J Nanopart Res (2014) 16:2414
DOI 10.1007/s11051-014-2414-2
RESEARCH PAPER
Behavior of gold nanoparticles in an experimental
algal–zooplankton food chain
Kyle D. Gilroy • Svetlana Neretina
Robert W. Sanders
•
Received: 30 June 2013 / Accepted: 9 April 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract The release of engineered nanomaterials
offers a significant concern due to their unexpected
behavior in biological systems. In order to establish
the level of threat from releasing nanomaterials into
ecosystems, simplified food webs are an effective
method to determine toxicity and bioassessment. A
study is presented examining the behavior of citratecapped gold nanoparticles (AuNPs) introduced into a
model food chain consisting of a phytoplankton food
(Ankistrodesmus falcatus) and a zooplankton grazer
(Daphnia magna). UV–Vis spectroscopy is used to
monitor the behavior of AuNPs in the presence of
algae (Ankistrodesmus) and Daphnia over the span of
5 days. Transmission electron microscopy shows the
attachment of gold aggregates to the surface of the
Ankistrodesmus. Bright field microscopy shows significant accumulation of AuNPs in the gut of Daphnia
via uptake of contaminated Ankistrodesmus and
directly from water. No toxicity was evident for
Electronic supplementary material The online version of
this article (doi:10.1007/s11051-014-2414-2) contains supplementary material, which is available to authorized users.
K. D. Gilroy (&) S. Neretina
College of Engineering, Temple University, Philadelphia,
PA 19122, USA
e-mail: [email protected]
R. W. Sanders
Department of Biology, Temple University, Philadelphia,
PA 19122, USA
Daphnia exposed to AuNPs at the concentration used
(880 lg L-1).
Keywords Gold nanoparticles Daphnia
magna Ankistrodesmus Nanotoxicity Discrete dipole approximation Environmental
effects
Introduction
The field of nanotechnology involves engineering of
materials at an atomic scale in order to achieve sizeand shape-dependent physical properties. Engineered
nanomaterials may achieve desired properties in an
industrial setting, however, little is known regarding
the fate of nanomaterials in biological systems (MacCormack and Goss 2008). As research in nanotechnology continues to grow, it is important to examine
the ecotoxicological and environmental implications
for this valuable scientific technology. There have
been several studies documenting the trophic transfer
of metallic (Ferry et al. 2009; Unrine et al. 2010; Li
et al. 2010) and semiconducting nanoparticles (Bouldin et al. 2008; Holbrook et al. 2008; Zhu et al. 2010;
Wanga et al. 2008) that suggest freshwater organisms
exposed to nanomaterials are prone to adverse effects.
In one study, transmission electron microscopy indicated that AuNPs bind to the cell wall of the freshwater
alga, Scenedememus subspicatus (Renault et al. 2008),
while another investigation found that exposure of
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Daphnia magna to AuNPs resulted in changes to
swimming behavior and an increase in both molting
and death rate (Li et al. 2010). Exposure of Daphnia
and its algal food to AuNPs and other nanoparticles
yields important information about toxicity and also
the consequences of the transfer of these particles
through the food web (Holbrook et al. 2008). For
example, food chain transport of polystyrene NPs
(1–100 nm) from algae to zooplankton and finally to
fish had adverse effects on the fish’s behavior and fat
metabolism (Cedervall et al. 2012).
In aquatic ecosystems, micro-algae are the base of
many food webs and can be strongly impacted by organic
and metallic pollution due to their small sizes and
consequently high surface-to-volume ratios. Microalgae are preyed upon by many organisms, including
Daphnia and other zooplankton. The major focus of this
paper is to demonstrate how AuNPs move through a
simplified freshwater food web from the water column to
an alga, Ankistrodesmus falcatus, and then to Daphnia
magna, a model organism for toxicity studies and a
prominent type of filter-feeding zooplankton in lakes and
ponds. UV–Vis spectroscopy is used to monitor the
plasmonic response of the gold nanoparticles suspended
in standard synthetic fresh water (SSF) over the course of
5 days. The plasmonic nature of the AuNPs makes the
local surface plasmon resonance (LSPR) peak sensitive
to changes in size, shape, aggregation, and local
environment (Kreibig and Vollmer 1995; Kelly et al.
2003; Jensen et al. 1990; Berciaud et al. 2005; Mulvaney
1996). Transmission electron micrographs are used to
characterize how AuNPs bind to the surface of the
Ankistrodesmus. We demonstrate significant bioconcentration of AuNPs in the digestive tract of Daphnia, which
is readily visible via optical microscopy, both in the
presence and absence of algal food.
J Nanopart Res (2014) 16:2414
from Alfa Aesar (Ward Hill, Massachusetts). The
solution was stirred rapidly during the injection and
for an additional 15 min, after which the solution was
stored at room temperature (RT) overnight and then
characterized using UV/Vis spectroscopy. The nanoparticles used throughout this study had an initial
localized surface plasma resonance (LSPR) peak of
524 nm with an ALSPR:A450 ratio of 1.74. This ratio
corresponds to an average diameter of 21 nm (Haiss
et al. 2007; Tullman et al. 2007). The average particle
diameter based on transmission electron microscopy
was 17.01 nm (Fig. S1). Discrepancy between diameters of AuNP measured using UV–Vis versus TEM
could be attributed to variation in AuNP shape,
polydispersity as well as the local chemical environment (Daniel and Astruc 2004). The AuNPs were then
suspended in standard synthetic fresh water (SSF)
used for the maintenance of Daphnia (EPA 2002) and
analyzed using UV–Vis spectroscopy to test for
significant changes to the LSPR response due to a
change in solvent. The LSPR peak position, intensity,
and full width half maximum (FWHM) from SSFsuspended AuNPs showed significant change over
1 week. Within an hour after the addition of AuNPs, a
second peak at a higher wavelength arose that is
attributed to AuNP aggregation (Lee and Ranville
2012) (Fig. 1c). This phenomenon is induced by the
high ionic strength (hardness) of the SSF water (Lee
and Ranville 2012). The stock solution used had a
concentration of 3.80 9 10-10 M, which is equivalent
to 2.29 9 1014 AuNP L-1 or 18.6 mg Au L-1.
UV–Vis Spectroscopy
Materials and methods
UV–Vis extinction spectra were acquired using 1 cm
optical path length cuvettes on JASCO’s UV–Vis
V560 spectrophotometer (Easton, MD). All measurements were performed at RT and blanks consisted of
SSF water.
Synthesis and characterization of AuNPs
Discrete dipole approximation (DDA)
AuNPs were synthesized using a variation of a
protocol developed by Frens (1973). The synthesis
called for the injection of 2 mL of 1 % trisodium
citrate dihydrate into a boiling solution of 1 mM
aqueous hydrogen tetrachloroaurate(III) hydrate. Trisodium citrate dihydrate (99 %) and hydrogen tetrachloroaurate(III) hydrate (99.999 %) were purchased
DDA is a method that utilizes finite element methods
to numerically solve for particles response to electromagnetic radiation (Draine and Flatau 1994, 2012). In
this case, DDSCAT (Version 7.2) was used to solve for
the extinction spectra of gold nanoparticles in three
distinct situations. Each situation was modeled using
LAMMPS Molecular Dynamics Simulator (Plimpton
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Fig. 1 UV–Vis extinction spectra of Ankistrodesmus, AuNPs
in stock solution and AuNPs in SSF at different concentrations.
a Labeled peaks of algae alone (0.68, 1.36 and 2.04 9 105
cells mL-1, blue, red and green lines, respectively); labeled
peaks correspond to extinction at 442 nm (CA1), 480 nm
(CR1), and 686 nm (CA2). b Local surface plasmon resonance
LSPR (524 nm) of AuNPs alone at three concentrations (0.36,
0.73, and 1.09 9 1013 AuNP L-1, blue, red and green lines,
respectively). c Spectral transformation after AuNPs are
suspended in SSF water. The graph shows the spectra of the
AuNP stock solution compared to (i) day 1 (1 h after injection
into SSF), (ii) day 3 (48 h after injection), and (iii) day 5 (96 h
after injection), represented by the purple, blue, red and green
lines, respectively. TEM images in a and b are of Ankistrodesmus (scale bar 5 lm) and AuNPs (scale bar 50 nm), respectively. (Color figure online)
1995) where the AuNPs were represented simply by a
sphere of diameter 20 nm (Fig. S2). The surface of
the Ankistrodesmus was simulated as a cube with
an edge length 60 nm and index of refraction of
n = 1.43 (Huang and Levitt 1977). In all simulations,
greater than 104 dipoles were used in order to ensure
accuracy. The dielectric function for gold was taken
from Johnson and Christy (1972).
electron microscope was used to image Ankistrodesmus
before and after the addition of gold nanoparticles.
Samples were prepared by dropping 40 lL of solution
on a 50 nm TiOx grid purchased from Ted Pella Inc.
Culture conditions and microscopy
Daphnia magna is a common freshwater cladoceran
zooplankton species and a model organism in toxicological studies. Daphnia were maintained at 18 °C
with a 14:10 light:dark cycle in standard synthetic
fresh water (SSF) with Ankistrodesmis as food.
Ankistrodesmus falcatus, a nonmotile chlorophyte
alga with widespread distribution in lakes and ponds,
was batch cultured in the same incubator as Daphnia
in modified MBL medium without silica (Williamson
and Butler 1987). For experimental setups and photographs, individual adult Daphnia of similar size
(*2.8 mm) without eggs or embryos were selected.
Daphnia magna were transferred with minimal culture
water into fresh SSF and then into the experimental
beakers using wide-bore plastic pipets.
Daphnia were observed and photographed at 409
magnification in spot plates using a Nikon Eclipse 6400
light microscope (Melville, NY) and a Luminera Infinity 2
camera system (Ottawa, ON). FEI Tecnai 12 transmission
AuNP exposure experiments
There were two series of experiments including (i) Ankistrodesmus in the presence of AuNPs, and (ii)
Daphnia in the presence of Ankistrodesmus and AuNPs.
For all exposure experiments, the AuNP stock solution
was added to 40 mL of SSF water in 50 mL glass
beakers, yielding a concentration of 1.09 9 1013
AuNP L-1 (880 lg Au L-1). This is below the lethal
concentration or LC50 for Daphnia magna (Li et al.
2010) (70 mg L-1) and slightly above the lowest
observable effect concentration of 20 nm colloidal
gold (500 lg L-1) (Lovern et al. 2008). Experimental
beakers were kept at the same temperature and light
conditions as the stock cultures. For the algae-only
experiments, a series of concentrations of Ankistrodesmus ranging from 6.8 9 104 to 2.0 9 105 cells mL-1
were prepared, and AuNPs added. Algal abundances in
this range are observed in spring blooms in eutrophic
temperate systems. Samples were taken for UV/Vis
analysis at noon daily for a period of 5 days. Prior to
each sampling, beakers were gently mixed to disperse
any algae that had settled toward the bottom of the
beaker. For the experiment with Daphnia, Ankistrodesmus in SSF (final concentration of 1.75 9 105
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cells mL-1) was prepared and distributed into beakers with AuNPs as above, followed by introduction of
Daphnia to create a series with increasing numbers of
zooplankton (0, 2, 4, 6, or 8 Daphnia per beaker). The
increasing number of Daphnia created range of grazing
impact on the algae. On succeeding days, a total of
1 9 105 Ankistrodesmus (0.5 mL of stock culture) was
added as food for the Daphnia. A 2 mL sample for UV/
Vis analysis was taken at the beginning of the
experiment and both before and immediately after
addition of food on subsequent days. All analyses were
corrected for changes in volume in the beakers.
Results and discussion
Spectral interpretation and analysis
Extinction spectra were measured for three concentrations of Ankistrodesmus in SSF water and there
were three prominent peaks of interest, CA1 (442 nm),
CR1 (480 nm), and CA2 (686 nm) (Fig. 1a). These
correspond to peaks for chlorophyll (CA1 and CA2)
and carotenoids (CR1). The relative intensity of the
peaks indicates qualitatively the amount of algae
present in the sample; higher peaks corresponded to
increased concentrations of Ankistrodesmus (Fig. 1a).
Preliminarily studies indicated that in the absence of
grazing, the population of Ankistrodesmus stays
relatively constant over a 5-day period (less than
15 % change) for a population of 2.4 9 106
cells mL-1 (Fig. S3). Changes in the population
density were also monitored using the height of the
CA2 peak which is independent of AuNPs LSPR. The
LSPR spectrum (Fig. 1b) of the AuNP stock solution
has a single peak narrow at 524 nm, representing a
near-monodisperse solution of AuNPs (Fig. S4).
Ankistrodesmus series
In order to examine the interaction of dispersed AuNPs
and Ankistrodesmus, extinction efficiency was monitored over a five-day period for the three algal
concentrations (0.68, 1.36, and 2.04 9 105 cells mL-1)
in the presence of an initial AuNP concentration of
1.09 9 1013 AuNPs L-1 (880 lg L-1). For each algal
concentration, the CA2 peak remained relatively constant throughout the 5 days decreasing \15 %, while
the intensity of the LSPR peak position (524 nm)
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J Nanopart Res (2014) 16:2414
decreased relative to both the CA2 peak (Fig. 2) and the
CA1 peak. The observed decrease and red shift of the
LSPR peak was expected due to AuNP aggregation
events similar to those that took place when the AuNPs
are suspended alone in SSF water (Yang et al. 2007;
Majzik et al. 2010). AuNPs suspended in SSF water
alone continually aggregate and result in a broad peak
around 700 nm by Day 5 (Fig. 1c). However, the peak
profile of AuNPs in the presence of the highest
Ankistrodesmus concentration increased in the region
from 550 to 650 nm (Fig. 2c) and resembled that of the
original pure Ankistrodesmus peak (i.e., without added
AuNPs, Fig. 1a); the subsequent rise of a broad peak at
700 nm noted for AuNPs alone (Fig. 1c) was not
observed. The relatively sharp peak and small change of
height in the area of the CA2 peak suggests that there are
few large AuNP aggregates free in the water and also an
interaction between the AuNPs and the Ankistrodesmus. Conversely, the peak profile for the lowest
Ankistrodesmus concentration showed a prominent
shoulder between 550 and 650 nm at day 5 and more
closely resembled the profile of AuNPs alone in SSF
water (Fig. 1c). But, the CA2 stayed relatively constant,
indicating that the LSPR peak did not shift as far to the
right as was observed when AuNPs were suspended in
SSF alone. These differences are indicative of an
Ankistrodesmus-AuNP interaction even at the lower
algal concentrations. In general, a shift in the LSPR
peak can occur for three main reasons: (i) the AuNPs
aggregate together and the LSPR shifts to the right and
decreases in intensity (Liu and Lu 2006), (ii) the AuNPs
stick to the cell wall of the Ankistrodesmus (either
singly or as aggregates) or (iii) AuNPs embed into the
cell wall and undergo a red shift due to a change of index
of refraction (Malinsky et al. 2001). To examine the
possibilities of AuNPs sticking to or imbedding in the
cell wall of the algae, we simulated the interaction using
Discrete Dipole Approximation simulations to determine feasibility.
Discrete dipole approximation simulation
DDA was used to simulate AuNPs in a three separate
states, (i) solution suspended, (ii) bound to the
membrane, and (iii) embedded in the membrane. The
AuNP (20 nm) suspended in a dielectric media with
n = 1.33 (Fig. 3a) resulted in an LSPR peak located at
525 nm (ALSPR: A450 = 1.89), consistent with our
experiment (stock solution). The index of refraction for
J Nanopart Res (2014) 16:2414
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Fig. 2 Normalized extinction spectra for three concentrations
of Ankistrodesmus on a day 1; b day 3; and c day 5. The initial
concentration of AuNPs was fixed at 880 lg L-1. The blue,
yellow, and red lines represent concentrations of 0.68, 1.36, and
2.04 9 105 Ankistrodesmus mL-1); d the ratio of A524 to ACA2
as a measure of relative shift in AuNP LSPR to algal
abundances. (Color figure online)
a bilayer membrane (Huang and Levitt 1977), like
those found in algae and other eukaryotic organisms, is
estimated to be n = 1.43. The extinction spectra of
both attached and embedded AuNPs (separation of
1 nm) in a bilipid layer of n = 1.43 was calculated
using DDA (Fig. 3b, c). A total red shift of 67 nm was
demonstrated for embedding the AuNPs as compared
to the isolated AuNP case. Previous studies supporting
this result found that AuNP aggregation along a surface
demonstrated significant red-shifting along with subsequent decrease in the extinction efficiency (Majzik
et al. 2010; Kim et al. 2005). We suggest that the
aggregation of AuNPs along the surface of Ankistrodesmus is similar to the AuNP binding seen on the
surface of another chlorophyte alga Scenedesmus
(Renault et al. 2008). In our experiment, a prominent
shoulder was formed at 550–650 nm (Fig. 2, notably
for the lowest concentration of Ankistrodesmus), which
is consistent with the simulation where AuNPs
attached to the cell wall had a peak at 575 nm and
592 nm for embedded AuNPs. Simulations were
designed after transmission electron micrographs
which confirm that AuNP aggregates bind heavily to
the sides of the Ankistrodesmus.
Daphnia series
In order to determine the behavior of AuNPs in a more
dynamic setting, AuNPs were added to beakers
containing Daphnia and Ankistrodesmus. The CA2
peak (686 nm), which corresponds to the relative
amount of the Ankistrodesmus in solution, flattens
over time for beakers containing Daphnia, but not for
the treatment without Daphnia. This indicates heavy
feeding on algae by the zooplankton—even when the
zooplankton are present at low densities (Fig. 4). After
5 days, UV–Vis spectra showed that beakers containing Daphnia had a single peak (524 nm) suggesting
the removal of AuNP aggregates, Ankistrodesmus and
Ankistrodesmus contaminated with AuNPs, by the
zooplankton. In this experiment, AuNPs could leave
the solution by (i) AuNPs binding to the surface of the
Daphnia, (ii) Daphnia ingesting AuNPs directly, (iii)
AuNP or AuNP aggregates binding to Ankistrodesmus,
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Fig. 3 Diagrammatic representation of theoretical scenarios
for AuNPs position and theoretical LSPR shift calculated using
discrete dipole approximation (DDA). a AuNP suspended in
pure water (n = 1.33); b AuNPs bound to the surface of the
J Nanopart Res (2014) 16:2414
algal cell membrane (n = 1.43); c AuNPs imbedded in the cell
membrane (n = 1.43); d extinction efficiency calculated by
DDA for scenarios in a, b, and c, yielding LSPR of 525, 575 and
592 respectively
Fig. 4 Extinction spectra of
samples with 1.09 9 1013
AuNP L-1, Ankistrodesmus
and increasing numbers of
Daphnia present. a Spectra
after transfer of Daphnia
into beakers (measured 1 h
after addition); b spectra on
day 5. A concentration of
Ankistrodesmus
2.04 9 105 mL-1 was used
for this series
which are then ingested by the Daphnia, and (iv) a
combination of these mechanisms. All of mechanisms
likely contributed to the removal of AuNPs in our
experiments. The slightly purple color of the Daphnia
exposed to AuNPs (Fig. 5d) suggests some binding to
the animals’ carapaces, while direct ingestion of AuNPs
was reported by Lovern et al. 2008 and indicated in our
studies by light microscopy (see below). However,
Daphnia are much less efficient at capturing even
bacteria-sized particles relative to algae (Jürgens et al.
1994) and we suggest that a much greater proportion of
AuNPs would be ingested via their attachment to larger
food particles such as algae.
Light and electron microscopy
Daphnia fed with Ankistrodesmus alone have a greencolored digestive tract indicative of feeding on the algae
(Fig. 5b). In the treatments with Ankistrodemus pre-
123
exposed to AuNPs or in the presence of AuNPs alone,
the gut of Daphnia was a red-brown color indicating the
presence of AuNPs in the gut (Fig. 5d). Daphnia with
red-brown guts from exposure to AuNPs regained a
completely green digestive system within 4 h of being
transferred to a beaker with fresh, uncontaminated
Ankistrodesmus, indicating that the bulk of the AuNPs
were moved through the digestive system in the
presence of abundant food. These results are consistent
with transmission electron microscopic observations
indicating that AuNPs were not significantly absorbed
into the gut tissues of Daphnia, even at concentrations
much higher than used in our experiments (Lovern et al.
2008). Ankistrodesmus unexposed to AuNPs were
dropped onto a TiOx TEM grid and examined for
comparison to algae exposed to AuNPs over 3 days
(Fig. 5). The large dark structures in Fig. 5a are
‘‘lysosome-like structures’’ (Churro et al. 2009). These
also were present in the Ankistrodesmus exposed to
J Nanopart Res (2014) 16:2414
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Fig. 5 Interactions of AuNPs with Ankistrodesmus and Daphnia. TEM micrographs of Ankistrodesmus growing in the
absence (a) and presence (c) of AuNPs (Note the darker top
region represents the body of the Ankistrodesmus whereas the
bottom white region is the TiOx substrate). The yellow, orange,
and red circles indicate, respectively, the binding of single,
small clustered, and larger aggregate AuNPs bound to Ankistrodesmus. Light micrographs of a Daphnia that fed on
Ankistrodesmus with AuNPs (d) and another animal 4 h after
being transferred from an experimental beaker with AuNPs to
AuNP-free Ankistrodesmus (b). (Color figure online)
AuNP, but are not visible in the higher magnification
used in Fig. 5c. Single AuNPs and small aggregates
(2–12 AuNPs clusters) adhered the alga are clearly
visible in Fig. 5c, which is consistent with our
interpretation of the UV–Vis data.
(Wetzel 2001), it provides a potential pathway for
AuNPs and other nanomaterials to enter into the diet of
humans with unknown consequences.
Conclusion
Little is known about the effects from potential
contamination of the natural environment with
nanomaterials (Handy et al. 2008). Previous studies
have shown bioaccumulation of nanomaterials from
water in ciliate-rotifer (Holbrook et al. 2008) and algaclam (Renault et al. 2008) and algae–zooplankton–fish
(Cedervall et al. 2012) food chains. After our sampling
for AuNP analyses was completed, Daphnia were left
in the AuNPs suspension for 21 days with no signs of
behavioral changes and with considerable reproduction, despite obvious accumulation of AuNPs in their
guts as seen in Fig. 5 and visible by eye.
Our results indicate that once AuNPs enter into the
SSF water, they aggregate and bind to the surface of
algae, which are bioaccumulated in the gut of Daphnia
magna. Additional routes for uptake by Daphnia
include direct ingestion of AuNPs, which also was
observed in this study, and binding of nanoparticles
directly to the Daphnia (Li et al. 2010). Although
AuNPs can be cleared from the digestive system of
Daphnia if not continuously ingested (Lovern et al.
2008) (Fig. 5), fish that ingest AuNP-contaminated
Daphnia could themselves accumulate AuNPs. Since
planktivorous fish are major consumers of Daphnia
Acknowledgments The work has benefited from the facilities
available through Temple University’s College of Science and
Technology. Temple University’s Owl’s Nest high-performance
computing cluster was used to carry out DDA simulations. KDG
acknowledges support received through a Temple University
Graduate Student Fellowship. RWS acknowledges support for
supplies from Temple University’s Indirect Cost Recovery
Program.
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