Nanotoxicology

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Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: The influence of silver behaviour in solution Fabianne Ribeiro, Julián Alberto Gallego-Urrea, Rhys M. Goodhead, Cornelis A. M. Van Gestel, Julian Moger, Amadeu M. V. M. Soares & Susana Loureiro To cite this article: Fabianne Ribeiro, Julián Alberto Gallego-Urrea, Rhys M. Goodhead, Cornelis A. M. Van Gestel, Julian Moger, Amadeu M. V. M. Soares & Susana Loureiro (2015) Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: The influence of silver behaviour in solution, Nanotoxicology, 9:6, 686-695, DOI: 10.3109/17435390.2014.963724 To link to this article: http://dx.doi.org/10.3109/17435390.2014.963724

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Date: 09 October 2017, At: 03:17

http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, 2015; 9(6): 686–695 ! 2014 Informa UK Ltd. DOI: 10.3109/17435390.2014.963724

ORIGINAL ARTICLE

Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: The influence of silver behaviour in solution Fabianne Ribeiro1, Julia´n Alberto Gallego-Urrea2, Rhys M. Goodhead3, Cornelis A. M. Van Gestel4, Julian Moger5, Amadeu M. V. M. Soares1, and Susana Loureiro1

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1

Department 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

Keywords

Raphidocelis subcapitata is a freshwater algae species that constitutes the basis of many aquatic trophic chains. In this study, R. subcapitata was used as a model species to investigate the kinetics 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 study were provided in a suspension of 1 mg Ag/l, with an initial size of 3–8 nm and coated with an alkane material. Algae was exposed for 48 h to both AgNP and AgNO3 and sampled at different time points to determine their internal Ag concentration over time. Samples were collected and separated into different sized fractions: total (Agtot), water column Ag (Agwater), small particulate Ag (Agsmall.part.) and dissolved Ag (Agdis). At AgNO3 exposures algae reached higher bioconcentration 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 small particulate 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 by algae only in its dissolved form. In addition, algae images obtained by Coherent Anti-stokes Raman Scattering (CARS) microscopy demonstrated large aggregates of nanoparticles external to the algae cells with no evidence of its internalisation, thus providing a strong suggestion that these AgNP were not able to penetrate the cells and Ag accumulation happens through the uptake of Ag ions.

Bioconcentration factor, Raphidocelis subcapitata, silver nanoparticles, toxicokinetics

Introduction Algae play a vital role in aquatic ecosystems, due to their major function as primary producers at the bottom of the trophic chain. Consequently, it is likely that any alteration of the algae community may be reflected at higher trophic levels and accordingly impact on the functioning of the ecosystem. For this reason, algae are often used as a model indicator species in the risk assessment of chemicals (Lewis, 1990; Pe´rez et al., 2010, 2011). As the nanotechnology market expands, the production of nanomaterials and nanoparticles (NP) is rapidly increasing to supply the growing demand (Keller et al., 2013; Roco, 2011). Therefore, it is a natural assumption that nanoparticles and their transformation products, e.g. silver sulphide and silver chloride (Levard et al., 2012), will be present in the environment at some point, from production and application of nanoparticle-containing

Correspondence: Fabianne Ribeiro, Department of Biology & CESAM, University of Aveiro, Campus Universita´rio de Santiago, 3810-093 Aveiro, Portugal. E-mail: [email protected] Susana Loureiro, Department of Biology & CESAM, University of Aveiro, Campus Universita´rio de Santiago, 3810-093 Aveiro, Portugal. Email: [email protected]

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

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 as colloidal silver, i.e. in a size range between 1 and 1000 nm and eventually result in a suspension containing metallic silver particles and Ag ions (Bhatt & Tripathi, 2011). Moreover, silver nanoparticles will likely be transformed into silver sulphide (Ag2S) and silver chloride (AgCl) under environmental conditions (Levard et al., 2012; Wang et al., 2012). Today AgNP are among the most widely applied nanoparticles on the market 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 metals present in aquatic ecosystems, even at the low concentrations found in natural waters (Ratte, 1999; Seltenrich, 2013). These aspects have drawn attention to the toxicity of AgNP and Ag+ to model species in aquatic ecotoxicology. There are many ecotoxicological studies demonstrating that AgNP induce negative effects 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 size of pores across the cell wall as well as the state of aggregation of

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DOI: 10.3109/17435390.2014.963724

Uptake and elimination kinetics of silver nanoparticles by R. subcapitata

the nanoparticulate Ag chemical species in the natural water. Both chlorides and sulphates play important roles in the mechanisms of Ag speciation, potentially reacting with Ag and creating complexes 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 polysaccharides. These EPS represent a dynamic source of interaction with NP in solution, causing nanoparticles to aggregate, stabilise or dissolve. EPS may also act as binding ligands to ions released from NP, thus reducing their bioavailability (Miao et al., 2009). Evidence of interactions between NP and EPS was described by Zhang et al. (2013), who showed that amino-functionalised quantum dots (QDs) had a tendency to aggregate in the presence of EPS secreted by Thalassiosira pseudonana, thus becoming less likely to interact with the cells. In another study, it was observed 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 behaviour of nanoparticles in the media (e.g. EPS, chlorides and sulphates), in the present study, we aimed at investigating how the behaviour of Ag in solution (which here will be considered as an indication of Ag speciation) would influence Ag concentration in the algae Raphidocelis subcapitata (previously known as Pseudokirchneriella subcapitata). A comparison between AgNP and AgNO3 exposures provides supporting information on the risk assessment of NP to the algae. Furthermore, we also aimed at studying whether AgNP were entering the cells as nanoparticles or in ionic/dissolved form, by using coherent anti-Stokes Raman scattering (CARS) microscopy. Bioconcentration was used as endpoint in this study as it relies on the chemical fractions that are available to the organism, thus as appropriate tool to assess nanoparticle 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 (Ło´dz´, Poland). The AgNPs were supplied dispersed in ultra-pure water at an initial concentration of 500 mg Ag/l, and kept in the dark at

Figure 1. Schematic representation of silver fractionation in AgNP and AgNO3 exposure media. See ‘‘Methods’’ section for a more detailed description of each Ag fraction.

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room temperature before use. AgNP test suspensions were prepared immediately before using by dilution of the initial dispersion of AgNP in algae media (MBL, Woods Hole, MA) to the desired concentration. The AgNO3 test solutions were prepared by diluting a 5 mg/l Ag+ stock solution in algae media to the final test-concentrations prior to testing. Bioconcentration tests Algae were exposed in 500 ml flasks containing MBL media spiked with AgNP or AgNO3. Two concentrations for both AgNP and AgNO3 were tested: 15 mg and 30 mg Ag/l, which correspond approximately to the EC10 and EC50, respectively, for growth inhibition 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 elimination phase. Test conditions were 21  C (±1  C) under a constant light source with replicates placed on an orbital shaker at 70 rpm. Algae and 50 ml water samples were taken from each replicate at 0, 6, 12, 24 and 48 h of the exposure phase to assess both the Ag concentration and the different Ag fractions in the exposure media. Time zero sample was considered as the control. At each sampling point, several sub-samples of the mixture were taken. ‘‘Total Ag’’ stands for the mixture suspension of algae cells and all sized fractions of Ag. After centrifugation at 2862g for 3 min, another sample was taken and named water column Ag (Agwater) that contains all sized fractions 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 which particles smaller than 0.45 mm were present. Finally, the fourth fraction was obtained by centrifuging 10 ml of the water column Ag at 2862g for 30 min using53 kDa AMICON (Merck Millipore, Darmstadt, Germany) centrifugal filters, and this fraction was named the dissolved Ag (Agdis). A scheme of this separation method is presented in Figure 1. Both 0.45 mm and 3 kDa filters were pre-treated with a solution of 0.1 M Cu(NO3)2 to avoid losses of Ag to the filter (Cornelis et al., 2010). To measure Ag elimination from the algae, the remaining algae in the Erlenmeyer flasks were centrifuged, washed three times with Milli-Q water and re-suspended in freshly prepared, un-dosed MBL media for the 48-h elimination phase.

50mL Total Fraction Contains algae cells + water column Ag

Centrifugation 3min, 3500 RPM

Dissolved Ag: Contains soluble Ag species, Including ionic Ag+

Small particulate Ag: Contains dissolved Ag, ionic Ag and small particulate Ag (i.e. AgNP, AgCl and Ag macromolecule complexes below 450nm)

Water column Ag: Contains dissolved Ag and particulate Ag (i.e. AgNP, AgCl and Ag macromolecule complexes)

Algae pellet: washed 3x with pure water (measured) – contains algae without external Ag

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Coherent Anti-stokes Raman Scattering microscopy

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In order to investigate whether Ag was taken up by the algae in its nanoparticle form or in the dissolved or ionic form (Ag+), another experimental section, apart from the bioconcentration assay was performed. Different treatments were set up to be imaged by CARS microscopy. Algae was previously exposed to both AgNP and AgNO3 at 15 mg Ag/l and sampled accordingly. The treatments consisted of: (1) suspended algae was sampled and centrifuged at 2862g for 3 min, then washed three times with Milli-Q water (exactly the same procedure that was used for the bioconcentration tests); and fixed in a single-strength glutaraldehyde fixative (4%) in cacodylate buffer at room temperature for 4 h; (2) algae was sampled then centrifuged at 2862g for 3 min (without pellet washing) and proceeded to fixation; (3) suspended algae cells were straight placed in the fixative (without centrifugation or washing). After the fixation period, all different algae treatments were placed on a microscope slide with a cover slip. CARS microscopy Coherent Anti-stokes Raman Scattering microscopy has emerged as a powerful optical microscopy technique with several advantages over conventional biological imaging techniques; label-free contrast, increased depth penetration and reduced phototoxicity (Evans & Xie, 2008; Rodriguez et al., 2006). Additionally, as shown by Moger et al. (2008), CARS has exceptional capabilities for locating NP within biological samples with 3-D sub-cellular resolution. CARS microscopy uses the chemical composition of a biological sample to exploit the different vibrational resonance of molecular bonds to generate image contrast. In the same way that nanoscale materials exhibit unique chemical properties, NPs also have extraordinary optical properties which give greatly enhanced optical responses (Moger et al., 2008; Wang et al., 2011). NPs generate large CARS signals that are independent of the vibrational frequency being probed and this difference in visualisation between frequency independent NP and specifically tuned vibrational frequencies for biological structures, allows the differentiation of signals from NP from those probed in the sample that is being imaged. As a final advantage the label-free nature of the technique eliminates the chemical perturbation seen when using fluorescent labelling of either NP or staining of surrounding tissues, both of which modify the cellular uptake and observed cytotoxicity of NP. Particle characterisation An aggregation experiment in algae media was performed using dynamic light scattering (DLS) in a Zetasizer nano ZS (Malvern instruments Ltd, Worcestershire, UK) and Zetasizer software 6.20 and processed with the multiple narrow modes algorithm (high resolution) to elucidate multiple peaks in the intensity-based particle size distribution (PSD). Short- and long-term experiments were conducted. For the short-term experiments (minutes), the initial AgNP stock suspension was diluted with the MBL media to the desired concentrations in polystyrene cuvettes (Malvern instruments Ltd, Worcestershire, UK) and inserted immediately in the instrument. The measurement was started at a fixed attenuator and measurement position to avoid the optimisation time, the correlation time was set to 2 s and 120 data points were generally obtained. For the long-term experiments (days), the first measurement (day zero) was obtained by creating an average result from the short-term data points. The cuvettes were stored in the dark and three measurements were performed (three runs of 20 s each) in the following days. To evaluate the effect of particle sedimentation, the samples were shaken after performing the

Nanotoxicology, 2015; 9(6): 686–695

measurement and a new measurement was done. Derived count rates were included in the long-term experiments to compare the capacity of the remaining particles (large and small) to scatter light. For transmission electron microscope (TEM) imaging, an initial suspension of AgNP (1000 mg Ag/l) was diluted to 100 mg Ag/l in MBL media and a drop of this suspension was deposited onto a holey carbon-coated Cu-TEM grid and dried at room temperature for several hours before examination. Experiments were 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 with 0.28 ml of H2O2 and 1.35 ml of HCl, bringing the concentrations to 1% of H2O2 and 5% of HCl for 24 h. This procedure was aimed to break down organic matter in the samples and ensure that any silver adsorbed to the sample holder’s wall was released as soluble AgClx complexes. After 24 h, samples were transferred to Teflon beakers (25 ml volume capacity) and allowed to evaporate on a hotplate over 45– 50  C (without boiling) until 1–1.5 ml of the sample remained in the beakers. Samples were then mixed with 1 ml HNO3 (65% trace analysis) and 3 ml of HCl (37% trace analysis) before being heated for 1 h. All samples were transferred to plastic graduated tubes and diluted with a 1% HCl to a final volume of 45 ml. Three replicates of un-dosed MBL media and three replicates of a known concentration of Ag (50 g Ag/l in Milli-Q water) were digested together with all other samples to be used as blank controls and as a recovery material for Ag measurements, respectively. Total silver was measured on a Perkin Elmer 5100 (PerkinElmer, Waltham, MA) Graphite Furnace Atomic Absorption Spectrophotometer (GF-AAS). Tissue After a three time-cycle of washing with Milli-Q water and centrifugation, the algae pellet was dried at 50  C, weighed and transferred to Teflon beakers for digestion. Three milliliters of HNO3 (65% trace analysis) were added to the beakers, which were heated (with lids on) on a hotplate at 50  C for approximately 30 min (or until the tissue dissolved). Samples were allowed to cool down at room temperature and mixed with 1 ml HCl (37% trace analysis) before being replaced on the hotplate with the lids on and heated for another 30 min. After 30 min, the lids were removed and samples were allowed to evaporate (without boiling) until approximately 1 ml remained. In addition, three replicates of the reference material DOLT-4 (Dogfish Liver Certified Reference Material for Trace Metals) were digested with the other tissue samples. The dilution and Ag measurement steps followed the same procedure as described for water samples. Toxicokinetic modelling In the present study, a one-compartment model was used to describe the kinetics of the bioaccumulation of Ag from AgNP and AgNO3 in R. subcapitata. This model was recently used by Piccapietra et al. (2012) in a similar study, and describes the fate of Ag based on mass balance equations. Assuming that the concentration of exposure would remain constant, two equations were used to model uptake and elimination of AgNP and AgNO3 in the algae:   k1 ð1Þ Uptake: QðtÞ ¼  Cexp  1  eðk2 tÞ k2

Uptake and elimination kinetics of silver nanoparticles by R. subcapitata

DOI: 10.3109/17435390.2014.963724

Elimination: QðtÞ ¼

k1  Cexp  ð1  eðk2 ðttcÞÞ  eðk2 tÞ Þ k2 ð2Þ

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where, Q(t) is the concentration in the algae at time t; tc is the time (hours) at which algae were transferred to uncontaminated media; k1 is the uptake rate constant (l/g/h); k2 is the elimination rate constant (1/h); Cexp is the concentration of exposure (Ag water column concentration measured at time zero); e stands for the exponential function. The bioconcentration factor (BCF) was calculated by the ratio of k1 and k2. The concentration of exposure included in the model was considered as the Ag water column concentration measured at time zero. Considering the decrease in concentration of silver in the media during exposure, a decay rate constant (kdec) was modelled by fitting the following equation to the concentrations of Ag measured at different points in time (Widianarko & VanStraalen, 1996) AgðtÞ ¼ ½Ag  eðkdec tÞ This decay constant was included in the uptake model (Equation (1)) for different fractions to read: QðtÞ ¼

k1  Cexp  eðkdec tÞ  eðk2 tÞ k2  kdec

ð3Þ

where, kdec is the Ag concentration decay rate constant (1/h). Statistical analysis Statistical differences in concentration of Ag in the water for all fractions were detected by a two-way ANOVA, with significant differences established at p50.05. The kinetics parameters standard 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 in water measured at 1 mg/l was 106 nm and showed to be reasonably stable for the duration of the experiment (Table 1 and Figure S1). The minimum concentration that provided a reliable signal from DLS for this dispersion was 1 mg/l, and Figure S1 and Table 1 present the variation of this suspension in MBL during short- and long-term experiments to simulate the exposure conditions. The agglomerate size varied between 200 and 250 nm in the long-term experiments. Ag fractionation in the exposure media Ag concentration in the AgNO3-spiked media decreased until 6 h of exposure, regardless the concentration and the fraction (Agwater and Agsmall part) F4,29 ¼ 2.4 (p40.05) (Figure 2).

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As expected, dissolved (Agdis) concentrations were statistically higher in the media containing AgNO3 (two-way ANOVA F1,19 ¼ 87.4, p50.0001). However, the pattern of decay in concentrations of dissolved Ag arising from AgNO3 was different for different concentrations: at 15 mg Ag/l, dissolved Ag concentration decreased rapidly over the first 6 h of exposure, after which it slightly increased and remained constant until the end of the exposure, whilst at 30 mg/l, concentration decrease remained constant until 12 h of exposure before following a similar pattern as the 15 mg/l treatment (Figure 2). For the media containing AgNP, Ag concentration decreased for both concentrations and fractions (Agwater and Agsmall part.) until 24 h of exposure, after which there seemed to be an increase in concentration again. The small particulate fraction (Agsmall part.) of the 30 mg Ag/l differed, as it remained constant throughout the experiment (Figure 2B). The dissolved Ag concentrations in the AgNP exposure media showed a rapid decrease from zero to 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 slowly at 30 mg/l, whilst at 15 mg/l, it appeared to remain constant. At both exposure concentrations, dissolved Ag concentration showed a slight increase after 24 h. Table 2 present the percentage of Ag loss caused by the washes of the pellet fraction. Those losses were higher in the AgNO3 media, as it is related to the precipitated Ag and/or Ag bound to large organic ligands. Constants of Ag concentration decay in solution are presented in Table 3, isolated for each fraction. For AgNP exposure, the larger the particle size within a fraction, the lower the decay rate constant was, with Agsmall part at 15 mg Ag/l being the fraction with the highest decay rate constant. For AgNO3, the Ag concentration decay rate constants in the exposure media were higher than for AgNP. For both the Agwater and Agsmall part fractions, concentration decay rate constants increased with increasing concentration. On the other hand, for Agdis, a reverse pattern could be observed, with the highest decay rate constant being found at 15 mg/l compared to 30 mg/l. Furthermore, after 24 h of exposure, Agdis from AgNP and AgNO3 reached similar concentrations. Toxicokinetics and Ag internalization Uptake and elimination kinetics of Ag in R. subcapitata were modelled according to the one-compartment model, in which the rate constants of uptake from water (k1) and elimination (k2) were calculated for exposures to AgNP and AgNO3. Assuming that Ag concentration remained constant during the uptake phase, i.e. when uptake was based on the concentration measured in the water column at time zero of exposure, algae accumulated approximately 50 mg/g (dw) of Ag when exposed to AgNP, in comparison to 100 mg/g (dw) upon exposure to AgNO3 at the EC10 concentration level (Figures 3 and 4). In the AgNO3 exposure media, the maximum Ag burden in algae (average) reached 140 mg/g (dw) (SE ¼ 16.6) at 15 mg Ag/l after 12 h, whereas at 30 mg/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 0 1.3 3.0 3.0 (after shaking)

Average Z-hydrodynamic diameter, nm

SD, nm

PDI

Peak 1, nm

Peak 2, nm

Peak 3, nm

Derived count rate, kcps

SD, kcps

106.3 178.0 242.3 204.4

9.2 7.3 26.7 11.9

0.209 0.371 0.348 0.351

111.2 195.6 197.5 201.3

50.4 53.7 37.5 1065.0

1221.0 25.0 24.1 0.0

1625.9 2368.8 1082.7 1082.6

220.4 74.6 64.8 51.1

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

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Ag concentration (µg/L)

30 (A)

30

(B)

AgNP

25

25

20

20

15

15

10

10

5

5

AgNO3

Water column 15µg/L Water column 30µg/L Small particulate 15µg/L Small particulate 30µg/L

0

0 0 1.0

6

12

18

24

30

36

42

0

48 10

(C) Dissolved 15µg/L

0.8

6

12

18

24

30

36

42

48

6

12

18

24

30

36

42

48

(D)

8

Dissolved 30µg/L

0.6

6

0.4

4

0.2

2 0

0.0 0

6

12

18

24

30

36

42

0

48 Time (hours)

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 between figures C and D. Table 2. Silver loss percentages at each sampling time. Nominal Ag (mg/l)

Time (hours)

Ag Loss (%) AgNP

Ag Loss (%) AgNO3

15

6 12 24 48

17 37 21 14

62 67 68 55

30

6 12 24 48

21 37 31 40

64 62 56 84

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

342 mg/g (dw) (SE ¼ 40.9). For AgNP, the Ag burden in algae reached 45.0 mg/g (dw) (SE ¼ 1.10) after 24 h of exposure to 15 mg/l and 93.7 mg/g (dw) (SE ¼ 8.49) after 24 h of exposure to 30 mg/l. When related to time zero water column concentration of exposure, uptake and elimination rate constants were lower in the AgNP treatments compared to AgNO3 (Table 4). After including Ag concentration decay rate constants in the model, the pattern observed for nominal concentrations remained, i.e. for all fractions (Agwater, Agsmall part and Agdis) BCF values were higher for AgNO3 than for AgNP exposure (Table 4). See supporting information (S2 and S3) for kinetics curves from each Ag fraction. Strictly focusing on BCF values calculated based on the small particulate fraction (Agsmall part), the difference between AgNP and AgNO3 was less for the other fractions.

Regarding CARS images interpretation, reconstructing multiple series of images separated by 0.25 mm in the z-plane into a three-dimensional image (Figures 6 and S4) provided no evidence of AgNP internalisation neither in the washed algae nor in the unwashed algae samples. The AgNP agglomerates changed in size according to the sample preparation method, with unwashed preparations displaying larger visible aggregates outside the cell.

Discussion This work focused on understanding the uptake and bioconcentration of Ag from different sources in a freshwater algae species R. subcapitata by comparing exposures to AgNP and to AgNO3. In order to better understand how the chemical reactivity of Ag with other constituents of the media and test conditions could influence uptake, the toxicokinetics were modelled by taking into account different sizes of Ag complexes, from large particle agglomerates/aggregates to the dissolved species. The aggregation rates of the AgNP in MBL were low and they were expected to be lower in the actual algae exposure because the number of concentrations are two orders of magnitude lower, leading to fewer collisions. Moreover, the organic nature of the nanoparticle coating material ameliorated their stability by preventing ionisation with the constituents of the algae media. From the fractionation analysis of AgNP-spiked exposure media, it was found that larger sized fractions decreased in concentration up to 24 h of exposure. In AgNO3 spiked media, however, those same fractions decreased rapidly during the first 6 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 increased exponentially as shown in Figures 3 and 4. Therefore, it can be assumed that this decrease in Ag concentration was due to both

Uptake and elimination kinetics of silver nanoparticles by R. subcapitata

DOI: 10.3109/17435390.2014.963724

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Table 3. Decay constants (kdec; mg/l/h) of Ag in MBL media, according to the fraction of AgNP and AgNO3 along a 48 h of exposure period. AgNP Agwater 15 mg/l 30 mg/l

Agsmall

0.01 (0.007) 0.02 (0.007)

AgNO3 Agdissolved

Agwater

0.18 (0.08) 0.14 (0.09)

0.24 (0.08) 0.31 (0.09)

part

0.03 (0.009) 0.04 (0.007)

Agsmall

Agdissolved

part

0.34 (0.08) 0.72 (83)

0.42 (0.08) 0.30 (4.7)

Standard errors of the parameters are presented in the parenthesis.

EC10

[Ag] algae (µg/g dw)

AgNO3

175

175

150

150

125

125

100

100

75

75

50

50 25

25

0

0 0

24

48

72

96

120

144

0

168

24

48

72

96

120

144

168

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

EC50

AgNP

AgNO3

450 450

400

400 350 [Ag] algae (µg/g dw)

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AgNP

350

300

300

250

250

200

200

150

150

100

100

50

50 0

0 0

24

48

72

96

120

144

168

0

24

48

72

96

120

144

168

Time (hours)

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

settling of Ag agglomerates/aggregates to the bottom of the experimental vessel and uptake of Ag into the algae cells. Nonetheless, considering the system dynamics, the decrease in the small particulate Ag (Agsmall part.) concentrations was more pronounced for the AgNO3 treatment than for AgNP, reaching 2 mg/l after 6 h. As Agsmall part contained dissolved Ag, Ag complexes and Ag bound to macromolecules (such as EPS), the rapid decrease in Agsmall part concentration in the AgNO3

treatment is likely to be associated with a decrease in its bioavailability to algae. This was not observed for the AgNP exposure media due to the presence of small particles (5450 nm) in the Agsmall part fraction, which could have been internalised by the algae. According to Visual Minteq speciation calculations at low dissolved Ag concentrations, chloride is the most important ligand from all components in the media and it is only after 70 mg/l

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Table 4. Kinetic parameters for Ag uptake and elimination in R. subcapitata estimated by Equation (3) for each fraction of AgNP and AgNO3. 15 mg/l Agtime

zero

Agwater

Agsmall

30 mg/l part.

Agdis

Agtime

zero

Agwater

Agsmall

part.

Agdis

AgNP k1 k2 BCF

0.14 (0.5) 0.02 (0.4) 7

0.51 (0.2) 0.13 (0.8) 3.9

0.42 (0.2) 0.07 (0.08) 5.8

0.58 (0.2) 0.01 (0.008) 75.0

0.15 (0.5) 0.02 (0.3) 7.5

0.39 (0.2) 0.07 (0.9) 5.7

0.41 (0.2) 0.05 (0.9) 7.6

0.53 (0.2) 0.01 (0.07) 45.9

AgNO3 k1 k2 BCF

6.34 (9.7) 0.81 (2.3) 7.8

3.02 (0.5) 0.03 (0.02) 100

3.76 (0.5) 0.03 (0.02) 125

4.40 (0.5) 0.02 (0.02) 220

4.1 (4.7) 0.47 (1.1) 8.7

4.32 (0.6) 0.03 (141) 144

7.87 (0.6) 0.02 (83) 393

4.79 (0.4) 0.03 (4.7) 159

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Agtime zero was calculated based on the average concentrations at the initial phase of exposure. The kinetics parameters and BCF values of the Agtime zero are related to the Figures 4 and 5. Agwater was based on the total fraction, Agsmall part. was obtained by the 0.45 mm-filtered fraction (small particulate 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 kinetics curves for the other fractions are presented in the supplementary material.

Ag+ concentration that AgCl(s) starts to form. Unfortunately, due to a lack of information regarding the exudates from the algae, it is not possible to elucidate the relative importance of the latter as ligands for Ag+. Similarly, it has been shown previously that different types of natural organic matter (NOM) interact with the surface of NP with different mechanisms leading to further stabilisation (Gallego-Urrea & Holmberg, 2014), dissolution (Hering, 1995) or aggregation (Zhang et al., 2009) depending on the NOM/NP ratio. In addition, as demonstrated by Lee et al. (2004), the uptake of Ag by R. subcapitata was not influenced by the concentration of chloride in the media, meaning that algae can readily internalise AgCl complexes. Dissolution or release of dissolved silver species was also measured over time for both exposure media containing AgNP and AgNO3. Dissolution was observed to occur faster for the AgNO3 media compared to AgNP, as shown in Figure 2. For both treatments at 15 mg/l, the concentration of dissolved silver decreased rapidly in the first 6 h before levelling off. However, at 30 mg Ag/l, dissolved Ag from AgNP and AgNO3 continued to decrease 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 of dissolved Ag released from AgNP increased exponentially during the first 6 h of exposure. However, that study was performed in deionised water, whilst our experiment used a culture media in the presence of algae. Considering that the internal concentration of Ag in R. subcapitata increased for both treatments during the first hours of exposure (Figures 3 and 4), it is possible that the algae were readily taking up most of the released Ag in its dissolved form, therefore leading to the decrease in Ag concentrations in the test media. The decay of dissolved Ag correlated with the maximum Ag measured in algae in the AgNO3 media, where the concentration of Agdis decreased with time until the 6th hour of exposure (at EC10 level), while a maximum concentration in algae was reached after 12 h of exposure. This may indicate a transfer of Agdis from the media to the algae. Moreover, algae dosed with AgNO3 seemed to have a faster uptake of Ag during the first hours of exposure and started to eliminate Ag already during the uptake phase. This indicates that equilibrium between uptake and elimination was not reached and that Ag was continually being internalised and eliminated by the algae at varying rates before the cells were transferred to Ag-free media. Assuming that Ag uptake followed a first-order kinetics model and 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 Ag fractions. These parameters, presented in Table 4, will hereafter be used to guide our interpretation of the Ag toxicokinetics in algae. The uptake rate constants related to all Ag fractions remained higher in the AgNO3 exposures, while the elimination rate constants based on Agwater and Agsmall part. were lower for AgNO3. It has been demonstrated that the mechanism behind Ag internalisation in algae is related to accidental cation transport, which is believed to occur through the same mechanism as the internalisation of essential cations (i.e. Na+, K+). This is due to the inability of the system to distinguish between Cu+ and Ag+, given that both metals share some chemical characteristics (Lee et al., 2004; Solioz & Odermatt, 1995). Additionally, AgCl0 is likely to be internalised through passive diffusion from the external cell environment to the cytosol, over protein channels in membranes (Lee et al., 2004). Either mechanism could explain the higher k1 values obtained for AgNO3 exposures as, in this case, Ag is more likely to be present as dissolved and consequently in a form that could be easily internalised by the algae. On the other hand, lower elimination constants indicate that the algae failed to completely excrete Ag either as dissolved or small particulate forms. Ag is known to be associated with metallothioneins (MT) (Robinson, 1989), thus it is likely that dissolved Ag in algae is sequestrated and bound or stored in such a way that it cannot be eliminated anymore, or it is eliminated in a rather slow rate. Such binding to MT is less expected for Ag complexes or particles. The BCF of AgNP was the highest when we considered the dissolved fraction, which corroborates the hypothesis that algae would take up dissolved Ag and/or Ag complexes from the media rather than Ag particulate forms. Moreover, the highest BCF value was obtained at the lowest AgNP concentration (EC10 level) indicating that dissolution rates of AgNP were more efficient at lower concentrations and consequently at lower aggregation rate of particles probably driven by a steeper concentration gradient and larger surface area exposed (less aggregation). This was also reported by Kittler et al. (2010), who observed that PVP-coated AgNP showed a higher dissolution at 0.05 g/l when compared to 0.1 g/l, after approximately 100 days in a long-term dissolution experiment. The lower BCF values obtained when using the water column and small particulate fractions from AgNP were interpreted as an indication of the behaviour of AgNP in the exposure media. As mentioned before, algae is known to secrete exudates that are mainly composed of organic matter, rich in polysaccharide molecules, which in turn can induce aggregation of AgNP,

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

altering their bioavailability to algae (Joshi et al., 2012). Based on our results, we can speculate that in this instance, exopolymeric substances produced by algae may have played a key role in decreasing the bioavailability of AgNP to the cells. By using the CARS microscope imaging technique, R. subcapitata exposed to AgNP showed no evidence of internalisation of the nanoparticles into the algal cells used in this experimental setup. Aggregates varying in size could be visualised externally, with the size of the aggregate being reduced in 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 were shown to be from outside the cell after 3D image sectioning with CARS, confirming that no particles were up taken by the algae (Figures 6 and S4). This suggests that internalised Ag was in the form of ionic or dissolved Ag. However, the nanoparticle agglomerates nearby the algae cells may induce a physical effect on algae, depending on agglomerate size and may interfere with the algae growth and/or lead to a faster sedimentation of cells. In addition, shading effects and induced higher local concentration could also play a vital role in toxicity, depending on the ability of the nanoparticle to attach to the cell surface. NP may thus interfere with photosynthesis and/or by their chemical characteristics the nanoparticles may induce the formation of a turbid media, which can suppress the light absorbance by algae (Aruoja et al., 2009; Schwab et al., 2011). Thus, our data highlight the importance, when predicting the potential risk of nanoparticle

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

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

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Conclusions In conclusion, we have demonstrated that the separation into different size fractions revealed to be a trustworthy tool to study silver chemical behaviour in our test media (MBL) and helped to estimate the relationship between silver behaviour and its bioavailability to algae. The amount of silver taken up by the algae 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 from AgNP, a higher value was obtained in comparison to the other Ag-sized fractions, indicating that upon AgNP exposure silver was internalised as Ag+ or dissolved Ag rather than in its particulate form. This was also confirmed by the CARS images showing that AgNP used in this study were unable to cross algae cell walls, which lead us to conclude that bioconcentration of AgNP is probably mediated by the internalisation of dissolved or/and ionic Ag.

Acknowledgements This study was partly supported by the project NanoFATE, financed by the FP7 Programme, European Commission (CP-FP 247739 NanoFATE), and by funding FEDER through COMPETE and Programa Operacional Factores de Competitividade and by the Portuguese National funding through FCT – Fundac¸a˜o para a Cieˆncia e a Tecnologia, within the research project FUTRICA – Chemical Flow in an Aquatic TRophic Chain (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 paper received support from the QNano Project (http://www.qnano.ri.eu), which is financed by the European Community Research Infrastructures under the FP7 Capacities Programme (Grant No. INFRA-2010-262163), and its partner the University of Exeter, within the UOE-TAF-42: The internalisation of Ag and ZnO NPs in aquatic and terrestrial organisms (Susana Loureiro). The authors would also like to thank Mr Rudo Verweij for the AAS measurements and the anonymous reviewers for their relevant comments, which highly improved the quality of this paper.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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

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