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Environmental Chemistry DOI 10.1002/etc.468
CHANGES IN AGGLOMERATION OF FULLERENES DURING INGESTION AND
EXCRETION IN THAMNOCEPHALUS PLATYURUS
Manomita Patra†, Xin Ma
‡, Carl Isaacson
‡, Dermont Bouchard
‡,
Helen Poynton§, James M. Lazorchak
§, Kim R. Rogers
†*
†U.S. Environmental Protection Agency, Las Vegas, NV
‡U.S. Environmental Protection Agency, Athens, GA
§U.S. Environmental Protection Agency, Cincinnati, OH
Running Head: Agglomeration of Ingested Fullerenes
Corresponding Author:
K.R.Rogers
U.S. Environmental Protection Agency
944 E. Harmon Ave.
Las Vegas, NV 89119
Tel: (702) 798-2299
[email protected]
To whom correspondence should be addressed ([email protected])
© 2011 SETAC Submitted 6 January 2010; Returned for Revision 1 November 2010; Accepted 15 November 2010
Environmental Toxicology and Chemistry
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Abstract: The crustacean Thamnocephalus platyurus was exposed to aqueous suspensions of
fullerenes C60 and C70. Aqueous fullerene suspensions were formed by stirring C60 and C70 as
received from a commercial vendor in deionized water (termed aqu/C60 and aqu/C70) for
approximately 100 d. The Z-average diameters of aqu/C60 and aqu/C70 aggregates as measured
by dynamic light scattering were 517 ± 21 nm and 656 ± 39 nm (mean ± 95% confidence limit),
respectively. Exposure of T. platyurus to fullerene suspensions resulted in the formation of dark
masses in the digestive track visible under a stereo microscope (40 x magnification). Fullerene
ingestion, over one hour exposure was quantitatively determined after extraction and analysis by
HPLC-MS. One hour exposures (at 3 mg/L and 6 mg/L) resulted in aqu/C60 burdens of 2.7 ± 0.4
µg/mg and 6.8 ± 1.5 µg/mg wet weight, respectively. Thin section TEM images of aqu/C60
exposed T. platyurus showed the formation in the gut of fullerene agglomerates (5 to 10 µm),
which were an order of magnitude larger in size than the suspended fullerene agglomerates.
Upon excretion, the observed fullerene agglomerates were in the 10 to 70 µm size range and
settled to the bottom of the incubation wells. In contrast to the control polystyrene microspheres,
which dispersed after depuration, the aqu/C60 agglomerates (greater than two orders of
magnitude larger than the suspended fullerenes) remained agglomerated for up to six months.
When exposed to fullerenes, crustacean T. platyurus shows the potential to influence the
agglomerate size and may facilitate movement of these nanoparticles from the water column into
the sediment.
Key words: Nanomaterials, Fullerenes, C60, C70, Thamnocephalus platyurus.
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INTRODUCTION
Nanotechnology is a rapidly growing industrial enterprise encompassing a diverse array
of engineered nanomaterials, developed and applied in fields such as medicine, plastics, energy,
electronics, and cosmetics. The characteristics of the nanomaterials, as well as concerns
involving nanoparticle transport, fate, exposure, dose and potential effects on ecosystems, have
been reviewed in the literature [1]. Because of the potential for nanomaterials to contaminate
various environmental settings (e.g., water soils, etc.) and contribute to ecosystem and human
exposure and risk, the United States Environmental Protection Agency (U.S. EPA) views these
materials as potential and emerging contaminants.
Fullerenes as a class of carbon nanomaterials are used in a wide range of commercial
products and are expected to be one of the major contributors in future product development
[2 (http://www.wilsoncenter.org/index.cfm?topic_id=166192&fuseaction=topics.home)]. Many
environmental studies with fullerenes to date have used solvent exchange techniques for creating
fullerene suspensions of C60 in water [3-5]. For these studies, fullerenes were dissolved in an
organic solvent, usually toluene or tetrahydrofuran (THF), the solution was dispersed in water,
and the organic solvent removed by distillation. Another approach to suspending fullerenes in
water is simply to stir them for extended time periods (> 100 days). Both stirring in water and
organic solvent-assisted techniques result in the formation of kinetically stable suspensions [6,7];
however, colloidal fullerene aggregates formed using solvent exchange and those formed in
water-only systems differ with respect to size, shape, charge, polydispersity and also in the toxic
effects they elicit [6,8-10]. The remaining THF in C60 suspensions has been found to contribute
to the toxicity effect for microorganisms such as Bacilus subtilis [11] and to the toxicity of a
mouse macrophage cell line [12]. Extended stirring in water without the aid of organic solvents
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was used in the present study to create colloidal suspensions of aqu/C60 and aqu/C70 that
contained fewer preparation artifacts and that are more representative of natural environmental
systems [7,13].
Aquatic environments may be particularly vulnerable to contamination of nanomaterials,
such as fullerenes, that can form colloidal suspensions. Factors that contribute to the formation of
these suspensions include the relative size of fullerene agglomerates as well as their morphology
and surface zeta potentials which are a function of their media environments as well as how they
were prepared in suspension [7,13,14]. In addition, the transport behavior and ecotoxicity of
fullerenes in aqueous environments also significantly depend on the properties of these
agglomerates [15,16]. Other important factors that limit the understanding of potential
ecosystem impacts include uncertainties concerning ingestion, bioavailability and
bioaccumulation of these materials in indicator species such as crustacean zooplankton.
Planktonic invertebrates are particularly important with respect to contamination of the
aquatic environment because they are the bridge in the food chain between pollutants bound to
suspended particulates, algae and fish [17,18]. Organisms such as Daphnia magna,
Thamnocephalus platyurus, and Brachiomus clyciflorus have been widely used as indicator
species for both ecosystems and human health [19]. Microcrustaceans have also been shown to
ingest and accumulate of carbon nanoparticles [20]. Exposure of D. magna to fullerene C60 and
carbon nanotubes have resulted in the presence of dark masses in the gut of these organisms
[17,21]. The use of radiolabelled nanotube agglomerates has also allowed the observation of
uptake and elimination kinetics under specific conditions [22].
A rapid screening assay that is sensitive to a wide range of pollutants in environmental
matrices has been reported using T. Platyurus. This assay is based on the accumulation of inert
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polystyrene microspheres which can be observed in these transparent organisms. Compounds
that induce a toxic stress response inhibit the accumulation of the indicator spheres [23,24].
Based on this microsphere accumulation behavior, this organism was chosen for the present
study.
Although C60 fullerenes have been observed to accumulate in the gut cavity of micro-
crustaceans such as D. magna, with the exception of Tervonen et al., changes in agglomeration
prior to and during ingestion and after excretion have not been well documented [20]. The
objective of the present study is to characterize the uptake of aqu/C60 and aqu/C70 fullerenes into
T. platyurus and measure changes in agglomeration that may occur during ingestion and
excretion of these materials. The implications these processes may have on the transport and fate
of these materials are also discussed.
MATERIALS AND METHODS
Particle characterization
Preparation of fullerene suspensions. The C60 (purity 99.9%) and C70 (purity 99.0%)
fullerenes were purchased from MER Corp. (Tucson, AZ, USA). Rapidtoxkits™ ( the source for
T. platyurus cysts) were purchased from Strategic Diagnostics (Newark, Delaware, USA). All
other chemicals were of reagent grade and purchased from Sigma-Aldrich (St. Louis, MO,
USA). For fullerene suspensions used for T. platyurus exposure, 100 mg of C60 or C70 were
added to 400 ml double de-ionized water (DDI) (>18 M Ohms-cm). The suspensions were stirred
using a magnetic stir plate for approximately 100 d to form nanometer-sized fullerene aggregates
that have been shown to be stable with respect to size and charge [7,13]. For particle size
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characterization prior to the exposure experiments, the suspensions were allowed to settle by
gravity for 1 h and the suspensions were sampled at 2-cm below the surface.
Mass quantitation. The aqu/C60 and aqu/C70 were quantified using a combination of
previously published methods [13,25,26]. For mass quantification, aqu/C60 and aqu/C70 were
dissolved into a toluene/methanol mixture (8:2 by volume) then quantified directly using a 13
C60
internal standard with liquid chromatography-atmospheric pressure photoionization mass
spectrometry. The fullerenes were separated with a 95% Toluene : 5% Methanol mobile phase
on a Accela liquid chromatograph (Thermo Fisher, West Palm Beach, FL) with a Cosmosil
column (4 x 150 mm) connected inline with a guard column (3 x 4 mm), both from Phenomenex
(Torrance, CA). Detection was provided by a Quantum Ultra Mass Spectrometer operating in
negative mode with a 10 eV krypton atmospheric pressure photoioization lamp. The mass
spectrometer was set to select molecular ions for C60 (m/z 720) C70 (m/z 840) and 20-30% 13
C
enriched C60 (m/z 734) as no fragment ions were detected.
Characterization of fullerenes by dynamic light scattering (DLS). Changes in fullerene
aggregate size were examined by DLS (ZetaSizer Nano ZS) [13]. Six measurements (12 runs
per measurement) were acquired from each sample. Instrument performance was verified using
National Institute of Standards and Technology (NIST)-traceable polystyrene microsphere
standards. The autocorrelation function was analyzed by the cumulant method to obtain the
moments of the aqueous fullerene aggregate size distribution. The fluctuations of scattered light
from particles can be mathematically correlated to the diffusion coefficient. The intensity
average (Z-average) hydrodynamic diameter was calculated from measured diffusivities using
the Stokes-Einstein equation. For surface charge characterization, zeta ( ) potentials of the
suspensions were monitored using a ZetaSizer Nano ZS instrument (Malvern Instruments,
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Worcestershire, UK). This instrument uses phase analysis light scattering (PALS) to measure
the electrophoretic mobility of charged particles. The Smoluchowski equation was used to
calculate -potential from electrophoretic mobility. For electron microscopic analysis, the
fullerenes were analyzed from aqueous suspensions. Fullerene suspensions (120 mg/L) were
placed drop-deposited onto a 200 mesh Fomvar-coated copper grids. Images were acquired at
300 kV using a Tecnai transmission electron microscope (TEM) (FEI, Hillboro, OR, USA) with
a 2k Gatan camera [27]. The imaged aggregates were also analyzed by energy dispersive X-ray
spectroscopy for elemental composition using an EDEX detector. Thin section images were
acquired using an FEI/Philips CM-10 TEM at 300 kV.
TEM and chemical analysis of the gut contents. For thin section TEM analysis, after
exposure to aqu/C60, the T. platyurus were fixed in 2% glutaraldehyde then exchanged into 1%
OsO4 in 0.1 M cacodylate buffer pH 7.4. The organisms were then dehydrated by stepwise
ethanol exchange (10, 30, 50, 90, 100% ethanol) then further exchanged from propylene oxide
into bisphenol A-epichlorhydrin (EPON) 828 resin. The resin was then polymerized for 48 hr at
60º C prior to thin sectioning and TEM analysis.
Chemical analysis of gut contents. After lyophilization, 50 of the exposed organisms
were placed in the extraction vessel. A 1-ml volume of toluene was then added to the extraction
vessel, the vessel was vortexed several seconds and then placed horizontally on an orbital shaker
at 200 rpm for 30 min. After removal from the shaker, the sample phases were allowed to
separate for 15 min before approximately 800-µlwas removed from the toluene supernatant layer
for analysis by HPLC-UV. The samples were then placed in a freezer at 25 C for 2 h to freeze
the aqueous layer and thus facilitate decantation of the remaining toluene layer. Following
decantation of the toluene layer, the aqueous layer was allowed to melt and a second 1-ml
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volume of toluene was added to the extraction vessel. The aqueous phase was then re-extracted
as described above. A total of three sequential extractions were performed using this technique
and the fullerene mass extracted in each sequential extraction was determined by HPLC [13].
Using this technique, the recovery and 95% confidence interval for the T. Platyurus extraction
was 57.3 ± 12.1%.
Thamnocephalus platyurus assays.
The relative rate of ingestion of colored polystyrene microspheres (5 µm) in T. platyurus
has been used as the basis for a rapid toxicity screening assay [18]. Due to this uptake behavior
as well as the visibility of these microspheres in the gut cavity of T. platyurus, these
microspheres were used as a comparative control for exposure of this organism to aqu/C60
fullerenes (which can be visibly differentiated by optical microscopy).
Exposure conditions. Thamnocephalus platyurus were hatched from cysts following the
Rapidtox™ protocol. The cysts were hydrated with moderately hard reconstituted water (CaSO4,
0.06 g/L; MgSO4, 0.06 g/L; NaHCO3, 0.096 g/L; KCl, 0.004 g/L) and incubated at 25º C under
4000 lux illumination for 30 to 45 h. After hatching, the larval suspensions were transferred into
double-deionized (DDI) water and mixed (1:1) with gently vortexed fullerene suspensions and
incubated for 1 hr prior to addition of the colored indicator microspheres (5 µm red polystyrene)
into the fullerene-organism mixtures. The organisms were exposed to the microspheres for 15
min prior to fixation with Lugol solution (final concentration, 1% KI, 0.5% I2) and final
observation.
Visual quantitation of fullerene uptake. For the fullerene visual uptake quantitation
experiment, T. platyurus were exposed to fullerenes at concentrations of 1.9, 3.7, 7.5, 30, and 60
mg/L for one h prior to fixation with Lugol solution and quantitative observation. Organisms
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with greater than 20% of their gut occupied by dark matter were counted and the percent of total
(number) of organisms accumulating fullerenes determined. For the algae accumulation
experiment, algae (Selenastrum capriconutum at 100,000 cells/ml) were added after 1 h exposure
to the fullerenes (60 mg/l). Algae stocks were maintained as previously described [28]. Fifteen
min after addition of algae, the organisms were fixed with Lugol Solution for observation. For all
visible microscopy observations, organisms were observed using a stereo microscope at 40 x
magnification and images recorded using Motic Image Plus 2.0 software.
For the experiments involving imaging of gut content by thin section TEM, T. platyurus
were exposed to aqu/C60 at 69 mg/L for 1 h prior to separation, preservation and analysis. For
chemical analysis of gut contents, the organisms were washed 3 times with DDI water by
centrifugation (3000xg, 3 min). After centrifugation, the supernatant was removed each time and
the pelleted organisms re-suspended in DDI water using a pasture pipette. For the sample blank,
aqu/C60 was added after the control organisms were fixed to account for the non-ingested
particles that were adhered to the surface of the organisms or carried over in the washes. The
pelleted organisms were then placed onto a pre-weighed weigh boat and the excess liquid
removed using the corner of a small paper towel. The organisms were then separated into groups,
frozen, lyophilized.
RESULTS AND DISCUSSION
Aqu/C60 and Aqu/C70 fullerene preparations.
The Z-average diameters for the aqu/C60 and aqu/C70 aggregates prior to feeding showed
size distribution averages of 517 ± 21 nm and 656 ± 39 nm (mean ± 95% confidence limits),
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respectively. Similar to the results by DLS, analysis of the vortexed aqu/C60 suspensions by TEM
showed aggregates that ranged from about 200 to 500 nm in length (Fig. 1A, B). Zeta ( )
potentials for the aqu/C60 and aqu/C70 suspensions were -27.5 ± 0.7 mV and -47.5 ± 5.4 mV,
respectively. Generally, colloidal suspensions of particles with absolute zeta potentials greater
than 30 mV will be stable and will not further aggregate or precipitate as long as background
solution parameters remain constant [29]. In the present study, the background solution ionic
environment did not change as all experiments were conducted in DDI. While the aqu/C70 zeta
potential was significantly more negative than -30 mV, the aqu/C60 zeta potential was
significantly higher, approaching the rule of thumb (-30mV) value.
Exposure of T. platyurus to fullerene suspensions.
The experimental design primarily involved exposure of T. platyurus to aqueous
suspensions of fullerenes (aqu/C60 and aqu/C70) and combinations of fullerenes with
microspheres or algae. In the latter case, the organisms were exposed to fullerenes prior to the
microspheres or algae to determine whether or not the accumulation of the smaller fullerene
agglomerates could exclude the larger indicator beads or algae (typical food supply). The relative
size distribution of the fullerene agglomerates were monitored prior to, during ingestion and after
excretion.
Because T. platyurus are semi-transparent organisms, exposure and accumulation of the
red indicator beads could be visualized as a red opaque outline of the gut cavity (Fig. 2A). When
T. platyurus were exposed to aqu/C60 or aqu/C70 fullerenes prior to exposure to the indicator
beads, they accumulated the fullerenes as dark masses in their digestive tract (Fig. 2B). The
fullerenes appeared to compete with the indicator microspheres with respect to accumulation in
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the gut. Although some of the organisms’ digestive tracks contained only red microspheres or
fullerenes, many of them showed segments of each (Fig. 2B). After exposure of T. platyurus to
aqu/C60 or aqu/C70 with no addition of indicator microspheres, the dark masses visible in the
digestive tract also appeared to be segmented (with intervening light areas) indicative of the
formation of excretion pellets (Fig. 2C).
The observed percentage of organisms accumulating the dark masses (in 20% of the gut
tract length) was concentration dependent for both aqu/C60 and aqu/C70 fullerenes (Fig. 3). After
a 1 h exposure, about 50% of the organisms accumulated observable fullerene masses in their gut
at an initial aqu/C60 concentration of 7.5 mg/L. At an initial concentration of 30 mg/L, about
80% of the organisms had accumulated these dark masses (Fig. 3). Visual observation did not
reveal whether or not the accumulated fullerenes were coating the inside of the digestive track or
filling the lumen. However, these observations may suggest that the fullerenes coalesced in
specific areas and forming the observed banding pattern.
When the T. platyurus were exposed to fullerenes followed by unicellular algae, the
ingested algae could also be observed as green bands between the dark masses of aqu/C60 or
aqu/C70 fullerenes, similar to the results observed with the red indicator spheres. Contrast
between the green and black bands could not be observed well in the photographs and these data
are not shown.
Quantitative Assessment of Uptake
To further confirm the uptake of fullerenes, the accumulation of aqu/C60 in T. platyurus
was quantified by solvent extraction and analysis by HPLC-MS. The fullerene agglomerate
burden was measured for two initial fullerene concentrations (3 mg/L and 6 mg/L) over a 60 min
time course (Fig 4). The uptake of aqu/C60 between 0 and 10 min was rapid for both initial
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exposure concentrations. However, after the initial uptake period, the C60 concentration per unit
mass of organism did not change significantly over the remaining 60 min measurement period.
One hour exposures (at 3mg/L and 6 mg/L) resulted in aqu/C60 burdens of 2.7 ± 0.4
µg/mg and 6.8 ± 1.5 µg/mg wet weight, respectively. The amount of aqu/C60 that bound to the
organisms after they had been fixed with Lugol solution was about 15% of the total uptake. A
comparison of the amount of aqu/C60 per organism for the two exposure levels after 60 min
differed by a factor of 2.5. This level of increase for the quantitative extraction and measurement
of accumulated aqu/C60 averaged for a group of 50 organisms was similar to results shown in ` 3
where an increase in exposure level from 3.7 mg/L to 7.5 mg/L resulted in a 2.7 times increase in
the percentage of organisms observed to accumulate dark masses (in 20%) of gut length. This
result suggests that the fullerene uptake behavior for individual organisms was similar. The
herein reported uptake values for aqu/C60 into T. platyurus were similar to the value of 4.5
µg/mg wet weight reported for the uptake of C60 into D. magna after a 24 h exposure at a
fullerene concentration of 2 mg/l [20].
Structure and composition of dark masses in the gut.
Electron micrographs of the transverse cross-sections of T. platyurus exposed to aqu/C60 and
polystyrene beads show the microvilli-lined lumen of the mid-gut containing aqu/C60
agglomerates (Fig. 5 A, B). Compared to the aqu/C60 agglomerates used for the exposure
experiments (200-500 nm by TEM), these aqu/C60 agglomerates appeared to be significantly
larger (5 to 10 µm) and composite in structure. These larger clusters observed in the gut were
presumably assembled from smaller agglomerates that were ingested by the organisms. The
micrograph in Figure 5B shows one of these agglomerates (a) next to one of the polystyrene
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microspheres (b). Part of this agglomerate appears to have coalesced into a larger structure. The
dense fullerene agglomerates did not appear to cross the gut lining in any of the numerous thin
section electron micrographs that we analyzed (not shown).
Exposure of T. platyurus to aqu/C60 for several hours resulted in the formation of pellets
which were excreted and settled to the bottom of the incubation wells. Analysis of this material
by light microscopy showed a population of agglomerates ranging in size from about 10 µm to
70 µm (Fig. 6A). Observation of the agglomerates from the excreted material by TEM also
showed the presence of micrometer sized agglomerates of aqu/C60 (Fig. 6B). These larger
aqu/C60 agglomerates from the excretion pellets appeared to be composed of smaller
agglomerates in the 200 to 500 nm size range that had coalesced into larger structures. The
excreted C60 agglomerates were stored in DDI water at room temperature and did not appear to
disperse even after 6 months. When the indicator microspheres were added in the absence of
fullerenes, they accumulated in the gut as evidenced by the red color in the gut track (see Fig.
2A). After exposure of the indicator spheres for several hours, there did not appear to be any
aggregates of these particles. Although unmodified polystyrene microspheres (5 µm) will settle
from suspension after several days, they tend to remain suspended and monodispersed for several
hours. Differences in the post-excretion dispersion behavior of aqu/C60 and polystyrene
microspheres may be due, in part, to their surface charge. It has been suggested that, in general,
particles with an absolute zeta potential of greater than 30 mV will form stable suspensions due
to their strong electrostatic repulsion [7]. Unmodified polystyrene microspheres have a zeta
potential of -70.8 mV [30], whereas the herein reported zeta potential for the aqu/C60 is -27.5
mV. Although the aqu/C60 used as the source material for the current exposure measurements
remained in suspension (without stirring) for six months after the experiment, it may be the case
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that the forced particle proximity resulting from the confined space of the gut resulted in the
observed coalescing of the aqu/C60 particles which showed a zeta potential less than typically
indicated for stable suspensions.
Table 1 shows size distributions visible microscopy and electron microscopy as well as
hydrodynamic size distributions by DLS before and after ingestion by T. platyurus. The relative
differences in size between the DLS and TEM measurements for the aqu/C60 and aqu/C70
suspensions prior to ingestion are likely to be due to inherent differences in these techniques.
More specifically, DLS measures a signal intensity-weighted hydrodynamic diameter
distribution, where as TEM measures a number-weighted population size distribution. Because
the larger particles tend to dominate the population size distribution measured by DLS, we
believe that the measurements by these two techniques are consistent. Negative potentials are
reported for the aqu/C60 and aqu/C70 and similar values have been previously observed for these
aqueous fullerene suspensions [7].
Potential ecological implications
Although aquatic crustacean filter-feeders can feed on a range of biological particulate
materials, they typically ingest unicellular algae in the micrometer size range with a preferred
size for food particles of >1 µm. In studies using micro spheres, D. cucullae have been shown to
accumulate polystyrene beads in the 5 to 35 µm size range [31]. Fairy shrimp (anostracans) have
been grown in the laboratory on diets ranging from bacteria to baker’s yeast (1 to 10 µm) [32].
For each of these examples, the sizes of food sources are typically greater than 1 µm. The
observation that zooplankton such as D. magna can accumulate relatively small polystyrene
spheres (50 nm) which were expected to pass through the filter combs has lead researchers to
propose mechanisms of accumulation of very fine particles based on particle adhesion as well as
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physical filtration [33]. We observed that in addition to the 5 µm indicator spheres, T. platyurus
accumulated the smaller C60 agglomerates suggesting that the fullerenes may also have, to some
extent, adhered to the organism’s filtering apparatus through particle adhesion mechanisms.
The basis of the T. platyurus feeding inhibition assay involves the organism’s sensing the
presence of toxins and subsequently slowing or stopping its feeding behavior [34]. Nevertheless,
T. platyurus appeared to ingest aqu/C60 to a similar extent as the indicator microspheres (see Fig
2B) suggesting that the fullerene agglomerates were mistaken as food [18]. Our observations are
consistent with reports that D. magna accumulates C60 agglomerates which are not acutely toxic
even at relatively high concentrations (5 mg/L) [17]. It has further been suggested, however, that
even though ingestion of nanoparticles may not be acutely toxic to aquatic filter feeders, the
depletion of energy reserves (due to nanoparticle competition) during periods of low food
supply, may be detrimental to these populations [35].
Filter-feeding organisms such as T. platyurus and D. magna play important roles in the
ecosystem due to their rapid filtration rate and have been shown to have significant impacts on
water turbidity and algal composition [36]. Filella et al. recently illustrated the potential for
Daphnia sp. to impact the size distribution of inorganic colloids, in fresh water lakes [37]. The
tendency for D. magna to filter and excrete carbon-based nanomaterials in an agglomerated state,
potentially change the size distribution and fate of these nanoparticles in the environment has
been suggested by Tervonen et al. [20]. We suggest that anostracans such as T. platyurus may
also show this potential to alter the size distribution of fullerene agglomerates.
Crustacean filter feeders have been shown to accumulate smaller fullerene agglomerates
from relatively low concentration suspensions. For example, D. magna have been shown to
ingest and accumulate agglomerates of C60 at sizes below 0.45 µm [17]. D. magna have also
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been shown to ingest and accumulate carbon nanotubes with an average length of < 200 nm [38].
These authors also reported that the nanotubes aggregated in the gut and were not efficiently
eliminated without continuous exposure to dispersed nanotubes or when additional algae were
available in the media. In another experiment reported by Roberts et al. [21], lipid-coated
nanotubes were ingested by D. magna and after accumulation in the gut, the nanotubes were
reported to have been excreted by the organisms as aggregated material that was stripped of the
original lipid coating that allowed the nanotubes to remain dispersed in suspension [38].
CONCLUSION
When T. platyurus are exposed to polystyrene indicator spheres, the spheres are initially
concentrated in the gut then disperse after passing through the organism. However, the
aqu/fullerenes form larger agglomerates during the process of ingestion and excretion. The
aqu/C60 agglomerates used for exposure were in the 200 nm to 500 nm size range. These
agglomerates remained in suspension prior to ingestion by T. platyurus. In the mid-gut, these
agglomerates appeared to coalesce into larger agglomerates that were in the 5 to 10 µm size
range. Upon excretion, the pellets appeared (by light microscopy) to form agglomerates in the 10
to 70 µm size range. These agglomerates were large enough to settle to the bottom of the flask
and remained stable for up to six months without stirring.
The present study illustrates that anostacans such as T. platyurus show the potential to
influence aqu/C60 fullerene agglomerate size. As a result, these organisms may facilitate the
movement of fullerene agglomerates from the water column and into the sediment potentially
shifting exposure to benthic organisms in the sediment.
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Acknowledgement - We thank Dr. Marisol Sepulveda and Debby Sherman (Purdue University).
Manomita Patra gratefully acknowledges a National Research Council Research Associateship
Award at the National Exposure Research Laboratory, Human Exposure and Atmospheric
Sciences Division, Las Vegas, Nevada.
The United States Environmental Protection Agency (EPA), through its Office of
Research and Development (ORD), has funded and managed the research described here. It has
been subjected to the Agency’s administrative review and has been approved for publication.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Figure Legends
Figure 1. Transmission electron micrographs of aqu/C60 fullerene agglomerates prior to
ingestion by T. platyurus. Selected images were representative of observations. (A) Larger
agglomerate; (B) Smaller agglomerates, (bars = 200 nm).
Figure 2. Accumulation of aqu/C70 fullerene (observed as dark gray) and red (observed as light
gray) polystyrene indicator beads by T. platyurus. (A) Control organisms, 30 to 45 h nauplii
exposed to indicator beads only (15 min); (B) Nauplii exposed to aqu/C70 (60 mg/L) for 60 min
prior to addition of indicator beads; (C) Nauplii exposed to only aqu/C70, 60 mg/ml, 60 min.
Figure 3. Visual observation of uptake of aqu/C60 and aqu/C70 fullerenes by T. platyurus. Bars
represent percentage of organisms showing greater than 20% gut length occupied by
accumulated fullerenes. The organisms (20 to 40) were scored and averaged in 2 trials. Exposure
conditions were the same as for Fig. 2C.
Figure 4. Quantitative assessment of uptake of aqu/C60. Initial concentration of aqu/C60 were 3
mg/L ( ) and 6 mg/L ( ). Other exposure conditions were the same as for Fig. 2C. Extraction
and quantitation described in Methods.
Figure 5. Transmission electron micrographs of transverse thin cross-sections of T. platyurus
after exposure to aqu/C60 (60 min., 69 mg/ml). (A) Arrows indicate aqu/C60 agglomerates (bar =
2 µm); (B) (a) aqu/C60 agglomerate, (b) polystyrene microsphere (bar =1 µm).
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Figure 6: Light microscopy and TEM images from T. platyurus excreted material after exposure
to aqu/C60. (A) Light microscopy image of excretion pellets after 30 d in water (white circle = 70
µm). (B) TEM image of one of the large agglomerates from the same sample as (A) (bar =
0.5µm).
Figure 1(A).
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Figure 1(B).
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Figure 2(A).
Figure 2(B).
Figure 2(C).
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Figure 3.
0 0.9 1.9 3.7 7.5 15 30 60
0
10
20
30
40
50
60
70
80
% O
rgan
ism
s A
ccum
ula
tin
g D
ark
Masses
Fullerene Concentration (mg/L)
C-60
C-70
Figure 4.
0 10 20 30 40 50 60
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
3 mg/L
6 mg/L
C6
0 M
ass (
g/m
g)
Time (min)
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Figure 5(A).
Figure 5(B).
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Figure 6(A).
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Figure 6(B).
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Table 1. Changes in fullerenes after ingestion, accumulation and depuration by T. platyurus.
Sample Agglomerate size
by Microscopy
Z average size
(nm) (mean ± 95%
confidence limit)
Polydispersivity
index (PDI)
(mean ± 95%
confidence limit)
Z potential (mV)
(mean ± 95%
confidence limit)
Aqu/C60 prior to feeding 200-500 nm 517 ± 20 0.38 ± 0.01 -27.5 ± 0.7
Aqu/C60 mid-gut thin
section TEM
5-10 µm ND ND ND
Aqu/C60 after excretion 10-70 µm* ND ND ND
C70 prior to feeding 200-500 nm 656 ± 39 0.58 ± 0.04 -47.5 ± 5.4
*200 – 500 nm sub-structure
ND = not determined
