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Aquatic Toxicology
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a r t i c l e i n f o a b s t r a c t
Article history: Gold nanoparticles (nAu) have recently been studied and developed within the biological and photother-
Received 1 October 2015 mal therapeutic contexts. The major clinical interest is within the application of novel drug delivery
Received in revised form systems. Environmental exposure to nanoparticles can occur in different stages of the lifecycle of the
20 November 2015
product; from their synthesis, applications, product weathering and their disposal. Freshwater Daph-
Accepted 22 November 2015
nids, specically Daphnia magna, have been used since the 1960s as a standard species in acute and
Available online 25 November 2015
chronic aquatic toxicity testing. Visualization of the interactions and uptake of nAu by D. magna was
related to reproduction and molting patterns. Exposure to nAu was done using a chronic reproduction
Keywords:
Body burden
test performed for 14 days at six concentrations (0.5 mg/L, 2 mg/L, 5 mg/L, 10 mg/L, 15 mg/L and 20 mg/L).
CytoViva Microscopy was used to determine whether there was any uptake or interaction of nAu with daphnia.
Adhesion However the concentration of nAu in the media and the charge of particles played a role in the uptake
Molts and surface adsorption. As exposure concentrations of nAu increased it appeared that the nAu aggre-
gated onto the surface and in the gut of the organisms in higher concentrations. There was no evidence
of nAu internalization into the body cavity of the daphnia. Aquatic exposure to nAu resulted in increased
adhesion of the particles to the carapace of daphnia, ingestion and uptake into the gut of daphnia and
had no signicant effect on reproduction and molting patterns.
2015 Elsevier B.V. All rights reserved.
1. Introduction and geometry not only play a role in cellular uptake but also play
a role in the bioavailability and biokinetic fate within an organism
Gold nanoparticles (nAu) in aquatic media have unique proper- (Arnida et al., 2011; Hirn et al., 2011).
ties, which include size distribution, surface charge and the ability Freshwater daphnids, specically Daphnia magna and Daphnia
to aggregate, and therefore the uptake and tissue distribution will pulex, have been used since the 1960s as standard species in acute
also be unique to these properties (Goodman et al., 2004; Oh and and chronic toxicity testing (Persoone et al., 2009). Daphnids are
Park, 2014). For nanoparticles to cause toxicity, it is assumed that internationally recognized as a test organism for small scale toxi-
they are taken up and distributed within an organism, therefore a city testing where different end points may assessed (Jonczyk and
better understanding of how they behave in an in vivo system is nec- Gilron 2005; Persoone et al., 2009). As lter feeders, D. magna ingest
essary (Wepener et al., 2011). Since nAu are specically designed any material ranging between 240 and 640 nm within a water col-
for the therapeutic and drug delivery elds it is essential that they umn and are able to lter up to 400 mL of water per day (Wray
will be able to move across tissue barriers as well as cell membranes and Klaine, 2015). The ltering activity will cause water currents
(Boisselier and Astruc, 2009; Yah, 2013). In the case of free metal or resuspend sediments through their thoracic appendages making
ions they are taken up across the gill, intestine or body surface (Van settled particles available for uptake again (Gillis et al., 2006). Most
Dyk et al., 2009) but with nanoparticles, agglomerates are formed studies indicate that the ingestion of nanoparticles by D. magna
in the medium based on forces that exist between water molecules seem to remain in external compartments such as the gut or remain
and charges on the particle surface. Variations in surface properties on the external carapace surface (Garca-Cambero et al., 2013;
Wray and Klaine, 2015). Khan et al. (2014) demonstrated that 70%
of the nAu intestinal burden in D. magna was cleared after a period
Corresponding author. of one hour and no internalization was observed over the surface
E-mail addresses: tlbothanano@gmail.com, 26964546@nwu.ac.za (T.L. Botha). carapace and intestinal barrier. Lee and Ranville (2012) proposed
http://dx.doi.org/10.1016/j.aquatox.2015.11.022
0166-445X/ 2015 Elsevier B.V. All rights reserved.
T.L. Botha et al. / Aquatic Toxicology 170 (2016) 104111 105
that molting is not only responsible for development and repro- Behavioral changes were noted and only adults were transferred
duction but could also aid in controlling internal concentrations of to new beakers at each media change.
certain metals.
The aims of this study were (1) to visualize the interaction and 2.4. CytoViva dark eld hyperspectral imaging
uptake of nAu by D. magna and (2) to relate reproduction and molt-
ing patterns to nAu exposure. Molts were collected and processed throughout the duration
of the test. Upon the conclusion of the test adult D. magna and a
2. Materials and methods drop of exposure media was also collected for further analyses. One
drop of exposure media was placed onto a microscope slide allowed
2.1. Bioassay protocol to dry for ten minutes, covered with a cover slip and sealed with
Entellan . Exposure media slides were used to set up a baseline to
A stock culture of D. magna was maintained in culture medium in refer to in slides prepared of exposed organisms. Cryopreserve gel
2.5 L tanks and media was replaced three times per week. Cultures (Tissue-Tek OCTTM Compound) was used to mount adult daphnia
were kept at 2022 C and were fed 1.5 105 cells/mL unicellular and molts onto a microscope slide and stored at 80 C until pro-
algae (Pseudokirchneriella subcapitata) daily as well as a supple- cessing. Slides were carefully covered with a cover slip to prevent
mented diet of daphnia food prepared by grinding and settling out the gut content from being pushed out of the organism prior to
trout pellets, yeast and Spirulina pellets before using 5 mL of the imaging. Visualization was done using a CytoViva 150 Unit inte-
liquid phase for feeding. grated onto an Olympus BX43 microscope. Images of the exposure
media, molts and D. magna were captured using the Dagexcel X16
2.2. Exposure and characterization camera and DAGE Exponent software at 60X magnication.
Gold nanoparticle stock solutions were prepared and supplied 2.5. Statistical analyses
by MINTEK; a minerals processing and metallurgical engineer-
ing company in South Africa (14 2 nm nAu with product The daily counts of D. magna molts and juveniles released at each
code TMU14G, batch numbers 20130304FKP49b; 20130308FKP52; concentration over a period of fourteen days were noted. The data
20140905BM001). Characterization, which included hydrody- were tested for normality using DAgostino and Pearson omnibus
namic size distribution and, zeta potential measured by elec- normality test. For normal data an Oneway ANOVA was conducted
trophoretic mobility on the Zetasizer instrument (Dynamic and the Tukeys Multiple Comparison Test was used to determine
light scattering; Malvern Zetasizer Nano series, NanoZS), parti- signicance between groups. In the absence of data normality the
cle functionalization (Fourier Transform Infrared Spectrometery; KruskalWallis Test using Dunns Multiple Comparison Test was
PerkinElmer FTIR-spectrometer Spectrum 100), agglomeration pat- performed to determine signicance. Signicance was regarded as
terns (transmission electron microscopy; FEI Tecnai G2) and p < 0.05.
measured concentrations (inductively coupled plasma atom emis-
sion spectroscopy) is discussed in detail in Botha et al. (2015a,b). 3. Results
The following concentration range was selected and made up using
OECD 211 (Oecd, 1984) daphnia media: 0.5 mg/L, 2 mg/L, 5 mg/L, 3.1. Characterization of nAu
10 mg/L, 15 mg/L and 20 mg/L.
The characterization of Daphnia medium containing nAu
2.3. Daphnia magna reproduction test showed that as concentration increased so did the particle aggre-
gation and agglomeration from 14 nm in the initial sample to up
The D. magna reproduction test was performed using the OECD to 3043 nm in the highest concentration after 48 h (Table 1). The
211 guideline (1984) across the above mentioned concentrations zeta potential was negative across all exposure concentrations.
and medium to determine whether the nAu particle interaction The measured concentrations were generally 10% lower than the
had an effect on reproduction rates. The test was run for the mini- nominal concentration, except at 2 mg/L where a 50% lost in con-
mum period of 14 days (Oecd, 1984, 1421 days). The medium and centration was observed possibly due to the non-uniform size
concentrations were renewed three times per week at 48 h time distribution of particles within the percentage of sample analyzed
intervals and each beaker was supplied with a 1.5 105 cells/mL of for ICP-MS. The particle agglomeration patterns were discussed in
P. subcapitata with each media change. The temperature was main- Botha et al. (2015a,b).
tained between 18 and 22 C with a 16 h light: 8 h dark interval
and oxygen saturation was maintained above 60% for the duration 3.2. External adsorption of nAu to Daphnia magna
of the test. Ten adult individuals per concentration were pooled
together in 500 mL glass beakers and the number of offspring The accumulation of nAu in the gut occurred within hours of
produced as well as molts was counted daily for each exposure. exposure during both the reproduction and acute immobilization
Table 1
Characterization of nAu in Daphnia magna medium adapted and summarized from Botha et al., 2015a.
0h 48 h 0h 48 h 0h 96 h
0.5 mg/L 61 11.6 16.9 1.85 0.59 9.52 2.50 0.554 0.46
2 mg/L 76.4 32.1 120 52.7 8.74 2.91 2.03 1.1 1.07
5 mg/L 181 29.3 253 142 3.07 1.58 7.17 4.3 4.64 4.72
15 mg/L 148 12.9 221 8 8.13 9.72 16.1 12.7 12.3
25 mg/L 800 16.8 1061 25.9 17.2 22.9 8.5 0.92 20.6 19.8
Fig. 1. Adult Daphnia magna viewed under a light microscope (4 magnication) after a 48 h exposure to varying concentrations of nAu (a) 0.5 mg/L, (b) 2 mg/L, (c) 5 mg/L,
(d) 10 mg/L, (e) 15 mg/L and (f) 50 mg/L.
test (Botha et al., 2015a). It appeared as though the accumulation face, which resembled the purple colors observed in the exposure
in the gut increased as the exposure concentration increased, as medium after the citrate coated nAu in the stock solution function-
shown in Fig. 1 for 48 h exposure. At concentrations of 15 mg/L and alized with other anions in the exposure medium. At concentrations
above the organisms displayed a purple tinge to their outer sur- of 10 mg/L and above the purple outer coating observed at 24 h
T.L. Botha et al. / Aquatic Toxicology 170 (2016) 104111 107
Fig. 2. Daphnia magna exposed to nAu particles in aquatic medium viewed under CytoViva dark eld hyperspectral imaging showing (a) aquatic medium, (b) body surface,
(c and d) appendages, (e) mandibles, and (f) gut.
was no longer present in some organisms after 48 h. It was there- 3.3. CytoViva dark eld hyperspectral imaging of Daphnia
fore assumed that the nAu coatings were shed through molting and magna
consequently the molting patterns were assessed in combination
with the reproduction test. CytoViva dark eld hyperspectral imaging was used to further
assess the internal particle distribution in the daphnids. A medium
108 T.L. Botha et al. / Aquatic Toxicology 170 (2016) 104111
Fig. 3. Daphnia magna exposed to nAu particles in aquatic medium viewed under CytoViva dark eld hyperspectral imaging showing (a) terminal claw, (b) surface spines
and (cf) molts.
control on a slide was used to setup a baseline image. The presence studying the distribution and effect, the control benchmark sample
of nAu particles was conrmed to be bright red to maroon-colored was compared to exposed samples under dark eld hyperspectral
clusters oating at different size agglomerations (Fig. 2a). When imaging. There was an accumulation of nAu on the carapace of
T.L. Botha et al. / Aquatic Toxicology 170 (2016) 104111 109
Fig. 4. The mean SEM (standard error mean) number of (a) juveniles and (b) molts released per day. Each exposure concentration represents a pooled sample of ten D.
magna adults.
D. magna (Fig. 2b) and on the maxillae used for locomotion and D. magna to nAu agglomerations that had settled out prior to expo-
feeding. As the result of movement of the appendages the nAu sure in the container for 48 h and found that they were still able
become trapped on and between the lter setae (Fig. 2c and d). to ingest and maintain them in the gut suggesting that aggregation
A current is formed by the maxillae in the water and food moves reduces but does not eliminate bioavailability (Khan et al., 2012).
into their mouth opening across a pair of toothed or ridged grind- Therefore aggregation, size and shape did not seem to play a marked
ing mandibles. Some of nAu agglomerations were ground onto the role in particle uptake (Wray and Klaine, 2015).
mandibles before passing into the gut (Fig. 2e). Closer magnica- Charges present on the nAu surface play a role in tissue distri-
tion of the gut (Fig. 2f) indicated that the nAu particles stay within bution and effects (Fent et al., 2009; Gu et al., 2009). The nAu in this
the gut lining and pack onto one another as more food is ltered study had negative charges across the lower exposure concentra-
in. There was no evidence that nAu penetrated the gut wall. The tions except for the higher exposure concentrations (1525 mg/L)
large post-abdomen is found at the posterior end of the body and it where there seemed to be a mixture of positive and negative
contains two long abdominal setae, two terminal claws and a series charges present at 0 h. This however changed to negative charges
of lateral teeth (Fig. 3a). As nAu agglomerates are pushed out of the after 48 h across the full range of exposures (Botha et al., 2015a,b).
gut, while more were taken up it was evident that the agglomerates Khan et al. (2012) and Wray and Klaine (2015) showed that sur-
started accumulating between the lateral spines on the terminal face charge played a major role in distribution of nAu inside the
claw (Fig. 3a and b). The terminal claw is mostly used for cleaning gut. Negative nAu surface potentials were associated with or in
debris from the thoracic legs but may aid in locomotion. It would close proximity to the peritrophic membrane or the microvilli sit-
appear that particles accumulated between the lateral spines on uated beneath it, which seemed to inhibit their elimination. The
the terminal claw during this cleaning process. The locomotion of particleparticle interactions between nAu will also play in a role
D. magna resembled erratic up and down movement as daphnids in the ability for higher concentrations to have higher nAu body
swim up and oat down toward the bottom. The nAu agglomer- burdens (Wray and Klaine, 2015). As the daphnia feed and swim
ates tended to accumulate in higher densities around the posterior through an exposure medium they would cause particles to collide
portion (Fig. 3b). Those daphnid that appeared to have molted dis- and further attach onto one another and onto the body surface of
played a change in swimming patterns from slow, almost heavy the daphnids increasing the body burden over time.
movements to normal locomotion. When molts were observed In this study the nAu present in the gut, and adsorption on the
under hyperspectral imaging it was evident that the nAu agglom- carapace and appendages was observed to be proportional to the
erates were present in high densities across the carapace and were nAu exposure concentration in the daphnia media (Fig. 2). Zhu et al.
lost during molts, which included the thoracic legs (Fig. 3cf). (2009) suggested that this could lead to mechanical disruptions of
the feeding appendages or penetration of the gut wall. Mechani-
3.4. Daphnia reproduction test cal disruptions in the gut wall could affect the uptake of nutrients
and gaseous exchange across the external surface thereby causing
The number of D. magna juveniles released at 2 mg/L appeared cell death (Zhu et al., 2009). Gophen and Gold (1981) suggested
to increase when compared to the other concentrations, however that daphnids could preserve food in the gut during periods of
statistical analysis revealed that there was no signicant increase starvation, however since the chronic toxicity protocol applied in
over long term exposure. There was also no signicant increase this study (i.e., OECD 211) requires feeding of the D. magna, it is
in the number of molts at the different concentrations, all varying unclear whether this would hold true for the nAu exposures as well.
between two and four molts per day (Fig. 4). Therefore, nAu binding Manseld et al. (2015) showed that titanium dioxide nanoparticles
to organism surfaces did not lead to sub lethal effects. also accumulated in the gut of daphnia until visible to the naked
eye. These studies and the present study indicate that a number of
different types of nanoparticles are able to accumulate in the gut
4. Discussion but were not able to be transported across the gut wall and enter
the organisms tissues.
The exposure concentrations used in this study were far below Lewinski et al. (2008) found that nAu that accumulated in the
the LC50 values for nAu in D. magna calculated by Li et al. (2010), gut remained there even after daphnia were placed in clean water
which ranged between 65 and 75 mg/L. D. magna are lter feed- for 48 h, but when daphnia were fed on algae the nAu in the gut
ers that are able to lter particles of up to 640 nm (Khan et al., was eliminated due to the action of the ingested food particles.
2014; Wray and Klaine, 2015 Zhu et al., 2009). In this study the However, recently the adsorption of nanoparticles onto the surface
largest aggregates formed (as measured using a Zetasizer) were of algae in suspension has also been observed for CeO2 (Angel et al.,
3043 nm with sizes predominantly ranging between 20 nm and 2015) and nAu (Botha et al., 2015b) which further supports the
640 nm (Botha et al., 2015a,b). Lee and Ranville (2012) re-exposed
110 T.L. Botha et al. / Aquatic Toxicology 170 (2016) 104111
ingestion of particles together with food particles by daphnids in therefore nAu are unlikely to pose any risk to D. magna in the envi-
aquatic media. Wray and Klaine (2015) and Khan et al. (2014) found ronment.
rapid removal of nAu present in the gut at lower concentrations
(highest concentrations of 0.5 mg/L and 0.6 mg/L respectively) than Conict of interest
used in this study. For lower concentrations irrespective of food
availability, it was found that 75% of the elimination took place The authors declare that there is no conict of interests regard-
within a few hours (less than 24 h), while the remaining 25% of ing the publication of this paper.
nAu that was ingested remained in the hindgut and was eliminated
one hundred times slower. Garca-Cambero et al. (2013) found low
Acknowledgments
lethality in acute testing but the effect over an extended time period
may answer important questions about the long term effects of nAu
This research was funded by the South African Department of
in the gut.
Science and Technology (DST). The authors wish to thank MINTEK
Lovern et al. (2007) also reported changes in swimming
for supplying the nAu stock solutions, as well as Professor Mary
behaviour where they would swim on the surface at a faster rate
Gulumian at the National Institute for Occupational Health (NIOH)
and remain static at the bottom of the beaker, specically in the
for assistance with CytoViva microscopy.
higher exposure concentrations where daphnia appeared heavy
(Garca-Cambero et al., 2013). It was noted that in daphnids where
the swimming pattern had changed into a slower hopping move- References
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