolved cadmium dose (Figure 1). Thus one concentration
above this threshold was chosen for analyzing cellular size,
morphology, and comparative metal, metalloid, and biomacromolecule contents for the QD and Cd(CH3COO)2 treatments. Using lower resolution (i.e., 1000) phase contrast
microscopy, QD-grown cells were shorter as indicated by
lower aspect ratios (3.3 ( 0.2) compared to either cadmiumtreated (3.8 ( 0.3) or control (3.9 ( 0.2) cells. Using highresolution STEM, cells cultured with Cd(CH3COO)2 appeared
similar to no-metal controls except for electron dense
deposits in and near the membranes (Figure 2A vs B) that,
by EDXA, were cadmium-rich (data not shown). Cells cultured
with QDs also contained Cd- and Se-rich electron dense
intracellular deposits (Figure S8) but were highly disfigured
(Figure 2C). A total of 54 QD-grown and 46 Cd(CH3COO)2treated cells in STEM images were evaluated for membrane
damage, as defined by holes, blebbing, or detachment of the
plasma membrane from the cell wall (Figure S9). Most (81%;
n ) 44) of the QD-treated and fewer (33%; n ) 15) of the
Cd(CH3COO)2-treated cells had membrane damage (e.g.,
Figure 2). Blebbing (Figure S9B) was observed in QD-treated,
but not for Cd(CH3COO)2-treated cells.
Nanosized particles appeared throughout the QD-treated
cells (Figure 2C), while in the Cd(CH3COO)2-treated cells
particles were in the periplasmic space (Figure 2B). The mean
particle diameters differed (t test, P ) 0.00), with 8.02 ( 0.24
nm and 14.99 ( 0.54 nm for the QD and Cd(CH3COO)2
treatments, respectively. Because the nanosized particles in
the QD treatments were similar in size to the administered
QDs (5 nm), these particles were counted, resulting in an

average of 1.87 ( 0.16 105 nanoparticles per cell. Intra- and

extracellular quantum dot concentrations were calculated
using the latter counts as well as the measured extracellular
concentrations of total and dissolved cadmium (Supporting
Information 2.3). No further evaluation was made of the
cadmium-rich inclusions within Cd(CH3COO)2-treated cells.
Metal, Metalloid, and Biomacromolecule Contents. For
cultures grown with 75 mg/L cadmium, total intracellular
cadmium (by ICP-AES) at 24 h (early stationary phase) was
similar for QD- and Cd(CH3COO 2)2-treated cultures and
averaged 0.15 ( 0.02 pg cell-1, which was approximately 4%
of that administered. Assuming cell dimensions (from STEM
images) and counts (from epifluorescent microscopy), the
intracellular concentration of this dissolved Cd(II) was then
approximately 335 g/L, which was 4460 times higher than
the medium. Cells grown with QDs (75 mg/L) had an
intracellular Se content (by ICP-AES) of 0.08 ( 0.01 pg cell-1,
which appeared, by XANES, to be mainly Se0 or other reduced
organoselenium compounds (Figure S10). By both ICP-AES
and the Measure iT assay, all of the intracellular Cd(II)
appeared to be dissolved (Table S3). However, a relatively
weak XRD peak at 2 24 supported that cells contained
at least some intact CdSe crystals (Figure S11). This peak is
consistent with the dominant peak for CdSe QDs synthesized
according to the same methods that we used (29). However,
this peak was only slightly above background, and other
identifying peaks were likely obscured by the dominant Al
peaks.
QD and Cd(CH3COO)2-treated cells had greater amounts
of intracellular protein, DNA, and carbohydrates as compared
to control cultures (Supporting Information 2.4). Also,
compared to sterile controls, QDs in cultures were less
dissolved (68.5% vs 59.7%, respectively) after 24 h, which
implied some extracellular QD stabilization in cultures (Table
S3).
Intracellular ROS and Media ROS. After correcting for
background fluorescence (from DCFH, but not from either
cells or QDs which did not interfere), and converting DCFH
fluorescence to H2O2 equivalents, neither QDs nor cadmium
salts at 10 mg/L total cadmium resulted in measurable
intracellular ROS (after 15 h of growth). However, at cadmium
concentrations spanning 20 through 125 mg/L, cellular ROS
was significantly greater for QD- versus Cd(II)-grown cells
(Figure 3). Abiotic ROS generation was greater for QDs in LB
than for QDs in water (detailed in Supporting Information
2.5). LB alone also generated ROS, but at a much lower
concentration than for either LB or water plus QDs (Supporting Information 2.5).
After correcting for background fluorescence (as above)
and again converting fluorescence to H2O2 equivalents, sterile
LB broth plus DCFH yielded an ROS concentration of 534 (
183 mg/L H2O2. When added to either LB or to water, CdSe
QDs also generated ROS abiotically, albeit to a much lesser
extent in water as compared to LB broth (Figure S12) and to
a much greater extent than LB alone (above). Cadmium salts
added to either sterile LB or water did not result in ROS
formation. The pattern of ROS versus cadmium concentration
was similar when comparing the biotic (Figure 3) and abiotic
(Figure S12) QD treatments, with the exception of the
decrease at 125 mg/L cadmium for the abiotic treatments.

Discussion
Prior reports for CdSe QDs suggest that surface cap and
solution chemistry modulate toxicity from cadmium ions
(9, 15). Yet ZnS-capped, i.e., core shell, CdSe (15) and CdTe
(21) QDs that did not release cadmium ions were still toxic,
and PEG-modified CdSe QDs were toxic to mammalian cells
in a dose-dependent fashion corresponding to intracellularized QDs (23). In the environment, various abiotic and
biotic fate-related processes affecting ENM phase distribution
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FIGURE 2. STEM images of P. aeruginosa grown in LB either without metals (A), or with 75 mg/L total cadmium in the form of either
cadmium acetate (B) or CdSe QDs (C), where the latter treatment is associated with cells exhibiting damaged membranes. Scale bar
is 1 m.

FIGURE 3. H2O2 equivalent intracellular reactive oxygen species

(ROS) concentration for P. aeruginosa grown in LB with 75 mg/
L total cadmium in the form of either cadmium acetate or CdSe
QDs. ROS is normalized to intracellular DNA to account for
slight differences in culture yield.
could enable exposure to Cd(II) ions and intact ENMs
simultaneously. However, as emphasized in a recent review
(30), the relative contributions to toxicity of co-occurring
ENMs and their dissolved phases are mostly unknown. We
addressed this issue by growing a model bacterial strain with
either QDs or cadmium ions, separately studying QD
dissolution and metal/metalloid fates with cells, and relating
growth inhibition to the dissolved cadmium concentration.
While aqueous phase cadmium speciation was not directly
studied, published relationships encompassing the medium
pH (7.4) and cadmium concentrations (e150 mg/L) studied
here would suggest that dissolved cadmium was predominantly Cd(II) (31). Additionally, our report is one of the first
of effects of ENMs on an opportunistic pathogen.
CdSe QD toxicity to P. aeruginosa clearly exhibited two
phases, i.e., a low concentration phase that mimicked the growth
inhibition from cadmium salts, and a higher concentration
phase that inhibited growth more extensively relative to
cadmium salts. Previously, the minimum inhibitory concentration (MIC) for Cd(II) was reported as 500-700 mg/L for P.
aeruginosa in LB (32, 33). In our study, above approximately
50 mg/L total cadmium, cells intoxicated by cadmium salts
became resistant whereas cells treated with QDs did not, thus
implying that toxicity from intact QDs was quantitatively
important above this threshold. The dissolution data confirmed
that both intact QDs and dissolved Cd(II) were co-occurring,
and thus further examination of cellular effects and metalloid
fates was performed for the 75 mg/L QD and cadmium salts
treatment, i.e., for a concentration at which a nanoparticle2592

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specific effect was clearly implied. Particularly with uncapped

nanoparticles including bare CdSe QDs, and metal oxides such
as ZnO and CuO (34) that can also easily dissolve, the approach
used here for measuring dissolved cadmium, which was
adopted from Cho et al. (35), was essential to revealing a potent
nanoparticle effect of intact QDs.
Selenium, added as sodium selenite, was not observed to
be toxic to the strain studied here. Nevertheless, tracking the
oxidation state of cell-associated selenium by XANES provided
evidence of QD breakdown. It has been proposed (9) that
oxidation of CdSe QD surfaces leads to QD dissolution and the
formation of SeO2. Had a similar process occurred here with
P. aeruginosa, a mixture of Se2- and Se4+ would have been
observed by XANES. Instead, the predominant form of selenium
appeared to be elemental selenium, or a mixture of elemental
selenium and organoselenium compounds (Figure S10). It is
possible that the above proposed process is an extracellular
phenomenon, or that an undetermined biotic process converted
SeO2 to elemental selenium. This has been observed with P.
aeruginosa and Na2SeO3 (data not shown) but has yet to be
evaluated with SeO2.
At 75 mg/L total cadmium, ROS generation was enhanced
for QD- versus Cd(II)-grown bacteria. ROS formation and
subsequent cellular effects, an emerging paradigm for cellular
toxicity by nanoparticles (36), have been used to explain toxicity
from CdTe QD-treated mammalian cells (37) and E. coli bacteria
(21). ROS formation does not require irradiation (21, 37) and
occurs in rich growth media (38). In this study, the growth
medium interacted with QDs to initiate ROS formation.
However, QDs enhanced ROS concentrations significantly
above the medium-only controls and relative to abiotic and
biotic treatments with cadmium salts. ROS damaged membranes and facilitated QD entry, as described before (12), which
may also explain a prior report of similarly formulated QDs
entering E. coli (22). Membrane damage is widely observed
with other ENMs (30) such as MgO (8), ZnO (6, 39), Ag (7, 40, 41),
and single walled carbon nanotubes (SWNTs) (42). In many
cases, ENMs associate with membranes (8, 30, 39) and direct
contact is required for toxicity (5, 30, 42) because ROS is
produced on the cell surface (21, 30). However, direct association
of QDs with the cell envelope, a previously described prerequisite for ROS-mediated membrane damage by CdTe QDs (21),
was not observed in our STEM images.
While cadmium ions can enter bacteria passively (see
Introduction), CdSe QDs are too large to diffuse into cells
(12) and thus in the absence of receptor-specific QD
bioconjugates (12), membrane damage can facilitate QD entry
(30). Some QD entry is implied from the end points studied
here, but the same end points could also arise from
extracellular QD breakdown, uptake of metal/ metalloids,
and then reformation of nanocrystals in cells. Still, when

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comparing the estimated cadmium mass in visualized

intracellular 8 nm CdSe particles against the 20-fold higher
total cadmium concentration quantified by ICP-AES, few
cellular QDs were intact. Extensively broken-down cellular
QD material is also supported by the weak XRD signal, the
XANES showing selenium as Se(0) or organoselenium
compounds, the fact that the total and dissolved cellular
cadmium levels were quantitatively the same, and the fact
that overall cadmium enrichment in cells was 4460 when
intact QD enrichment appeared to be 30. Thus, we conclude
that QDs enter cells where they mostly decompose, through
oxidative, ROS-forming processes that enhance the toxicity
of intracellularized QDs relative to intracellular cadmium.
The impression here that QDs were more reactive, and
thus perhaps more damaging inside versus outside of cells,
is compelling and worthy of further evaluation. To evaluate,
we compared the levels of ROS per intact QD under abiotic
conditions (in culture media) versus the ROS per intracellular
QD. Using the ROS data in Figure 3, the measured intracellular
DNA (iDNA) per cell, and the estimated number of QDs per
cell, we calculated (for the 75 mg/L cadmium concentration)
that the intracellular ROS per cell-associated QD was
approximately 6.4 10-13 mg/L H2O2 equivalents per QD.
Then using dissolution data (Figure S6) to determine the
amount of QD cadmium in LB, and the estimated 109
cadmium atoms per QD (see Results, and Kasuya et al. (43))
plus the ROS per QD (Figure S12), we calculated that there
were 1.7 10-15 mg/L H2O2 equivalents per QD in sterile LB.
Comparing the two numbers, it would appear that ROS per
QD were nearly 400 times greater inside cells than outside.
The two numbers could converge either if our estimate of
intact intracellular QDs was low, or if intracellular Cd(II)
also generated ROS, the latter of which is possible based on
mammalian cell culture (44). But in our studies, intracellular
ROS from Cd(II) ions was very low (Figure 3) and thus there
were either more QDs in cells than we estimated from STEM
images or QDs were relatively more potent toxicants once
inside cells. Overall, a plausible scenario for the QDassociated toxicity observed here is that (1) cadmium ions
induced efflux pump-mediated resistance that became
overwhelmed, leading to intracellular Cd(II) ion accumulation, (2) QDs produced ROS extracellularly which damaged
membranes and allowed QDs to enter cells, (3) once inside
cells, QDs, and to a lesser extent Cd(II), continued to generate
ROS, thus exacerbating toxicity processes that began extracellularly but also facilitated QD breakdown inside cells, and
(4) membrane damage inhibited performance of higher
concentration-related cadmium resistance functions, thus
further impairing cells when intact QDs were present. Still
unknown, but of considerable interest in future research,
would be establishing how cells may participate in enhancing
intracellular ROS production from QDs, and if the process
also catalyzes QD decay in bacteria as observed here.
The cellular fates of QDs occurring in this study (Figure
4) may be environmentally relevant, particularly as they were
studied for an environmentally important opportunistic
pathogen. Cadmium ions hyperaccumulated in cells and QDs
appeared relatively broken down inside cells, but QDs were
marginally stabilized against extracellular dissolution. As
above, intact QDs were in higher concentration inside cells
relative to outside (Figure 4). Many bacteria are known to
hyperaccumulate heavy metals (45) including cadmium (46),
and it has been suggested that metal-laden bacteria could
mediate metal transport in the environment (47). In our prior
work with Cr(VI) and P. putida biofilms, extracellular DNA
and Cr(III) appeared to stabilize each another (27). Here,
protein, total carbohydrate as well as intracellular DNA were
elevated in bacteria grown with either QD or cadmium salts
(see Supporting Information) which may imply biomacromolecular involvement in intracellular, as well as extracellular

FIGURE 4. Model of citrate-stabilized CdSe quantum dot (QD)

interactions with planktonic P. aeruginosa. QDs dissolve
partially extracellularly, but dissolution is greater in the
absence of cells. QDs generate extracellular ROS that
permeabilize membranes, facilitating cellular entry, wherein
enhanced ROS production per QD is observed. QDs are
enriched 30 in cells compared to the extracellular
environment, but most QDs in cells decay to metal/metalloids
and cadmium enrichment is 4460.
(48), metal hyperaccumulation. A plausible environmental
scenario is that these bacteria stabilize QDs extracellularly,
but intracellularly sequester QDs, cadmium, and selenium.
These outcomes could reduce long-range transport of ENMs
and their constituents in the environment, but might allow
long-term effects on microbial communities (49-51).

Acknowledgments
This research was supported by the U.S. EPA STAR Awards
R831712 and R833323, by the University of California Toxic
Substances Research and Training Program: Lead Campus
program in Nanotoxicology, and by the Office of Science
(BER), U.S. Department of Energy, Grant DE-FG0205ER63949. Publication of this material is supported by the
National Science Foundation and the Environmental Protection Agency under Cooperative Agreement EF 0830117.
Any opinions, findings, and conclusions or recommendations
expressed in this material are those of the author(s) and do
not necessarily reflect the views of the National Science
Foundation or the Environmental Protection Agency. This
work has not been subjected to EPA review and no official
endorsement should be inferred. This work made use of UCSB
MRL Central Facilities supported by the MRSEC Program of
the National Science Foundation under award DMR05-20415.
Portions of this research were carried out at the Stanford
Synchrotron Radiation Laboratory, a national user facility
operated by Stanford University on behalf of the U.S.
Department of Energy, Office of Basic Energy Sciences.

Supporting Information Available

Detailed descriptions of the materials and methods, as well
as the complete results of the effects of CdSe QDs and
cadmium acetate on growth, the abiotic QD dissolution, and
biomacromolecule contents.This material is available free
of charge via the Internet at http://pubs.acs.org.

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Published on February 27, 2009 on http://pubs.acs.org | doi: 10.1021/es802806n

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ES802806N

Supporting Information
Prepared for Environmental Science and Technology

Effects of Soluble Cadmium Salts Versus CdSe Quantum Dots on

the Growth of Planktonic Pseudomonas aeruginosa

John H. Priester1, Peter K. Stoimenov2, Randall E. Mielke3, Samuel M. Webb4,

Christopher Ehrhardt5, Jin Ping Zhang6, Galen D. Stucky2, Patricia A. Holden1*

Donald Bren School of Environmental Science & Management, 2Department of

Chemistry and Biochemistry, 5Earth Sciences, 6Materials Research Laboratory,
University of California, Santa Barbara, CA 93106
3

Center for Life Detection, Jet Propulsion Laboratory, California Institute of Technology,
Pasadena, CA 91109
4

Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, Menlo

Park, CA 94025

Corresponding Author

Email: holden@bren.ucsb.edu
Tel: 805-893-3195
FAX: 805-893-7612

S1

Table of Contents
1. Materials and Methods
2. Supplementary Results
2.1 Effects on growth
2.2 QD dissolution
2.3 Intra and extra cellular QD concentrations
2.4 Biomacromolecule contents
2.5 Abiotic ROS generation
3. Literature Cited

S3
S6
S6
S6
S6
S6
S7
S18

Supporting Tables
Table S1: Growth parameters for P. aeruginosa cultures exposed to
varying cadmium acetate concentrations.
Table S2: Growth parameters for P. aeruginosa cultures exposed to
varying CdSe quantum dot concentrations.
Table S3: Distribution of total cadmium and dissolved Cd2+ across
intracellular and extracellular fractions for QD- and Cd(CH3COO))2 treated
P. aeruginosa PG201 grown in LB broth.

S8
S8
S9

Supporting Figures
Figure S1: Growth of P. aeruginosa versus time with varying
concentrations of total cadmium delivered as cadmium acetate.
Figure S2: Growth of P. aeruginosa versus time with varying
concentrations of total cadmium delivered as bare CdSe QDs.
Figure S3: Growth parameters for planktonic P. aeruginosa cultivated with
sodium citrate.
Figure S4: Time course of dissolution for 75 mg/L citrate-stabilized
CdSe QDs in de-ionized water.
Figure S5: Relationships between measured vs. added Cd(II) concentrations.
Figure S6: Relationship between cadmium ion mass and total cadmium
mass measured in P. aeruginosa cultures amended with the associated
total mass of cadmium as CdSe QDs.
Figure S7: Lag time and yield of planktonic P. aeruginosa grown either
without or with cadmium in the form of either cadmium acetate or CdSe
QDs.
Figure S8: Representative STEM image and line scan a of P. aeruginosa
PG201 cell grown in the presence of 75 mg/L CdSe QDs.
Figure S9: STEM micrographs of CdSe QD-cultured cells showing
membrane holes, blebbing, and cell wall detached from the plasma
membrane.
Figure S10: XANES spectra for cells amended with CdSe QDs.
Figure S11: XRD spectra for P. aeruginosa, QD, and P. aeruginosa + QD samples.
Figure S12: Reactive oxygen species produced abiotically with various
concentrations of CdSe QDs added to either sterile water or sterile LB broth.

S2

S10
S10
S11
S11
S12
S12

S13

S14
S15

S15
S16
S17

1. Materials and Methods

Quantum dot synthesis and washing. CdSe QDs were prepared according to Rogach et
al. (1) which yields fully-dispersed red (peak emission of 600 650 nm), citratestabilized bare QDs (mean dia. 5 nm) in water. QD suspensions were stored in the dark
at room temperature, and were dialyzed 24 h before each use in Spectra/Por 1, flat-width
10-mm membranes (MW cutoff 6,000 to 8,000; Fisher Scientific, Hampton, NH) against
4 mM sodium citrate to remove excess Cd(II) ions.
Culture conditions. Bacterial culturing was in Luria Bertani (LB) broth (200 L) under
aerobic conditions in the dark (200 rpm; 30 oC) within 96-well plates using a BioTek
Synergy 2 microplate reader (UV/vis/fluorescence; Winooski, VT) that automatically
measured optical density (OD, 600 nm) over time. Prior to each experiment, archived
bacteria (maintained at -80 oC in 70% LB plus 30% glycerol v/v) were freshly streaked
for inocula onto LB agar and incubated at 30 oC for 18 h.
Biochemical analysis. Culture subsamples were centrifuged (12,000 g for 30 min) to
separate cells from extracellular polymeric substances (EPS) as before (2). The EPScontaining supernatants were stored (-20 oC) until analysis. Pelletized cells were either
untreated for microscopy, dried under an N2 stream for XRD analysis, immediately
transported for XANES analysis, or resuspended in 0.5 ml of 1.0 N NaOH then lysed by
heating (80 oC, 1 h), neutralized with 0.5 ml 1.0 N HCl and stored (-20 oC) until analysis
for DNA, total protein, or dissolved Cd(II). Samples for ICP-AES were prepared by
dissolving in hot 10% (v/v) aqua regia. DNA was quantified by the PicoGreen method
(Invitrogen, Carlsbad, CA) using calf thymus DNA as the standard. Total protein was
quantified using the Bradford method (3) using Bio-Rad (Hercules, CA) reagents.
Cellular carbohydrates were quantified by the phenol-sulfuric acid method using glucose
as the standard (4).
DCFH-DA assay. DCFH-DA (Sigma Chemical; dissolved in ethanol at 1mM) was first
freshly deacetylated to dichlorofluorescein (DCFH) by reacting 2 mL with 8 mL 0.01 N
NaOH for 30 min. in the dark (5). The reaction was halted by adding 40 mL of 25 mM
sodium phosphate buffer (pH 7.2), and the solution immediately placed on ice in the dark
until use. Mid-exponential phase (15 hours for Cd acetate concentrations from 0 125
mg/L and QD concentrations from 0 75 mg/L; 27 hours for 125 mg/L QDs) P.
aeruginosa cells were harvested from cultures amended with either CdSe QDs or
cadmium acetate. Cell pellets were resuspended in 7.5 mL sterile 0.9% (w/v) NaCl, and
triplicate 88 L aliquots were deposited into a 96 well plate, then 167 L of the DCFH +
phosphate buffer solution was added immediately. Cultures were also subsampled (1
mL) to quantify intracellular DNA as a proxy for biomass. Abiotic controls (sterile LB
broth with QD and cadmium amendments as above) were similarly sampled and assayed
as were H2O2 and dichlorofluorescein (DCF) standards (10 concentrations each, prepared
in water, up to 750 and 0.5 mg/L, respectively). After incubating in the dark (30 min.),
well fluorescence was measured using the BioTek Synergy 2 plate reader
(excitation/emission = 485/528 20 nm).

S3

Abiotic QD dissolution. CdSe QDs were evaluated for abiotic dissolution in sterile
nanopure H2O under ambient light at room temperature, and also in the dark in either
sterile nanopure H2O, sterile LB, or filter-sterilized (0.22 m) supernatant from 24 hr LBgrown cultures, all in triplicate. For the former, 25 mL of a 75 mg/L (total cadmium)
CdSe QD suspension in nanopure water were dialyzed and immediately transferred to a
sterile test tube surrounding a Spectra/Por DispoDialyzer tube (MW cutoff 6,000 to
8,000; Fisher Scientific, Hampton, NH) containing 2.5 mL sterile 4 mM sodium citrate.
The dialysis tube contents were sampled (2.5 L) at regular times and measured for
dissolved Cd(II), as above. For the latter, individual 75 mg/L (total cadmium) QD
suspensions were prepared from freshly-dialyzed QDs using each aqueous condition
above. Each suspension was dialyzed in the dark against 4 mM sodium citrate for 24 h.
Total cadmium remaining in each dialysis tube was measured and compared to the
starting concentration to determine loss by dissolution.
XRD sample preparation. Cell pellets were washed 1 in sterile DI water to remove
excess growth media, then deposited onto Al foil-covered glass XRD slides and dried
overnight (O/N). Slides were placed in a Phillips Xpert Diffractometer and XRD profiles
were generated using Cu K radiation (1.5405) at 40 kV and 50 mA. Diffraction peaks
between 2 values of 2 and 70 were recorded and compared to those from a standard
slide prepared from dialyzed stock CdSe QDs.
XANES sample preparation. Cell samples were filtered into a concentrated spot of a 0.2
m polycarbonate filter. Standards used for Se oxidation state measurements were grey
metallic selenium (Se(0)), Na2SeO3 (Se(IV)), bulk CdSe (Se(-II)) and CdSe QDs (Se(II)). X-ray energy was selected using a Si(220) double-crystal monochromator, detuned
20% for harmonic rejection. Energy was calibrated by defining the first derivative peak
of a Se foil to be 12,658 eV. Samples were collected in fluorescence mode using a 30element Ge array detector. The total incoming count rates from fluorescent photons were
less than 30,000 s-1 per element, which is well within the linear response range of the
detector. Spectra were collected over the range from 12,640 to 12,670 eV using a step
size of 0.5 eV through the edge. Individual scans typically took 5 minutes for completion
to minimize radiation exposure to the sample. All spectra were averaged, background
subtracted, and normalized using SIXPACK (6).
STEM and EDXA. Specimen preparation involved resuspending cell pellets in 2% (v/v)
glutaraldehyde, storing (4 oC), washing fixed pellets 2 in DI, and dispersing washed cell
pellets in Noble agar. Staining was with 2% (v/v) osmium tetroxide and 2% (v/v) uranyl
acetate (1 h each), except for samples containing cadmium which were stained only with
2% (v/v) osmium tetroxide because of EDXA interferences between the Cd and U peaks
(data not shown). All samples were progressively dehydrated by ethanol (25%, 50%,
75%, 100% (v/v, 2); 15 min. each), followed by washes with 50:50 ethanol:acetone and
100% acetone (v/v, 1; 15 min. each), then incubated (O/N) in 50:50 acetone:Epon resin.
Embedding was in 100% (v/v) Epon resin (cured 24 hr at 60 oC), followed by sectioning
(60 nm) using an MT-X ultramicrotome with a 55o Diatome diamond knife (7). The
sections were placed on 400-mesh, carbon-coated copper grids (Ted Pella Inc., Redding,
CA) followed by vacuum drying O/N (60 oC). Images were acquired using either an FEI

S4

Tecnai G2 F30 FEG microscope (FEI Company, Hillsboro, OR) operating at 300 kV or
an FEI Nano600 FEG microscope operating at 30 kV accelerating voltage, each with a
STEM detector at a working distance of 6.7 mm. EDXA line scans were performed with
a sub-nanometer (0.5 nm or less) electron beam to verify the locally arranged QDs and
spectra were analyzed using Genesis software (EDAX, Inc., Mahwah, NJ).
Estimation of nanoparticle numbers per cell. The numbers and diameters for 25
nanoparticles (total) were scaled from three images per QD treatment, using 1 or more
cells per image. These data were used to estimate the amount of nanoparticulate, perhaps
QD-associated, cadmium that appeared in cellsas an additional estimate (besides
calculations using the differences between total and dissolved cadmium) of QDs in cells.
The total nanoparticle-associated cadmium mass with cells was calculated under two endmember scenarios, assuming: 1) QDs were only on cell surfaces, and 2) QDs were only
in the cell interior. To determine nanoparticle surface density, 3 square regions from
each cell were designated in Photoshop, and the total numbers of nanoparticles in each
region were counted. To estimate the number of cadmium atoms in one nanoparticle
(assuming CdSe composition), data from Kasuya et al. (8) was extrapolated to an 8 nm
diameter CdSe particle yielding an estimated 109 cadmium atoms per nanoparticle, which
was then used to calculate the total nanoparticulate cadmium mass per cell. The two endmember scenarios were compared to the measured amounts of cellular cadmium (by ICPAES) to estimate the percentage of total cadmium in the form of intact QDs.

S5

2. Supplementary Results
2.1 Effects on growth
The three growth parameters calculated (i.e. lag time, yield and growth rate calculated
from growth curves; Figures S1 and S2) were related to cadmium concentration and
appeared similar for the Cd(CH3COO))2 and QD treated cultures (Tables S1 and S2;
shown graphically in Figures 1 and S7). Consistently, total 24 h cell counts (by
epifluorescence microscopy) at 75 mg/L total cadmium were statistically similar (P=0.19)
and averaged 2.08 0.20 1010 per mL, yet were significantly less than total counts in
the LB control (5.18 0.75 1010, per mL; P=0.02). Culture tube versus microplate
formats yielded similar growth parameters (data not shown). Extensive damage to QD
treated cells was visible (Figure S9).
2.2 QD dissolution
The analytical approach for distinguishing nanoparticulate QD-cadmium from dissolved
Cd(II) involved measuring total cadmium by ICP-AES and dissolved cadmium by the
Measure iT assay, then calculating nanoparticulate QD-cadmium as the difference. By
this approach, CdSe QD dissolution in water was rapid and followed the Noyes
Whitney relation for sparingly soluble solids in aqueous solutions (9) (Figure S4). The
Measure iT assay was performed similarly in LB broth, spent (24 h) culture supernatant
and water (Figure S5), and the QD dissolution extent (by dialysis) was also similar across
these solutions (data not shown). As expected, higher initial Cd masses (as QDs) resulted
in higher dissolved Cd(II) concentrations (Figure S6). The amount of dissolved Cd(II)
from the QDs was also measured intra and extra cellularly in P. aeruginosa cultures
(Table S3).
2.3 Intra and extra cellular QD concentrations
Assuming a cell width of 1 m (Fig. 4), and the aspect ratio for QD-treated cells (above)
of 3.3, the cellular concentration of these intracellular QDs is then approximately
7.21019 QDs/liter. Cellular (i.e. a combination of membrane-associated and internallydistributed), QD-associated cadmium was then estimated, assuming 109 cadmium atoms
per nanoparticle by extrapolating from Kasuya et al. (8), to be 0.0083 pg of cadmium per
cell. The presence of intracellular QDs is supported by the observed co localization of
Cd and Se inside cells (Figure S8). Using this same density of cadmium atoms per
nanoparticle (109) and the dissolution data (Fig. S5) for QDs in LB broth, then the
extracellular concentration of QDs is approximately 2.51018 QDs /liter, which is
approximately 30 fold less than the intracellular concentration.
2.4 Biomacromolecule contents
For the 75 mg/L (total cadmium) concentration, intracellular DNA was similar (P=0.31)
for the QD-treated (2.52 0.11 10-6 ng/cell) and Cd(CH3COO))2-treated (2.03 0.88
10-6 ng/cell) cultures but significantly (P=0.00) greater than the no-metal controls (1.12

S6

0.18 10-6 ng/cell). Intracellular protein contents for the QD and Cd(II) treatments were
similar and averaged 92.6 9.8 pg/cell which was significantly (P = 0.01) greater than
the controls (51.6 5.1 pg/cell). Similarly, intracellular carbohydrate contents for the
QD and Cd(II) treatments were similar and averaged 4.6 0.3 pg/cell which was
significantly (P = 0.01) greater than the controls (1.7 0.1 pg/cell).
2.5 Abiotic ROS generation
After correcting for background fluorescence (as above) and again converting
fluorescence to H2O2 equivalents, sterile LB broth plus DCFH yielded an ROS
concentration of 534 183 mg/L H2O2. When added to either LB or to water, CdSe QDs
also generated ROS abiotically, albeit to a much lesser extent in water as compared to LB
broth (Fig. S12) and to a much greater extent than LB alone (above). Cadmium salts
added to either sterile LB or water did not result in ROS formation.

S7

Supporting Tables

Table S1. Growth parameters for P. aeruginosa cultures exposed to varying cadmium
acetate concentrations. Like letters in each column indicate no significant difference (Ttest, p > 0.05).
Cd(II) (mg/L)

Lag Time (h)

Specific Growth Rate (h-1)

Max. OD600

0.0
5.0
10.0
20.0
37.5
75.0
115.0
150.0

3.69 0.13a
5.43 0.14b
5.56 0.11b
5.24 0.37b
7.15 0.07c
9.93 0.84d
10.55 0.14d
13.24 0.07e

0.92 0.02a
0.67 0.04b
0.63 0.02b
0.51 0.01c
0.46 0.02d
0.44 0.00d
0.29 0.02e
0.24 0.02f

1.60 0.02a,b
1.70 0.02c,d
1.60 0.02a
1.58 0.07a,b,d
1.47 0.05b,e
1.45 0.07a,e
1.21 0.03f
1.24 0.06f

Table S2. Growth parameters for P. aeruginosa cultures exposed to varying CdSe
quantum dot concentrations. Shown are both the total Cd(II) concentration, as well as the
Cd(II) ion concentration at 6 hours. Like letters in each column indicate no significant
difference (T-test, p > 0.05).
Total Cd (mg/L) Cd(II) at 6 h (mg/L)

Lag Time (h)

Specific Growth Rate (h-1)

0.0
10.0
20.0
37.5
50.0
75.0
100.0
125.0

3.69 0.13a
4.89 0.15b
5.67 0.08c
6.97 0.08d
7.41 0.06e
7.96 0.11f
8.41 0.06g
27.66 3.87h

0.92 0.02a
0.63 0.02b
0.69 0.03b
0.55 0.04c
0.49 0.04c,d
0.43 0.02d
0.31 0.03e
0.27 0.05e

0.0
4.9
8.6
14.6
18.2
23.6
29.1
33.1

S8

Max. OD600

1.60 0.02a
1.54 0.01a
1.84 0.07b
1.76 0.04b
1.68 0.05a,b
1.41 0.05c
1.04 0.02d
0.51 0.01e

Table S3. Distribution of total cadmium (by ICP-AES) and dissolved Cd2+ (by Measure
iT assay) across intracellular (Cells) and extracellular (Supernatant) fractions for QD- and
Cd(CH3COO))2 treated P. aeruginosa PG201 grown in LB broth. Controls were
Sterile LB amended with either Cd(C2H3O2)2 or CdSe QDs at a total Cd(II)
concentration of 75 mg/L. Assays were performed immediately after inoculation (t = 0 h)
and at the end of exponential phase (t = 24 h). Experiment volume was 10 mL with all
treatments performed in triplicate.

Initial (t = 0 h)

Final (t = 24 h)

Total Cd (mg)

Cd2+ (mg)

Total Cd (mg)

Cd2+ (mg)

Cd(II) Sterile
Cd(II) Cells
Cd(II) Supernatent*

0.79 0.04
~
0.73 0.03

0.75 0.07
~
0.66 0.04

0.70 0.03
0.03 0.00
0.55 0.04

0.62 0.05
0.03 0.00
0.57 0.03

QD Sterile
QD Cells
QD Supernatent*

0.77 0.01
~
0.76 0.01

0.18 0.04
~
0.24 0.07

0.73 0.01
0.02 0.01
0.70 0.01

0.50 0.01
0.03 0.00
0.40 0.01

*At 0 h of growth all Cd was in the media/supernatant.

S9

Supporting Figures

Figure S1. Growth of P. aeruginosa versus time with varying concentrations of total
cadmium delivered as cadmium acetate.

Figure S2. Growth of P. aeruginosa versus time with varying concentrations of total
cadmium delivered as bare CdSe QDs

S10

Figure S3. Growth parameters for planktonic P. aeruginosa cultivated with sodium
citrate amended at varying concentrations, showing that citrate is unlikely to affect
growth when cultures are amended with citrate-stabilized bare QDs (i.e. Figure S2).

Figure S4. Time course of dissolution for 75 mg/L (total cadmium basis) citratestabilized CdSe QDs in de-ionized water. Predicted values are modeled using the Noyes
Whitney relation for sparingly soluble solids in aqueous solutions 57, as described in the
Results.

S11

Figure S5. Relationships between measured vs. added Cd(II) concentrations where
cadmium acetate was dissolved into three sterile aqueous solutions relevant to this study,
showing that dissolved Cd(II) is measured equivalently across these three conditions at
all relevant concentrations.

Figure S6. Relationship between (dissolved) cadmium ion mass and total (QD +
dissolved) cadmium mass measured in P. aeruginosa cultures amended with the
associated total mass of cadmium as CdSe QDs. The line of regression and fit parameter
suggest a linear relationship, and the slope supports that most QDs are undissolved after 6
h (entry into exponential phase).

S12

Figure S7. Lag time (A) and yield (by maximum OD, B) of planktonic P. aeruginosa
grown either without (control, dark circle) or with cadmium in the form of either
cadmium acetate (open square) or CdSe QDs (open diamond). QDs appear to cause
increased lag times and, above 50 mg/L (total cadmium basis), lower yields relative to
cadmium acetate when each form is expressed as dissolved Cd(II) concentration at the
end of exponential phase (6 h).

S13

Figure S8. Representative STEM image of P. aeruginosa PG201 cell grown in the
presence of 75 mg/L CdSe QDs (top). The EDS spectra, acquired for the line scan across
the cell (white horizontal line), are for Cd (middle) and Se (lower). Vertical arrows
indicate regions rich in Cd and Se; the horizontal arrows show cell envelope damage
(blebbing).

S14

Figure S9. STEM micrographs of CdSe QD-cultured cells showing membrane holes
(A), blebbing (B), and cell wall detached from the plasma membrane (C). White arrows
indicate the region of damage.

Figure S10. XANES spectra for cells amended with CdSe QDs (Q.D.s + cells) and all
relevant standards, showing that the oxidation state of selenium associated with cells
grown with CdSe QDs is more similar to elemental selenium, Se(0), as compared to the
other relevant standards tested here.

S15

Figure S11. XRD spectra for P. aeruginosa control (top), CdSe QD control (middle),
and P. aeruginosa + CdSe QD (bottom) samples. The weak peaks at 2 24o in the QD
control and P. aeruginosa + QD samples are shown as insets (middle and bottom).

S16

Figure S12. Reactive oxygen species (ROS, as H2O2 equivalents per liter) produced
abiotically with various concentrations of CdSe QDs added to either sterile water or
sterile LB broth. ROS is related to either A) total cadmium concentration in the form of
QDs or B) dissolved cadmium after 6 hours, i.e. the time of entry into exponential phase
when cells are present. Similar measurements were made for cadmium acetate in both
aqueous conditions, but no ROS were produced across the same cadmium concentration
range (not shown).

S17

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