Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: The influence of silver behaviour in solution

http://informahealthcare.com/nanISSN: 1743-5390 (print), 1743-5404 (electronic)

Nanotoxicology, Early Online: 1–10! 2014 Informa UK Ltd. DOI: 10.3109/17435390.2014.963724

ORIGINAL ARTICLE

Uptake and elimination kinetics of silver nanoparticles and silver nitrateby Raphidocelis subcapitata: The influence of silver behaviour in solution

Fabianne Ribeiro1, Julian Alberto Gallego-Urrea2, Rhys M. Goodhead3, Cornelis A. M. Van Gestel4, Julian Moger5,Amadeu M. V. M. Soares1, and Susana Loureiro1

1Department of Biology & CESAM, University of Aveiro, Aveiro, Portugal, 2Department of Chemistry and Molecular Biology, University of

Gothenburg, Gothenburg, Sweden, 3Department of Biosciences, Ecotoxicology and Aquatic Biology Research Group, College of Life and

Environmental Sciences, University of Exeter, Devon, UK, 4Department of Ecological Science, Faculty of Earth and Life Sciences, VU University.

De Boelelaan, Amsterdam, The Netherlands, and 5Department of Physics, College of Engineering, Mathematics and Physical Sciences,

University of Exeter, Exeter, UK

Abstract

Raphidocelis subcapitata is a freshwater algae species that constitutes the basis of many aquatictrophic chains. In this study, R. subcapitata was used as a model species to investigate thekinetics of uptake and elimination of silver nanoparticles (AgNP) in comparison to silver nitrate(AgNO3) with particular focus on the Ag sized-fractions in solution. AgNP used in this studywere provided in a suspension of 1 mg Ag/l, with an initial size of 3–8 nm and coated with analkane material. Algae was exposed for 48 h to both AgNP and AgNO3 and sampled at differenttime points to determine their internal Ag concentration over time. Samples were collected andseparated into different sized fractions: total (Agtot), water column Ag (Agwater), smallparticulate Ag (Agsmall.part.) and dissolved Ag (Agdis). At AgNO3 exposures algae reached higherbioconcentration factor (BCF) and lower elimination rate constants than at AgNP exposures,meaning that Ag is more readily taken up by algae in its dissolved form than in its smallparticulate form, however slowly eliminated. When modelling the kinetics based on the Agdis

fraction, a higher BCF was found. This supports our hypothesis that Ag would be internalised byalgae only in its dissolved form. In addition, algae images obtained by Coherent Anti-stokesRaman Scattering (CARS) microscopy demonstrated large aggregates of nanoparticles externalto the algae cells with no evidence of its internalisation, thus providing a strong suggestion thatthese AgNP were not able to penetrate the cells and Ag accumulation happens through theuptake of Ag ions.

Keywords

Bioconcentration factor, Raphidocelissubcapitata, silver nanoparticles,toxicokinetics

History

Received 19 March 2014Revised 12 August 2014Accepted 25 August 2014Published online 13 October 2014

Introduction

Algae play a vital role in aquatic ecosystems, due to their majorfunction as primary producers at the bottom of the trophic chain.Consequently, it is likely that any alteration of the algaecommunity may be reflected at higher trophic levels andaccordingly impact on the functioning of the ecosystem. Forthis reason, algae are often used as a model indicator species inthe risk assessment of chemicals (Lewis, 1990; Perez et al., 2010,2011). As the nanotechnology market expands, the production ofnanomaterials and nanoparticles (NP) is rapidly increasing tosupply the growing demand (Keller et al., 2013; Roco, 2011).Therefore, it is a natural assumption that nanoparticles and theirtransformation products, e.g. silver sulphide and silver chloride(Levard et al., 2012), will be present in the environment at somepoint, from production and application of nanoparticle-containing

products to their final use and disposal (Benn & Westerhoff,2008; Nowack et al., 2011). The entrance of silver nanoparticles(AgNP) into the environment is predicted to commonly occur ascolloidal silver, i.e. in a size range between 1 and 1000 nm andeventually result in a suspension containing metallic silverparticles and Ag ions (Bhatt & Tripathi, 2011).

Moreover, silver nanoparticles will likely be transformed intosilver sulphide (Ag2S) and silver chloride (AgCl) under environ-mental conditions (Levard et al., 2012; Wang et al., 2012). TodayAgNP are among the most widely applied nanoparticles on themarket due to their inherent antimicrobial properties (Kim et al.,2007; PEN – Project on Emerging Technologies, 2014).

Before the advent of large-scale usage of nanotechnology,silver was already considered as one of the most toxic metalspresent in aquatic ecosystems, even at the low concentrationsfound in natural waters (Ratte, 1999; Seltenrich, 2013). Theseaspects have drawn attention to the toxicity of AgNP and Ag+ tomodel species in aquatic ecotoxicology. There are many eco-toxicological studies demonstrating that AgNP induce negativeeffects in key species, such as algae (Oukarroum et al., 2012;Ribeiro et al., 2014), zooplankton (Wang et al., 2012; Zhao &Wang, 2010, 2011;) and fish (Choi et al., 2010; Farkas et al.,2011). The interaction of Ag with algae cells depends on the sizeof pores across the cell wall as well as the state of aggregation of

Correspondence: Fabianne Ribeiro, Department of Biology & CESAM,University of Aveiro, Campus Universitario de Santiago, 3810-093Aveiro, Portugal. E-mail: [email protected] Loureiro, Department of Biology & CESAM, University ofAveiro, Campus Universitario de Santiago, 3810-093 Aveiro, Portugal. E-mail: [email protected]

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the nanoparticulate Ag chemical species in the natural water. Bothchlorides and sulphates play important roles in the mechanisms ofAg speciation, potentially reacting with Ag and creatingcomplexes that are not available for algae cells (Lee et al.,2005). Moreover, algae produce exo-polymeric substances (EPS)that are mainly composed of organic matter rich in polysacchar-ides. These EPS represent a dynamic source of interaction withNP in solution, causing nanoparticles to aggregate, stabilise ordissolve. EPS may also act as binding ligands to ions releasedfrom NP, thus reducing their bioavailability (Miao et al., 2009).Evidence of interactions between NP and EPS was describedby Zhang et al. (2013), who showed that amino-functionalisedquantum dots (QDs) had a tendency to aggregate in the presenceof EPS secreted by Thalassiosira pseudonana, thus becomingless likely to interact with the cells. In another study, it wasobserved that EPS stabilised dust-derived iron-nanoparticle(FeNP) aggregates and enhanced dissolution of Fe from FeNP(Kadar et al., 2014).

Considering that several factors can influence the behaviourof nanoparticles in the media (e.g. EPS, chlorides and sulphates),in the present study, we aimed at investigating how thebehaviour of Ag in solution (which here will be considered asan indication of Ag speciation) would influence Ag concentrationin the algae Raphidocelis subcapitata (previously known asPseudokirchneriella subcapitata). A comparison between AgNPand AgNO3 exposures provides supporting information on the riskassessment of NP to the algae. Furthermore, we also aimed atstudying whether AgNP were entering the cells as nanoparticlesor in ionic/dissolved form, by using coherent anti-Stokes Ramanscattering (CARS) microscopy. Bioconcentration was used asendpoint in this study as it relies on the chemical fractions that areavailable to the organism, thus as appropriate tool to assessnanoparticle bioavailability to algae.

Methods

Materials

Silver nitrate was purchased from Sigma-Aldrich (St. Louis, MO)as a crystalline powder, 99% purity CAS 7761-88-8. AgNP (3–8 nm) with an alkane coating were supplied by AMEPOX (Łodz,Poland). The AgNPs were supplied dispersed in ultra-pure waterat an initial concentration of 500 mg Ag/l, and kept in the dark at

room temperature before use. AgNP test suspensions wereprepared immediately before using by dilution of the initialdispersion of AgNP in algae media (MBL, Woods Hole, MA) tothe desired concentration. The AgNO3 test solutions wereprepared by diluting a 5 mg/l Ag+ stock solution in algae mediato the final test-concentrations prior to testing.

Bioconcentration tests

Algae were exposed in 500 ml flasks containing MBL mediaspiked with AgNP or AgNO3. Two concentrations for both AgNPand AgNO3 were tested: 15 mg and 30mg Ag/l, which correspondapproximately to the EC10 and EC50, respectively, for growthinhibition of algae exposed to both silver forms (Ribeiro et al.,2014). Three replicates were performed for each concentration.The exposure phase lasted for 48 h, followed by a 48 h eliminationphase. Test conditions were 21 �C (±1 �C) under a constant lightsource with replicates placed on an orbital shaker at 70 rpm.Algae and 50 ml water samples were taken from each replicate at0, 6, 12, 24 and 48 h of the exposure phase to assess both the Agconcentration and the different Ag fractions in the exposuremedia. Time zero sample was considered as the control. At eachsampling point, several sub-samples of the mixture were taken.‘‘Total Ag’’ stands for the mixture suspension of algae cells andall sized fractions of Ag.

After centrifugation at 2862g for 3 min, another sample wastaken and named water column Ag (Agwater) that contains all sizedfractions of Ag except algae cells, and large nano-Ag aggregates,or nano-Ag adsorbed to the algal cell surfaces.

A third fraction was filtered with a 0.45 mm polystyrene filter,and defined as the small particulate Ag (Agsmall part), in whichparticles smaller than 0.45 mm were present. Finally, the fourthfraction was obtained by centrifuging 10 ml of the water columnAg at 2862g for 30 min using53 kDa AMICON (Merck Millipore,Darmstadt, Germany) centrifugal filters, and this fraction wasnamed the dissolved Ag (Agdis). A scheme of this separationmethod is presented in Figure 1.

Both 0.45 mm and 3 kDa filters were pre-treated with a solutionof 0.1 M Cu(NO3)2 to avoid losses of Ag to the filter (Corneliset al., 2010). To measure Ag elimination from the algae, theremaining algae in the Erlenmeyer flasks were centrifuged, washedthree times with Milli-Q water and re-suspended in freshlyprepared, un-dosed MBL media for the 48-h elimination phase.

Figure 1. Schematic representation of silverfractionation in AgNP and AgNO3 exposuremedia. See ‘‘Methods’’ section for a moredetailed description of each Ag fraction.

Total Fraction

Water column Ag: Containsdissolved Ag and particulate Ag

(i.e. AgNP, AgCl and Agmacromolecule complexes)

50mL

Contains algae cells +water column Ag

Centrifugation3min, 3500 RPM

Small particulate Ag:Contains dissolved Ag, ionicAg and small particulate Ag

(i.e. AgNP, AgCl and Agmacromolecule complexes -

below 450nm)

Dissolved Ag:Contains soluble Ag

species,Including ionic Ag+

Algae pellet: washed3x with pure water

(measured) – containsalgae without external

Ag

2 F. Ribeiro et al. Nanotoxicology, Early Online: 1–10

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Coherent Anti-stokes Raman Scattering microscopy

In order to investigate whether Ag was taken up by the algae in itsnanoparticle form or in the dissolved or ionic form (Ag+), anotherexperimental section, apart from the bioconcentration assay wasperformed. Different treatments were set up to be imaged byCARS microscopy. Algae was previously exposed to both AgNPand AgNO3 at 15mg Ag/l and sampled accordingly. Thetreatments consisted of: (1) suspended algae was sampled andcentrifuged at 2862g for 3 min, then washed three times withMilli-Q water (exactly the same procedure that was used for thebioconcentration tests); and fixed in a single-strength glutaralde-hyde fixative (4%) in cacodylate buffer at room temperature for4 h; (2) algae was sampled then centrifuged at 2862g for 3 min(without pellet washing) and proceeded to fixation; (3) suspendedalgae cells were straight placed in the fixative (without centrifu-gation or washing). After the fixation period, all different algaetreatments were placed on a microscope slide with a cover slip.

CARS microscopy

Coherent Anti-stokes Raman Scattering microscopy has emergedas a powerful optical microscopy technique with several advan-tages over conventional biological imaging techniques; label-freecontrast, increased depth penetration and reduced phototoxicity(Evans & Xie, 2008; Rodriguez et al., 2006).

Additionally, as shown by Moger et al. (2008), CARS hasexceptional capabilities for locating NP within biological sampleswith 3-D sub-cellular resolution. CARS microscopy uses thechemical composition of a biological sample to exploit thedifferent vibrational resonance of molecular bonds to generateimage contrast. In the same way that nanoscale materials exhibitunique chemical properties, NPs also have extraordinary opticalproperties which give greatly enhanced optical responses (Mogeret al., 2008; Wang et al., 2011).

NPs generate large CARS signals that are independent of thevibrational frequency being probed and this difference invisualisation between frequency independent NP and specificallytuned vibrational frequencies for biological structures, allows thedifferentiation of signals from NP from those probed in thesample that is being imaged. As a final advantage the label-freenature of the technique eliminates the chemical perturbation seenwhen using fluorescent labelling of either NP or staining ofsurrounding tissues, both of which modify the cellular uptake andobserved cytotoxicity of NP.

Particle characterisation

An aggregation experiment in algae media was performed usingdynamic light scattering (DLS) in a Zetasizer nano ZS (Malverninstruments Ltd, Worcestershire, UK) and Zetasizer software 6.20and processed with the multiple narrow modes algorithm (highresolution) to elucidate multiple peaks in the intensity-basedparticle size distribution (PSD). Short- and long-term experimentswere conducted. For the short-term experiments (minutes), theinitial AgNP stock suspension was diluted with the MBL media tothe desired concentrations in polystyrene cuvettes (Malverninstruments Ltd, Worcestershire, UK) and inserted immediatelyin the instrument. The measurement was started at a fixedattenuator and measurement position to avoid the optimisationtime, the correlation time was set to 2 s and 120 data points weregenerally obtained. For the long-term experiments (days), the firstmeasurement (day zero) was obtained by creating an averageresult from the short-term data points. The cuvettes were stored inthe dark and three measurements were performed (three runs of20 s each) in the following days. To evaluate the effect of particlesedimentation, the samples were shaken after performing the

measurement and a new measurement was done. Derived countrates were included in the long-term experiments to comparethe capacity of the remaining particles (large and small) toscatter light.

For transmission electron microscope (TEM) imaging, aninitial suspension of AgNP (1000 mg Ag/l) was diluted to 100 mgAg/l in MBL media and a drop of this suspension was depositedonto a holey carbon-coated Cu-TEM grid and dried at roomtemperature for several hours before examination. Experimentswere carried out on a JEOL JEM 2010 200 kV instrument (JEOL,Tokyo, Japan).

Sample digestion

Water

Prior to digestion, all water samples (10 ml) were mixed with0.28 ml of H2O2 and 1.35 ml of HCl, bringing the concentrationsto 1% of H2O2 and 5% of HCl for 24 h. This procedure was aimedto break down organic matter in the samples and ensure that anysilver adsorbed to the sample holder’s wall was released as solubleAgClx complexes.

After 24 h, samples were transferred to Teflon beakers (25 mlvolume capacity) and allowed to evaporate on a hotplate over 45–50 �C (without boiling) until 1–1.5 ml of the sample remained inthe beakers. Samples were then mixed with 1 ml HNO3 (65% traceanalysis) and 3 ml of HCl (37% trace analysis) before being heatedfor 1 h. All samples were transferred to plastic graduated tubesand diluted with a 1% HCl to a final volume of 45 ml. Threereplicates of un-dosed MBL media and three replicates of aknown concentration of Ag (50 g Ag/l in Milli-Q water) weredigested together with all other samples to be used as blankcontrols and as a recovery material for Ag measurements,respectively. Total silver was measured on a Perkin Elmer 5100(PerkinElmer, Waltham, MA) Graphite Furnace AtomicAbsorption Spectrophotometer (GF-AAS).

Tissue

After a three time-cycle of washing with Milli-Q water andcentrifugation, the algae pellet was dried at 50 �C, weighed andtransferred to Teflon beakers for digestion. Three milliliters ofHNO3 (65% trace analysis) were added to the beakers, which wereheated (with lids on) on a hotplate at 50 �C for approximately30 min (or until the tissue dissolved). Samples were allowed tocool down at room temperature and mixed with 1 ml HCl (37%trace analysis) before being replaced on the hotplate with the lidson and heated for another 30 min. After 30 min, the lids wereremoved and samples were allowed to evaporate (without boiling)until approximately 1 ml remained. In addition, three replicates ofthe reference material DOLT-4 (Dogfish Liver CertifiedReference Material for Trace Metals) were digested with theother tissue samples. The dilution and Ag measurement stepsfollowed the same procedure as described for water samples.

Toxicokinetic modelling

In the present study, a one-compartment model was used todescribe the kinetics of the bioaccumulation of Ag from AgNPand AgNO3 in R. subcapitata. This model was recently used byPiccapietra et al. (2012) in a similar study, and describes the fateof Ag based on mass balance equations. Assuming that theconcentration of exposure would remain constant, two equationswere used to model uptake and elimination of AgNP and AgNO3

in the algae:

Uptake: QðtÞ ¼ k1

k2

� Cexp � 1� eð�k2�tÞ� �

ð1Þ

DOI: 10.3109/17435390.2014.963724 Uptake and elimination kinetics of silver nanoparticles by R. subcapitata 3

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Elimination: QðtÞ ¼ k1

k2

� Cexp � ð1� eð�k2�ðt�tcÞÞ � eð�k2�tÞÞ

ð2Þ

where, Q(t) is the concentration in the algae at time t; tc is thetime (hours) at which algae were transferred to uncontaminatedmedia; k1 is the uptake rate constant (l/g/h); k2 is the eliminationrate constant (1/h); Cexp is the concentration of exposure(Ag water column concentration measured at time zero); estands for the exponential function.

The bioconcentration factor (BCF) was calculated by the ratioof k1 and k2. The concentration of exposure included in the modelwas considered as the Ag water column concentration measured attime zero.

Considering the decrease in concentration of silver in the mediaduring exposure, a decay rate constant (kdec) was modelled byfitting the following equation to the concentrations of Ag measuredat different points in time (Widianarko & VanStraalen, 1996)

AgðtÞ ¼ ½Ag� � eð�kdec�tÞ

This decay constant was included in the uptake model(Equation (1)) for different fractions to read:

QðtÞ ¼ k1

k2 � kdec

� Cexp � eð�kdec�tÞ � eð�k2�tÞ ð3Þ

where, kdec is the Ag concentration decay rate constant (1/h).

Statistical analysis

Statistical differences in concentration of Ag in the water for allfractions were detected by a two-way ANOVA, with significantdifferences established at p50.05. The kinetics parametersstandard errors were obtained by a non-linear regression analysis,using the SPSS 20.0 statistical software (IBM corporation,Armonk, NY).

Results

Particle characterisation

The z-averaged hydrodynamic diameter of AgNP suspended inwater measured at 1 mg/l was 106 nm and showed to bereasonably stable for the duration of the experiment (Table 1and Figure S1). The minimum concentration that provided areliable signal from DLS for this dispersion was 1 mg/l, andFigure S1 and Table 1 present the variation of this suspension inMBL during short- and long-term experiments to simulate theexposure conditions. The agglomerate size varied between 200and 250 nm in the long-term experiments.

Ag fractionation in the exposure media

Ag concentration in the AgNO3-spiked media decreased until 6 hof exposure, regardless the concentration and the fraction (Agwater

and Agsmall part) F4,29¼ 2.4 (p40.05) (Figure 2).

As expected, dissolved (Agdis) concentrations were statisticallyhigher in the media containing AgNO3 (two-way ANOVAF1,19¼ 87.4, p50.0001). However, the pattern of decay inconcentrations of dissolved Ag arising from AgNO3 was differentfor different concentrations: at 15mg Ag/l, dissolved Ag concen-tration decreased rapidly over the first 6 h of exposure, after whichit slightly increased and remained constant until the end of theexposure, whilst at 30mg/l, concentration decrease remainedconstant until 12 h of exposure before following a similar patternas the 15 mg/l treatment (Figure 2).

For the media containing AgNP, Ag concentration decreasedfor both concentrations and fractions (Agwater and Agsmall part.)until 24 h of exposure, after which there seemed to be an increasein concentration again. The small particulate fraction (Agsmall

part.) of the 30 mg Ag/l differed, as it remained constant throughoutthe experiment (Figure 2B). The dissolved Ag concentrations inthe AgNP exposure media showed a rapid decrease from zeroto 6 h for both 15 mg/l and 30 mg/l (Figure 2D). From 6 h to 24 h,the dissolved Ag concentration continued to decrease slowlyat 30 mg/l, whilst at 15 mg/l, it appeared to remain constant.At both exposure concentrations, dissolved Ag concentrationshowed a slight increase after 24 h.

Table 2 present the percentage of Ag loss caused by the washesof the pellet fraction. Those losses were higher in the AgNO3

media, as it is related to the precipitated Ag and/or Ag bound tolarge organic ligands.

Constants of Ag concentration decay in solution are presentedin Table 3, isolated for each fraction. For AgNP exposure, thelarger the particle size within a fraction, the lower the decay rateconstant was, with Agsmall part at 15 mg Ag/l being the fractionwith the highest decay rate constant. For AgNO3, the Agconcentration decay rate constants in the exposure media werehigher than for AgNP. For both the Agwater and Agsmall part

fractions, concentration decay rate constants increased withincreasing concentration. On the other hand, for Agdis, a reversepattern could be observed, with the highest decay rate constantbeing found at 15 mg/l compared to 30mg/l. Furthermore, after24 h of exposure, Agdis from AgNP and AgNO3 reached similarconcentrations.

Toxicokinetics and Ag internalization

Uptake and elimination kinetics of Ag in R. subcapitata weremodelled according to the one-compartment model, in which therate constants of uptake from water (k1) and elimination (k2) werecalculated for exposures to AgNP and AgNO3. Assuming that Agconcentration remained constant during the uptake phase, i.e.when uptake was based on the concentration measured in thewater column at time zero of exposure, algae accumulatedapproximately 50 mg/g (dw) of Ag when exposed to AgNP, incomparison to �100mg/g (dw) upon exposure to AgNO3 at theEC10 concentration level (Figures 3 and 4). In the AgNO3

exposure media, the maximum Ag burden in algae (average)reached 140mg/g (dw) (SE¼ 16.6) at 15 mg Ag/l after 12 h,whereas at 30mg/l the maximum Ag burden after 12 h was

Table 1. Summary of the results obtained in the long-term variation of particle size with DLS.

Time of sample,days

Average Z-hydrodynamicdiameter, nm SD, nm PDI Peak 1, nm Peak 2, nm Peak 3, nm

Derived countrate, kcps SD, kcps

0 106.3 9.2 0.209 111.2 50.4 1221.0 1625.9 220.41.3 178.0 7.3 0.371 195.6 53.7 25.0 2368.8 74.63.0 242.3 26.7 0.348 197.5 37.5 24.1 1082.7 64.83.0 (after shaking) 204.4 11.9 0.351 201.3 1065.0 0.0 1082.6 51.1

Standard deviations (SD) between the measurements (120 for time 0 and 3 for the others) are presented. Polydispersity index (PDI) and the threeintensity peaks were obtained from re-analysis of all measurements done in one sample.

4 F. Ribeiro et al. Nanotoxicology, Early Online: 1–10

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342mg/g (dw) (SE¼ 40.9). For AgNP, the Ag burden in algaereached 45.0 mg/g (dw) (SE¼ 1.10) after 24 h of exposure to15mg/l and 93.7 mg/g (dw) (SE¼ 8.49) after 24 h of exposure to30mg/l. When related to time zero water column concentration ofexposure, uptake and elimination rate constants were lower in theAgNP treatments compared to AgNO3 (Table 4).

After including Ag concentration decay rate constants in themodel, the pattern observed for nominal concentrations remained,i.e. for all fractions (Agwater, Agsmall part and Agdis) BCF valueswere higher for AgNO3 than for AgNP exposure (Table 4). Seesupporting information (S2 and S3) for kinetics curves from eachAg fraction. Strictly focusing on BCF values calculated based onthe small particulate fraction (Agsmall part), the difference betweenAgNP and AgNO3 was less for the other fractions.

Regarding CARS images interpretation, reconstructing mul-tiple series of images separated by 0.25 mm in the z-plane into athree-dimensional image (Figures 6 and S4) provided no evidenceof AgNP internalisation neither in the washed algae nor in theunwashed algae samples. The AgNP agglomerates changed in sizeaccording to the sample preparation method, with unwashedpreparations displaying larger visible aggregates outside the cell.

Discussion

This work focused on understanding the uptake and bioconcen-tration of Ag from different sources in a freshwater algae speciesR. subcapitata by comparing exposures to AgNP and to AgNO3.In order to better understand how the chemical reactivity of Agwith other constituents of the media and test conditions couldinfluence uptake, the toxicokinetics were modelled by taking intoaccount different sizes of Ag complexes, from large particleagglomerates/aggregates to the dissolved species.

The aggregation rates of the AgNP in MBL were low and theywere expected to be lower in the actual algae exposure becausethe number of concentrations are two orders of magnitude lower,leading to fewer collisions. Moreover, the organic nature of thenanoparticle coating material ameliorated their stability bypreventing ionisation with the constituents of the algae media.

From the fractionation analysis of AgNP-spiked exposuremedia, it was found that larger sized fractions decreased inconcentration up to 24 h of exposure. In AgNO3 spiked media,however, those same fractions decreased rapidly during the first6 h of exposure, and remained at low levels until 48 h. Moreover,during the period of 0 to 12 h, Ag body burdens in algae increasedexponentially as shown in Figures 3 and 4. Therefore, it can beassumed that this decrease in Ag concentration was due to both

AgNO3

Dissolved 15µg/L

Dissolved 30µg/L

Time (hours)

Water column 15µg/LWater column 30µg/LSmall particulate 15µg/LSmall particulate 30µg/L

AgNP

Ag

conc

entr

atio

n (µ

g/L

)

0

5

10

15

20

25

30

0

2

4

6

8

10

0.0

0.2

0.4

0.6

0.8

1.0

0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48

0 6 12 18 24 30 36 42 480 6 12 18 24 30 36 42 480

5

10

15

20

25

30 (A)

(C)

(B)

(D)

Figure 2. Water column, small particulate (A and B) and dissolved (C and D) silver concentrations at each sampling time (hours) for AgNP and AgNO3

in MBL media. Error bars indicate the standard error from three measurements at one sampling time. Note the 10-fold difference on the X-axis betweenfigures C and D.

Table 2. Silver loss percentages at each sampling time.

NominalAg (mg/l)

Time(hours)

Ag Loss (%)AgNP

Ag Loss (%)AgNO3

15 6 17 6212 37 6724 21 6848 14 55

30 6 21 6412 37 6224 31 5648 40 84

The percentages indicate the amount of Ag that was lost throughout thepellet washes, and not quantified in the Ag concentration decrease in themedia.

DOI: 10.3109/17435390.2014.963724 Uptake and elimination kinetics of silver nanoparticles by R. subcapitata 5

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settling of Ag agglomerates/aggregates to the bottom of theexperimental vessel and uptake of Ag into the algae cells.

Nonetheless, considering the system dynamics, the decrease inthe small particulate Ag (Agsmall part.) concentrations was morepronounced for the AgNO3 treatment than for AgNP, reaching2mg/l after 6 h. As Agsmall part contained dissolved Ag, Agcomplexes and Ag bound to macromolecules (such as EPS), therapid decrease in Agsmall part concentration in the AgNO3

treatment is likely to be associated with a decrease in itsbioavailability to algae. This was not observed for the AgNPexposure media due to the presence of small particles (5450 nm)in the Agsmall part fraction, which could have been internalised bythe algae.

According to Visual Minteq speciation calculations at lowdissolved Ag concentrations, chloride is the most important ligandfrom all components in the media and it is only after �70 mg/l

350

400

450

200

250

300

[Ag]

alg

ae (

µg/g

dw

)

50

100

150

Time (hours)

0

350

400

450

200

250

300

50

100

150

0

0 24 48 72 96 120 144 1680 24 48 72 96 120 144 168

EC50AgNP AgNO3

Figure 4. Uptake and elimination kinetics of silver in R. subcapitata exposed to AgNP and AgNO3. The kinetic model curve was calculated byEquation (1) for the uptake phase and Equation (2) for the elimination phase, using algae body burdens the concentration of exposure measured in thewater column at time zero of exposure, which was 26mg/l for both AgNP and AgNO3. The elimination phase started at 48 h.

150

175

100

125

25

50

75

0

150

175

100

125

25

50

75

0

[Ag]

alg

ae (

µg/g

dw

)

Time (hours)

0 24 48 72 96 120 144 1680 24 48 72 96 120 144 168

EC10AgNP AgNO3

Figure 3. Uptake and elimination kinetics of silver in R. subcapitata exposed to AgNP and AgNO3. The kinetic model curve was calculated byEquation (1) for the uptake phase and Equation (2) for the elimination phase, using algae body burdens e concentration of exposure measured in thewater column at time zero of exposure, which was 12.5mg/l for both AgNP and AgNO3. The elimination phase started at 48 h.

Table 3. Decay constants (kdec; mg/l/h) of Ag in MBL media, according to the fraction of AgNP and AgNO3 along a48 h of exposure period.

AgNP AgNO3

Agwater Agsmall part Agdissolved Agwater Agsmall part Agdissolved

15mg/l 0.01 (0.007) 0.03 (0.009) 0.18 (0.08) 0.24 (0.08) 0.34 (0.08) 0.42 (0.08)30mg/l 0.02 (0.007) 0.04 (0.007) 0.14 (0.09) 0.31 (0.09) 0.72 (83) 0.30 (4.7)

Standard errors of the parameters are presented in the parenthesis.

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Ag+ concentration that AgCl(s) starts to form. Unfortunately, dueto a lack of information regarding the exudates from the algae, itis not possible to elucidate the relative importance of the latter asligands for Ag+. Similarly, it has been shown previously thatdifferent types of natural organic matter (NOM) interact with thesurface of NP with different mechanisms leading to furtherstabilisation (Gallego-Urrea & Holmberg, 2014), dissolution(Hering, 1995) or aggregation (Zhang et al., 2009) dependingon the NOM/NP ratio. In addition, as demonstrated by Lee et al.(2004), the uptake of Ag by R. subcapitata was not influenced bythe concentration of chloride in the media, meaning that algae canreadily internalise AgCl complexes.

Dissolution or release of dissolved silver species was alsomeasured over time for both exposure media containing AgNPand AgNO3. Dissolution was observed to occur faster for theAgNO3 media compared to AgNP, as shown in Figure 2. For bothtreatments at 15 mg/l, the concentration of dissolved silverdecreased rapidly in the first 6 h before levelling off. However,at 30 mg Ag/l, dissolved Ag from AgNP and AgNO3 continued todecrease in concentration until 12 h and 24 h of exposure,respectively. This is opposite to the trend observed by Lee &Campbell et al. (2005), who reported that the concentration ofdissolved Ag released from AgNP increased exponentially duringthe first 6 h of exposure. However, that study was performed indeionised water, whilst our experiment used a culture media in thepresence of algae. Considering that the internal concentration ofAg in R. subcapitata increased for both treatments during the firsthours of exposure (Figures 3 and 4), it is possible that the algaewere readily taking up most of the released Ag in its dissolvedform, therefore leading to the decrease in Ag concentrations in thetest media.

The decay of dissolved Ag correlated with the maximum Agmeasured in algae in the AgNO3 media, where the concentrationof Agdis decreased with time until the 6th hour of exposure (atEC10 level), while a maximum concentration in algae was reachedafter 12 h of exposure. This may indicate a transfer of Agdis fromthe media to the algae. Moreover, algae dosed with AgNO3

seemed to have a faster uptake of Ag during the first hours ofexposure and started to eliminate Ag already during the uptakephase. This indicates that equilibrium between uptake andelimination was not reached and that Ag was continually beinginternalised and eliminated by the algae at varying rates before thecells were transferred to Ag-free media.

Assuming that Ag uptake followed a first-order kinetics modeland considering that the algae cells behaved as one compartment,we were able to calculate uptake (k1) and elimination (k2) rate

constants and bioconcentration factors (BCF) for different Agfractions. These parameters, presented in Table 4, will hereafterbe used to guide our interpretation of the Ag toxicokinetics inalgae. The uptake rate constants related to all Ag fractionsremained higher in the AgNO3 exposures, while the eliminationrate constants based on Agwater and Agsmall part. were lower forAgNO3. It has been demonstrated that the mechanism behind Aginternalisation in algae is related to accidental cation transport,which is believed to occur through the same mechanism as theinternalisation of essential cations (i.e. Na+, K+). This is due tothe inability of the system to distinguish between Cu+ and Ag+,given that both metals share some chemical characteristics (Leeet al., 2004; Solioz & Odermatt, 1995). Additionally, AgCl0 islikely to be internalised through passive diffusion from theexternal cell environment to the cytosol, over protein channels inmembranes (Lee et al., 2004). Either mechanism could explainthe higher k1 values obtained for AgNO3 exposures as, in thiscase, Ag is more likely to be present as dissolved andconsequently in a form that could be easily internalised by thealgae. On the other hand, lower elimination constants indicate thatthe algae failed to completely excrete Ag either as dissolved orsmall particulate forms. Ag is known to be associated withmetallothioneins (MT) (Robinson, 1989), thus it is likely thatdissolved Ag in algae is sequestrated and bound or stored in sucha way that it cannot be eliminated anymore, or it is eliminated in arather slow rate. Such binding to MT is less expected for Agcomplexes or particles.

The BCF of AgNP was the highest when we considered thedissolved fraction, which corroborates the hypothesis that algaewould take up dissolved Ag and/or Ag complexes from the mediarather than Ag particulate forms. Moreover, the highest BCFvalue was obtained at the lowest AgNP concentration (EC10 level)indicating that dissolution rates of AgNP were more efficient atlower concentrations and consequently at lower aggregation rateof particles probably driven by a steeper concentration gradientand larger surface area exposed (less aggregation). This was alsoreported by Kittler et al. (2010), who observed that PVP-coatedAgNP showed a higher dissolution at 0.05 g/l when compared to0.1 g/l, after approximately 100 days in a long-term dissolutionexperiment.

The lower BCF values obtained when using the water columnand small particulate fractions from AgNP were interpreted as anindication of the behaviour of AgNP in the exposure media. Asmentioned before, algae is known to secrete exudates that aremainly composed of organic matter, rich in polysaccharidemolecules, which in turn can induce aggregation of AgNP,

Table 4. Kinetic parameters for Ag uptake and elimination in R. subcapitata estimated by Equation (3) for each fraction of AgNP and AgNO3.

15mg/l 30mg/l

Agtime zero Agwater Agsmall part. Agdis Agtime zero Agwater Agsmall part. Agdis

AgNPk1 0.14 (0.5) 0.51 (0.2) 0.42 (0.2) 0.58 (0.2) 0.15 (0.5) 0.39 (0.2) 0.41 (0.2) 0.53 (0.2)k2 0.02 (0.4) 0.13 (0.8) 0.07 (0.08) 0.01 (0.008) 0.02 (0.3) 0.07 (0.9) 0.05 (0.9) 0.01 (0.07)BCF 7 3.9 5.8 75.0 7.5 5.7 7.6 45.9

AgNO3

k1 6.34 (9.7) 3.02 (0.5) 3.76 (0.5) 4.40 (0.5) 4.1 (4.7) 4.32 (0.6) 7.87 (0.6) 4.79 (0.4)k2 0.81 (2.3) 0.03 (0.02) 0.03 (0.02) 0.02 (0.02) 0.47 (1.1) 0.03 (141) 0.02 (83) 0.03 (4.7)BCF 7.8 100 125 220 8.7 144 393 159

Agtime zero was calculated based on the average concentrations at the initial phase of exposure. The kinetics parameters and BCF values of the Agtimezero are related to the Figures 4 and 5. Agwater was based on the total fraction, Agsmall part. was obtained by the 0.45mm-filtered fraction (smallparticulate Ag) and Agdis was based on the dissolved Ag obtained by ultracentrifugation with 3 kDa membranes. k1 – uptake rate constant (l/g/h); k2 –elimination rate constant (per hour); BCF – bioconcentration factor. Standard errors of the parameters are presented in the parenthesis. The kineticscurves for the other fractions are presented in the supplementary material.

DOI: 10.3109/17435390.2014.963724 Uptake and elimination kinetics of silver nanoparticles by R. subcapitata 7

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altering their bioavailability to algae (Joshi et al., 2012). Based onour results, we can speculate that in this instance, exopolymericsubstances produced by algae may have played a key role indecreasing the bioavailability of AgNP to the cells.

By using the CARS microscope imaging technique,R. subcapitata exposed to AgNP showed no evidence ofinternalisation of the nanoparticles into the algal cells used inthis experimental setup. Aggregates varying in size could bevisualised externally, with the size of the aggregate being reducedin response to the washing process (Figure 5). Additionally,CARS images show a lack of association of AgNPs with the algae(Figure 5B and C; Figures 6 and S4). All AgNP signals wereshown to be from outside the cell after 3D image sectioning withCARS, confirming that no particles were up taken by the algae(Figures 6 and S4). This suggests that internalised Ag was in theform of ionic or dissolved Ag. However, the nanoparticleagglomerates nearby the algae cells may induce a physicaleffect on algae, depending on agglomerate size and may interferewith the algae growth and/or lead to a faster sedimentation ofcells. In addition, shading effects and induced higher localconcentration could also play a vital role in toxicity, depending onthe ability of the nanoparticle to attach to the cell surface. NP maythus interfere with photosynthesis and/or by their chemicalcharacteristics the nanoparticles may induce the formation of aturbid media, which can suppress the light absorbance by algae(Aruoja et al., 2009; Schwab et al., 2011). Thus, our data highlightthe importance, when predicting the potential risk of nanoparticle

Figure 5. CARS images of R. subcapitata. (A) Control. (B, C) dosed with AgNP at 15mg Ag/l, centrifuged and washed with Milli-Q water. (D) Dosedwith AgNP at 15mg Ag/l, centrifuged and unwashed. Arrows indicate nanoparticle signal. Figures B and C, which have been washed show smallagglomerates outside the cells and not attached to the cell surface, and figure D, representing the unwashed treatment shows large NP agglomeratesoutside the cells. Scale bars are 10mm.

Figure 6. Schematic 3-D reconstruction of R. subcapitata dosedwith AgNP at 15mg Ag/l centrifuged and unwashed. Largeaggregates of Ag show association but no penetrance into algae cells.An animated version of this image is available online on the supportinginformation.

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presence in the environment, of taking into consideration all thedifferent forms of nano-Ag (dissolved, suspended, aggregated andinternalised) interacting with phytoplankton.

Conclusions

In conclusion, we have demonstrated that the separation intodifferent size fractions revealed to be a trustworthy tool to studysilver chemical behaviour in our test media (MBL) and helped toestimate the relationship between silver behaviour and itsbioavailability to algae. The amount of silver taken up by thealgae was dependent on the uptake and elimination rate constants,which in the AgNO3 exposures resulted in higher BCF values.When BCF was calculated on the basis of dissolved Ag fromAgNP, a higher value was obtained in comparison to the otherAg-sized fractions, indicating that upon AgNP exposure silverwas internalised as Ag+ or dissolved Ag rather than in itsparticulate form. This was also confirmed by the CARS imagesshowing that AgNP used in this study were unable to crossalgae cell walls, which lead us to conclude that bioconcentrationof AgNP is probably mediated by the internalisation of dissolvedor/and ionic Ag.

Acknowledgements

This study was partly supported by the project NanoFATE, financed bythe FP7 Programme, European Commission (CP-FP 247739 NanoFATE),and by funding FEDER through COMPETE and Programa OperacionalFactores de Competitividade and by the Portuguese National fundingthrough FCT – Fundacao para a Ciencia e a Tecnologia, within theresearch project FUTRICA – Chemical Flow in an Aquatic TRophicChain (FCOMP-01-0124-FEDER-008600; Ref. FCT PTDC/AAC-AMB/104666/2008) and by a PhD grant awarded by FCT to Fabianne Ribeiro(SFRH/BD/64729/2009). Susana Loureiro was ‘‘Bolsista CAPES/BRASIL’’, Project No. 106/2013. The research presented in this paperreceived support from the QNano Project (http://www.qnano.ri.eu), whichis financed by the European Community Research Infrastructures underthe FP7 Capacities Programme (Grant No. INFRA-2010-262163), and itspartner the University of Exeter, within the UOE-TAF-42: Theinternalisation of Ag and ZnO NPs in aquatic and terrestrial organisms(Susana Loureiro). The authors would also like to thank Mr Rudo Verweijfor the AAS measurements and the anonymous reviewers for theirrelevant comments, which highly improved the quality of this paper.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of the paper.

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Supplementary materials available onlineSupplementary Figures S1–S3 and supplementary video S4.

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