Differential lethal and sublethal effects in embryonic zebrafish exposed to different sizes of silver nanoparticles

Environmental Pollution 248 (2019) 627e634

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Differential lethal and sublethal effects in embryonic zebrafish exposed to different sizes of silver nanoparticles* Xiaobo Liu a, 1, Eduard Dumitrescu a, 1, Ajeet Kumar a, Daniel Austin a, Dan Goia a, Kenneth N. Wallace b, Silvana Andreescu a, * a b

Department of Chemistry & Biomolecular Science, Clarkson University, 8 Clarkson Avenue, Potsdam, NY, 13699-5810, USA Department of Biology, Clarkson University, 8 Clarkson Avenue, Potsdam, NY, 13699-5805, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 September 2018 Received in revised form 20 February 2019 Accepted 24 February 2019 Available online 27 February 2019

Various parameters can influence the toxic response to silver nanoparticles (Ag NPs), including the size and surface properties, as well as the exposure environment and the biological site of action. Herein, we assess the intestinal toxicity of three different sizes (10, 40, and 100 nm) of Ag NPs in embryonic zebrafish, and describe the relationship between the properties and behavior of Ag NPs in the exposure medium, and induction of lethal and sublethal effects. We find that the composition of the medium and the size contribute to differential NPs agglomeration, release of Ag ions, and subsequent effects during exposure. The exposure medium causes dramatic reduction in silver dissolution due to the presence of salts and divalent cations, which limits the lethal potential of silver ions. Lethality is observed primarily for embryos exposed to medium sized Ag NPs (40 nm), but not to the supernatant originated from particles, which suggests that the exposure to particulate silver is the main cause of mortality. On the other hand, the exposure to 10 nm and 100 nm NPs, as well as Ag ions, only causes sublethal developmental defects in skeletal muscles and intestine, and induces a nitric oxide imbalance. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Zebrafish embryos Silver nanoparticles Nitric oxide Dissolution Intestinal defects

1. Introduction The rapid development of nanotechnology has stimulated the use of nanoparticles (NPs) in various fields. Due to their small size, NPs possess unique physicochemical and catalytic characteristics, compared to their bulk counterparts. These properties have attracted enormous scientific and technological interest for use in functional materials and devices. NPs have been successfully incorporated into consumer products, such as cosmetics and clothes (Lai, 2012), drug delivery vehicles (Mukhopadhyay et al., 2012), and microelectronics (Lai et al., 2014). Silver nanoparticles (Ag NPs) are some of the most widely used NPs in consumer applications (Lem et al., 2012), due to their well-known bactericidal effects (Morones et al., 2005; Rai et al., 2009; Marambio-Jones and Hoek, 2010). As of 2013, silver was accounted for about 24% of the commercial products containing at least one nanosized material (Vance et al., 2015). While largely used, the consequences of NPs

*

This paper has been recommended for acceptance by Baoshan Xing. * Corresponding author. E-mail address: [email protected] (S. Andreescu). 1 These authors contributed equally.

https://doi.org/10.1016/j.envpol.2019.02.085 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

release in the environment are still unknown and subject to debate. Ag NPs exposure takes place through skin, inhalation, or ingestion (Vance et al., 2015). Ag NPs may be discharged in the environment during synthesis, manufacturing, or use of silver-containing consumer products (Fabrega et al., 2011; McGillicuddy et al., 2017; Gottschalk et al., 2009), resulting in release and accumulation in the aquatic ecosystems (Fabrega et al., 2011; Moore, 2006; Johnston et al., 2010; Massarsky et al., 2014). Toxicity of Ag NPs has been investigated using a number of different model systems, from small biological models such as cells (AshaRani et al., 2008; Guo et al., 2016) to more complex models, like murine models (van der Zande et al., 2012). In spite of the amount of published research, the understanding of the mechanism of action is hindered by variability of the NPs characteristics used in the exposure studies, including size (Powers et al., 2011; Browning et al., 2013; Bar-Ilan et al., 2009), shape (George et al., 2012), coating (Powers et al., 2011), dissolution profile (Cunningham et al., 2013), surface interaction (Gupta et al., 2016), contact time (Barker et al., 2018). bioaccumulation (Ribeiro et al., 2017), and transformation in realistic environmental conditions. These factors may change the toxicity profile and the interpretation of the results (Badawy et al., 2010; Riaz Ahmed et al., 2017; Ki-Tae

628

X. Liu et al. / Environmental Pollution 248 (2019) 627e634

et al., 2013; Truong et al., 2012). In addition, the biological model used and the contribution from the silver speciation can also impact the toxicity outcome (Beer et al., 2012; Lubick, 2008). Previous research showed that Ag NPs toxicity could be attributed to either silver ions (van Aerle et al., 2013), particulate silver (Kim et al., 2009). or a mixed synergistic mechanism (Navarro et al., 2008; Kang et al., 2016; Ribeiro et al., 2014). Several studies identified the production of reactive oxygen and nitrogen species (ROS/ RNS) as possible contributors to toxicity (Kim et al., 2009; Wu and Zhou, 2013; Choi et al., 2010). As dissolution of silver ions and the existence of a particulate silver/silver ions mixture in the exposure medium have been shown to contribute to the Ag NP toxicity, a systematic study taking into account each of these factors in defined media conditions will help to elucidate the factors that are contributing to the lethal and sublethal effects of Ag NPs exposure. The goal of this paper is to study the effect of Ag NPs exposure and assess the localized lethal and sublethal effects of Ag NPs exposure in an aquatic organism model. We use zebrafish (Danio rerio) embryos as a testing organism that shares similar organ structure and function, as well as genetic information, with other vertebrates. To perform exposure studies, we selected Ag NPs of three different sizes (10, 40, and 100 nm) prepared using the same synthetic pathway. Studies were performed to assess: 1) mortality and lethal dose, 2) morphological defects and skeletal muscle structure of embryos through histology, and 3) oxidative stress through in vivo monitoring of nitric oxide (NO) release in the intestine. The physicochemical properties of the three Ag NPs were compared and their effects were related to the composition of the growth medium, formation of agglomerates, as well as the dissolution in the exposure medium. These results contribute to a better understanding of the involvement of particle size and dissolution in the exposure medium in the Ag NPs toxicity targeted at the intestinal level in zebrafish embryos. 2. Materials and methods Materials and Reagents. Carbon fibers (~5 mm diameter), copper wire, and Dumont #5 forceps were obtained from World Precision Instruments. Silver conductive epoxy was purchased from MG Chemicals. Pyrex® glass capillary tubes with the size of 1.5e1.8
100 mm were purchased from Corning Inc. Nonconductive Devcon® epoxy was obtained from ITW Devcon. Nafion (5% mixed in aliphatic alcohol), o-phenylenediamine (o-PD), 2-propanol, and potassium phosphate monobasic were purchased from Sigma-Aldrich. Sodium phosphate dibasic was purchased from Spectrum Chemical Mfg. Corp. Sodium nitrite and calcium chloride were purchased from Acros. Sulfuric acid, sodium chloride, magnesium sulfate, and potassium nitrate were purchased from Fisher Scientific. Potassium chloride and sodium hydroxide were purchased from LabChem Inc. Silver nitrate was purchased from Chemsavers. Deionized (DI) water from Millipore Direct-Q water purification system with a resistivity of 18.2 MU cm was used to prepare all solutions. For silver nanoparticles synthesis, high purity grade silver nitrate crystals were purchased from Ames Goldsmiths. Resorcinol, ammonium hydroxide 30% (Alfa Aesar), sodium hydroxide 10 N (Fisher Scientific Co.), ethyl alcohol (Pharmco Products Inc.), and arabic gum (Flutarom) were all used as received. Instrumentation. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed with an electrochemical analyzer (CH1030a, CH Instruments Inc.). All experiments were carried out with a three-electrode electrochemical cell equipped with a Ag/AgCl/3 M NaCl as the reference electrode and a platinum wire as the counter electrode. The working electrode was a single carbon fiber microelectrode (SCFME) with 5 mm diameter fabricated in our lab. A 3 mm diameter glassy carbon

electrode (CH Instrument) was also used to quantify the extent of Ag dissolution. All potentials were referred to the Ag/AgCl reference electrode. A Narishige PP-83 capillary puller was used in the fabrication of the SCFME. Synthesis of Ag NPs. Silver particles of different sizes were synthesized using a chemical precipitation process reported by Kumar et al. (2016). The detailed method is described in the Supplementary Material. All particles were synthesized using the same chemical precipitation process (Kumar et al., 2016), involving silver ammonia complex reduction with resorcinol in presence of Arabic gum. Tailoring the size of the particles was performed by increasing the addition rate of reactants as described (Kumar et al., 2016). The synthetic procedure yields stable Ag NPs and any potentially toxic solvents or by-products are removed. Characterization of Ag NPs. The size and morphology of Ag NPs were assessed by field emission scanning electron microscopy (FESEM). Samples of Ag NPs were dispersed and directly placed on a copper grid, followed by drying under vacuum. Their degree of dispersion was assessed by comparing the SEM size and the average particle size determined by dynamic light scattering (DLS) analysis. The surface charge of Ag nanoparticles was measured with a ZetaPals instrument (Brookheaven Instruments). Particles (1 mg/ml concentration) were analyzed in both deionized water and E3 medium for DLS size and zeta potential. The optical properties were monitored as a function of time with a PerkineElmer Lambda 35 UVeVis spectrophotometer. Small dispersion aliquots (1 ml) removed from the reaction vessel were first diluted in a 50 ml volumetric flask and then transferred in the 10-mm optical path length quartz cuvette. The scanning range used was from 200 nm to 1100 nm. The surface charge of Ag nanoparticles was measured with a ZetaPals instrument (Brookheaven Instruments). The structure of particles was investigated by X-ray diffraction (XRD) using a Bruker D8 instrument. For the diffraction pattern acquisition, the step width and period were 0.02 and 1.5 s respectively, while the source, sample, and detector slits were 2, 0.6, and 1 mm. The Scherrer equation was used to calculate the size of the constituent crystallites. Zebrafish Husbandry. Fish maintenance and mating were performed as previously described (Westerfield, 1993). Adult AB wild type zebrafish were maintained at 28.5  C on a 14 h light/10 h dark cycle in an Aquatic Habitats recirculating system. Water was purified through reverse osmosis and pH was adjusted to 7.0 with conductivity of 350 ms. Zebrafish eggs were collected right after fertilization and grown in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4, pH 6.9e7.2). At 24 h postfertilization (hpf), the embryos were manually dechorionated to facilitate direct exposure and ingestion of the NPs by the embryos without the protective effect of the chorion. Exposure experiments were performed after 24 hpf, when endodermal cells have recently migrated to the midline and have begun to organize into the future digestive tube. During the selected exposure period, the intestine is exposed to nanomaterials externally and internally, without affecting earlier phases of specification and assembly of the cells that will give rise to the organ. The healthy embryos were separated based on their developmental stage examined under an optical microscope (Nikon SMZ1000 Stereomicroscope) and divided into 20 per well in 6-well plates. 3 ml of NPs suspension was added to each well. Viable embryos were recorded every 24 h for four days, until 5 days post-fertilization (dpf). For each type of NPs, seven concentrations, 1, 5, 10, 50, 100, 150, and 200 ppm, were selected for the viability assay. The exposure concentrations are reported as number of particles per volume in Table S1, as calculated based on the primary size measured by FE-SEM. Ag NPs dispersions were replaced every 24 h with a fresh sample from the same stock to ensure consistency in exposure conditions. The LD50 value (the NPs

X. Liu et al. / Environmental Pollution 248 (2019) 627e634

concentration value that causes the death of 50% of the test sample) was calculated according to the method developed by Miller and Tainter (Randhawa, 2009). To assess toxicity of the released silver ions, NP dispersions in similar concentration range were centrifuged at 13400 rpm for 30 min in order to separate most of the particulate silver and the supernatant was used for viability assays. The centrifuged dispersions were replaced daily with newly centrifuged NP dispersion from the same stock. Alternatively, known concentrations of AgNO3 dissolved in E3 medium in the range from 0.2 to 1 ppm were used for comparative investigation and assessment of silver ions effects. To obtain statistically representative data, triplicate sets of experiments were carried out for each exposure condition. All animals were handled in strict accordance with good animal practice as defined by national (NIH Office of Laboratory Animal Welfare) and local (Clarkson University Institutional Animal Care and Use Committee) bodies, and all work was approved by the appropriate committee (Clarkson University IACUC #11-01). Electrochemical Measurement of Dissolved Silver Ions from Ag NPs. Silver ions were measured by anodic stripping voltammetry using a reported procedure (Jiao et al., 2015). Briefly, a threeelectrode electrochemical cell was used with a glassy carbon electrode as working electrode. Potassium nitrate was used as electrolyte in order to avoid silver precipitation as insoluble salt. The anodic stripping voltammetry protocol consisted of an accumulation step, when the electrode was kept at a constant potential of 0.55 V vs Ag/AgCl in a stirred solution, and a stripping step, when the potential was linearly swept between 0 and 0.6 V vs Ag/ AgCl, at 0.1 V/s. The oxidation peak observed corresponding to the oxidation of silver reduced on the electrode was quantified by measuring the corresponding charge. For calibration purpose, standard solutions of AgNO3 were prepared in DI water. Dissolution of Ag NPs was assessed at 0 h, 24 h, and 72 h in the supernatant of Ag NPs samples centrifuged at 13.4k rpm for 30 min. For assessment of silver ions concentrations in AgNO3 solutions prepared in E3 medium, selected samples were centrifuged at 13.4k for 30 min, and the supernatant was used for silver ions quantification. Histological Study. 5 dpf zebrafish embryos were fixed in 4% paraformaldehyde, dehydrated in methanol, and embedded in methacrylate using a JB-4 kit (Polysciences, Inc.). 5 mm thick sections were cut using a Leica microtome. Sections were stained in 0.1 mg/ml Azure II-methylene blue for 25e30 s and mounted in Permount (Fisher). The samples were observed using a Zeiss AX10 Lab A1 optical microscope and pictures were taken with the Lumenera Infinity Analyze software. In Vivo Measurement of Intestinal NO in Embryonic Zebrafish. In vivo measurement of intestinal NO was performed using € SCFMEs fabricated using a previously described protocol (Ozel et al., 2013). NO measurements and calibration of the modified SCFME were performed using DPV. All measurements were performed in E3 medium, the growth medium for embryonic zebrafish. To calibrate the sensor, NO standard solution of known concentration was added to the E3 medium and the NO oxidation current was measured using DPV in the potential range from 0.2 to 1.2 V (vs. Ag/AgCl), with a scan increment of 0.004 V. The pulse amplitude was 50 mV, the pulse width 50 ms, and the pulse period 200 ms. NO standards were synthesized based on a previously reported procedure (Diab and Schuhmann, 2001), using an adopted € experimental setup (Ozel et al., 2013). In vivo measurements were performed on live 5 dpf zebrafish embryos. Before measurements, the embryos were washed twice in E3 medium and immobilized on an agarose gel plate to minimize movement of embryos. NO measurements with the SCFME were performed with the electrode inserted into the middle segment of the intestine of the embryo. During the entire process of the in vivo measurement, no sedatives

629

are added to the embryo. Statistical Analysis. All the measurements and viability assay experiments were performed in triplicate. Reported data are replicates from three independent zebrafish cohorts measured in three wells per treatment. Results are expressed as mean ± standard deviation (n ¼ 3). Statistical significance was evaluated using oneway ANOVA with post-hoc Tukey HSD. Asterisks denote statistically significant results as a function of control experiments. One (*) and two (**) asterisks indicate statistical significance at p < 0.05 and p < 0.01 respectively. 3. Results Characterization of Ag NPs. Three different sizes of Ag NPs were studied to assess the biological and toxicological effects of nanosilver on embryonic zebrafish. Since all particles were obtained using the same procedure, we assume that they have the same surface coating, likely the Arabic gum, which provides protection against agglomeration. SEM images of the three types of Ag NPs reveal uniform spherical shape particles with sizes of 10, 40, and 100 nm (Figure S1). The UVevis analysis (Figure S2a) shows a sharp plasmon band and demonstrates that the particles are uniform and highly dispersed. X-ray diffraction analysis (Figure S2b) shows well defined peaks corresponding to the face centered cubic (FCC) structure of metallic silver (JCPDS File No. 04e0783). DLS measurements and zeta potential analysis of the NPs in DI water listed in Table 1 confirm the particle sizes and show a negative surface charge for all three types of NPs tested. Analysis performed in the E3 medium illustrates agglomeration of the NPs with a significant increase in size (2e6 fold) for all the three types of particles tested as compared to the particles in DI water. However, the Ag NPs in E3 medium maintain a high negative zeta potential for all sizes, confirming the stability of the particle dispersion in spite of the agglomeration effect. Furthermore, the variation of zeta potential in comparison with the Ag NPs in deionized water suggests transformation of particles surface when dispersed in E3 medium. Agglomeration of NPs in E3 medium was reported in previous studies (Ozel et al., 2014). The size distribution histograms measured by DLS (Figure S3) illustrate that the agglomeration is not complete and a proportion of the dispersion also contains particles with hydrodynamic diameters close to the primary size. Differential Dissolution of Ag NPs in E3 Medium versus DI Water. Dissolution of Ag ions from Ag NPs is believed to be one of the main mechanisms of toxicity (Powers et al., 2010). Due to size differences, there may be a variation of Ag ion concentrations dissolved from the different types of Ag NPs. To quantify the availability of Agþ due to dissolution in the exposure medium, a stripping electrochemical procedure was used (Jiao et al., 2015). The calibration curve with a linearity range from 0.5 ppb to 1 ppm is presented in Figure S4. Dissolution was followed over a period of 72 h, corresponding to the incubation of NPs in the E3 medium during embryo exposure. The amounts of silver ions detected for dissolution of 10 nm Ag NPs at any concentration were under the

Table 1 Comparison of primary particle size diameter measured by FE-SEM with the hydrodynamic diameter measured by DLS, and zeta potential values of various Ag NPs in DI water and E3 medium. Nanoparticles size

In deionized water

In E3 medium

Size (nm)

Zeta potential (mV)

Size (nm)

10 nm 40 nm 100 nm

10 ± 2 40 ± 2 100 ± 2

28 19 21

44.7 ± 3.9 18.8 ± 1.1 88.4 ± 0.5 28.3 ± 3.9 607.5 ± 20.5 35.2 ± 2.1

Zeta potential (mV)

630

X. Liu et al. / Environmental Pollution 248 (2019) 627e634

0.5 ppb detection limit of the electrochemical method, which could be explained by the transformation of NPs in the exposure media leading to limitation of dissolution. In comparison, 40 and 100 nm particles exhibit a time dependent dissolution (Fig. 1 a, c) during the first 3 days, in the low ppb range. A maximum concentration of ~35 ppb Agþ was observed for the highest NPs concentration of 200 ppm. Overall, the dissolution rate reached a maximum during the first 24 h. Interestingly, the dissolution profile of the NPs dispersed in DI water showed significantly higher release of Ag ions (Fig. 1 b, d) as compared to the particles in E3 medium. Dissolution in DI water was about 100 times higher for the 40 nm Ag NPs and 10 times for the 100 nm, at the highest NPs concentration tested. High concentrations of chloride and sulfate ions in E3 medium can determine formation of insoluble silver salts at the surface of NPs, preventing further dissolution of Ag ions. Preparation of a solution of Agþ by dissolution of AgNO3 in E3 medium created a visible precipitate, likely AgCl. When tested by electrochemistry using anodic stripping voltammetry, the measured concentration of Agþ in the AgNO3 solution prepared in E3 medium was significantly lower than the theoretical value (Figure S5). These results suggest the E3 medium significantly lowers the dissolution rate of Ag NPs. Effects of Ag NPs Exposure in Zebrafish: Viability Assay. Zebrafish embryos were exposed to the three different sizes Ag NPs to determine whether there is a relationship between lethality and

particle size. Zebrafish embryos were exposed to various concentrations of 10 nm, 40 nm, and 100 nm Ag NPs dispersed in E3 medium, beginning at 24 hpf and incubated for 4 days (Fig. 2). No significant lethality was observed for the 10 and 100 nm particles at any tested concentration; however, decreased viability was observed with 40 nm particles during the 4 days exposure (LD50 of 104.63 ± 0.43 ppm) beginning at concentrations of 100 ppm and higher. During the 40 nm NPs exposure, the only statistically significant period of embryo death was observed during the last 24 h (between 96 and 120 hpf), which is when the intestine opens to the exterior and embryos begin to ingest material from the environment. No significant lethality was observed for the soluble constituents of the 10 and 100 nm NPs dispersion, and a slight decrease for the 40 nm particles, after the solid particles were separated by centrifugation (Figure S6). Exposure to soluble ions in the concentration range similar to those released by Ag NPs in E3 medium also showed no significant decrease in viability (Figure S7). The detailed viability numbers are presented in Figure S8. Histological Comparison of Ag NP Exposure to Soluble Ag Ions during Embryonic Development. To identify the effect of Ag NPs on the intestinal lumen and annex tissue, embryos exposed to 10 nm, 40 nm, and 100 nm Ag NPs from 24 hpf to 120 hpf were cross-sectioned at the end of exposure protocol. Previously, accumulation of NPs within the intestinal lumen with different sizes of

Fig. 1. Dissolution profiles of the 40 nm (a, b) and 100 nm (c, d) Ag NPs in the E3 medium (a, c) and in DI water for comparison (b, d). The dissolution profile of 10 nm Ag NPs in E3 medium and DI water exhibited concentration values under the detection limit (0.5 ppb) for all concentrations and time points. The average percentage values were calculated from 3 replicate experiments. Error bar represents standard deviation for (n ¼ 3).

X. Liu et al. / Environmental Pollution 248 (2019) 627e634

Fig. 2. Day 5 viability of zebrafish embryos exposed to various size and concentrations of Ag NPs during 5-day embryogenesis. The average percentage values were calculated from 3 replicate trials of 20 embryos per each dose. Error bars represent standard deviation (n ¼ 3). One (*) and two asterisks (**) indicate p < 0.05 and p < 0.01, respectively.

nickel NPs was visualized in histological sections (Ispas et al., 2009). As the dye is used to stain the biological tissue, accumulation of silver nanoparticles or other insoluble particulate silver can be observed due to color contrast. Histological cross sections of 120 hpf embryo exposed to 10 and 40 nm Ag NPs (Fig. 3 g, h) reveal occasional agglomerates of insoluble silver particulates within the intestinal lumen, while 100 nm Ag NP exposures have no accumulation of exposure materials within the intestinal lumen (Fig. 3 d, i). These results suggest that the retention of particulate silver at the intestinal level is size-dependent, with the large agglomerates formed by the 100 nm Ag NPs in E3 medium being unable to access the intestine in comparison with the 10 nm and 40 nm Ag NPs. Histological cross-sections also identify defective organ development related to Ag NP exposure. While there are no observed defects in 40 nm Ag NPs exposures, there were developmental defects in intestine and skeletal muscle of embryos exposed to 10 nm and 100 nm NPs, as well as soluble Ag ions (Fig. 3). Skeletal muscles exhibit a rarefied structure, while the intestinal folds are not well-defined. Since exposure to 10 nm and 100 nm Ag NPs or Ag ions demonstrate no increase in lethality, the alteration in development of skeletal or intestinal organs appear to be sublethal defects. The lack of defects was particularly surprising in embryos exposed to 40 nm Ag NPs, which have statistically significant

631

lethality by 120 hpf. The structure of skeletal muscles is intact, as it was observed for control samples, and the intestinal folds do not show structural defects. Ag NPs and Silver Ions Alter Intestinal NO Levels in Exposed Embryos. To determine whether the increased toxicity observed with 40 nm Ag NP exposure is related to higher levels of oxidative € stress, a SCFME (Ozel et al., 2013) was used to measure the intestinal NO concentrations in the intestine of 5 dpf zebrafish embryos (Fig. 4a). NO is a marker for nitrosative stress and an indicator of € oxidative stress in nanotoxicity studies (Ozel et al., 2014). The NO sensor exhibits a linear detection range from 1 to 40 mM, with a sensitivity of 0.145 nA/mM (Figure S9). The normal physiological intestinal NO concentrations of 5 dpf zebrafish embryos is 1.701 ± 0.180 mM. Exposure to 1, 10, and 100 ppm of each size of Ag NPs shows variable effects on the intestinal NO concentration (Fig. 4 d-f). For exposure to the 1 ppm 10 nm, 40 nm, and 100 nm particles, an increased intestinal NO concentration was measured (37.3% for 10 nm, 18.5% for 40 nm, 27.9% for 100 nm). Similarly, significant NO level increase was observed for the 10 ppm 40 nm and 100 nm Ag NPs exposures (16.2% for 40 nm and 20.1% for 100 nm). While each of the different size Ag NPs exposure reveals a statistically significant increase in NO for the lowest concentrations tested, only the 40 nm particles demonstrate a statistically significant increase in NO for the highest concentration tested (2.249 ± 0.081 mM, 32.3% higher than normal physiological concentration). For comparison, the NO level was also measured in embryos exposed to 0.4 ppm Agþ nominal concentration. For this concentration, the amount of free Agþ ions measured was comparable with that dissolved from 40 nm to 100 nm NPs after 3 days. Exposure to Agþ induced a significant increase in intestinal NO concentration (2.126 ± 0.123 mM, 25% increase) (Fig. 4 c). Therefore, release of Ag ions may induce a statistically significant oxidative stress response within the exposed embryos.

4. Discussion In this report, we expose zebrafish embryos to variable size Ag NPs prepared using the same synthetic procedure. By employing the same reagents in synthesis, the level of toxicity is caused by differences in the size and dissolution profile, rather than the surface coating. Moreover, the preparation procedure involves materials like Arabic gum, which is known to be biocompatible and nontoxic (Roque et al., 2009). The particle size distribution in E3 medium for each type or Ag NPs shows a bimodal profile, with the

Fig. 3. Cross-sections of anterior intestine (aee), posterior intestine (fej) and skeletal muscle (keo) of control zebrafish embryos (a, f, k), embryos exposed to 100 ppm 10 nm Ag NPs (b, g, l), 100 ppm 40 nm Ag NPs (c, h, m), 150 ppm 100 nm Ag NPs (d, i, n), and 0.4 ppm Agþ (e, j, o). NPs accumulation in the intestinal lumen is indicated by red arrows. The scale bar in all pictures represents 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

632

X. Liu et al. / Environmental Pollution 248 (2019) 627e634

Fig. 4. a) In vivo electrochemical NO measurement with an implanted SCFME; b) Representative example of electrochemical DPV measurement; Intestinal NO concentrations measured in 5 dpf embryos exposed to c) 0.4 ppm silver ions, d) 10 nm, e) 40 nm and f) 100 nm Ag NPs measured in vivo using the SCFME sensor. Measurements are performed in triplicate using a similar experimental set up in independently experiments with different exposed embryos. Error bars represent standard deviation (n ¼ 3). One (*) and two asterisks (**) indicate p < 0.05 and p < 0.01, respectively.

majority of particles being found as agglomerates of large size. However, a proportion of Ag NPs are still present as primary particles with diameters as measured by FE-SEM. The significant lethality observed only for exposure to 40 nm Ag NPs (>100 ppm) indicates the involvement of non-agglomerated particles in the observed lethality outcome. We hypothesize that the increased lethality associated with the 40 nm Ag NPs exposure could be due to localization of NPs in the intestine of embryos. Agglomerates of Ag NPs or other particulate silver forms were observed within the intestinal lumen for embryos exposed to 40 nm Ag NPs. Mortality during the 40 nm Ag NP exposure primarily occurs between 96 and 120 hpf, which is when the digestive system opens to the exterior and embryos begin eating. This suggests that ingestion of 40 nm Ag NPs and exposure to the intestine may be one of the mechanisms triggering the observed lethality due to the 40 nm NPs becoming trapped within the intestinal lumen to a higher degree than 10 and 100 nm NPs in the later stages of exposure. Comparable trends as in our work were observed by Bowman et al. (2012) for a similar type of NPs which showed that medium-sized Ag NPs are more prone to exhibit lethality. Overall, Ag NPs with diameters <50 nm have lower LD50 values compared to high-diameter particles (Ki-Tae et al., 2013; Park et al., 2013). Lower LD50 values might be explained by the fact that particles with sizes <50 nm are more easily taken into cells through endocytosis in comparison to their bigger counterparts (Johnston et al., 2010; Chithrani et al., 2006). At the same time, NPs with medium size (~50 nm) enter cells more efficiently than NPs with smaller or larger size via endocytosis (Chithrani et al., 2006; Osaki et al., 2004). If the 40 nm NPs are able to reach the intestinal lumen and contact epithelial cells, it may be sufficient to contribute to the observed mortality. The dissolution of silver ions does not seem to play a role in the mortality observed for 40 nm Ag NPs. While the 40 nm NPs show higher dissolution than 10 or 100 nm Ag NPs, the amount of dissolved ions reaches a plateau in the first 24 h, which is not consistent with the mortality period in the later stages of exposure. Moreover, the viability experiments done using supernatant separated from the Ag NP dispersions confirm no mortality in presence of Agþ ions only. Silver dissolution starting with the earliest time of exposure is one of the factors contributing to existence of sublethal effects. Similar developmental defects in intestine and skeletal muscle are observed during 10 and 100 nm Ag NPs exposure and soluble Ag ion exposure. However, developmental effects are not observed in the case of 40 nm Ag NPs, most probably due to

particles inducing lethality before 120 hpf. Nitrosative stress was also observed to be a sublethal effect of exposure to Ag NPs. Several studies have indicated that oxidative/nitrosative stress, a redox disequilibrium state, is one of the possible effects resulting from NPs exposure (Wu and Zhou, 2013; Yong et al., 2013; Abdelhalim and Jarrar, 2012). High levels of ROS/RNS can cause severe cellular damage, including necrosis and apoptosis (Xia et al., 2006). Both Ag NPs and Agþ exposure induce moderate elevation of intestinal NO concentrations in 5 dpf zebrafish embryos. Similar effect was observed for both the Ag NPs and ions, which suggest that the silver ions are responsible for nitrosative stress generation. Similar findings have been reported before in several studies on zebrafish embryos (Massarsky et al., 2013) or other biological models (Choi et al., 2010; Carlson et al., 2008; Foldbjerg et al., 2009). We conclude that less severe sublethal effects are associated with the exposure to 10 or 100 nm Ag NPs and silver ions in comparison with 40 nm Ag NPs. The environment in which Ag NPs are suspended greatly influences Ag dissolution, surface composition and properties (e.g. formation of AgCl), and behavior of the NPs. As a result, the toxicity of Ag NPs is altered by the exposure medium. Several previous studies reported high dissolution rates for Ag NPs in aqueous media (Levard et al., 2011; Liu and Hurt, 2010; Kittler et al., 2010). We find that the dissolution rate of the Ag NPs in E3 medium is significantly lower than that in DI water. The dissolution process in E3 medium is time-dependent and the dissolution rate decreases drastically after the first 24 h. By comparison, the concentration of dissolved silver increases up to 10e100 times when NPs are dispersed in DI water. The 10 nm particles show no significant dissolution during the timeline of exposure in the E3 medium, while the 40 and 100 nm particles release soluble silver ions in a concentration dependent manner, with the highest concentration in the 30e40 ppb range after 3 days incubation. Dissolution of Ag NPs is likely to change again once ingested and passed through the embryonic zebrafish intestine. Moreover, the E3 medium also induces NPs agglomeration, regardless of the primary size, further reducing surface area and contributing to reduced dissolution. The E3 medium in which zebrafish embryos are grown contains chloride and sulfate anions, and divalent cations (Mg2þ and Ca2þ). It is known that Ag NPs agglomerate in the presence of high salt concentrations (Peterson et al., 2016). Several studies also reported that the presence of divalent cations, such as Mg2þ and Ca2þ, induces Ag NPs agglomeration (Jin et al., 2010; Zhang and Oyanedel-Craver, 2011;

X. Liu et al. / Environmental Pollution 248 (2019) 627e634

Anderson et al., 2014). In addition to agglomeration, other components in E3 medium may react with Ag ions to form insoluble products. Insoluble salts can rapidly form on the surface of the NPs, further hindering the dissolution process. Chloride anions are known for the strong reaction with silver ions, to form insoluble AgCl(s) (Ksp ¼ 1.77
1010) (Levard et al., 2012). It was also shown that silver ions can form silver chloride species through reaction 2 3 with Cl (AgCl(aq), AgCl 2 , AgCl3 , and AgCl4 ) and the speciation of silver chloride will depend on the Cl/Ag ratio in solution (Levard et al., 2013). Moreover, silver ions can also form an insoluble salt with sulfate (Ksp ¼ 1.20
105), also present in the E3 medium as MgSO4 (Levard et al., 2012). Such effects have been reported previously for Ag NPs dispersed in medium containing Cl and SO2 4 (Levard et al., 2013). Overall, these results indicate that the dissolution behavior of Ag NPs is affected by the exposure medium composition. In support of formation of insoluble Ag products in E3 medium, we find lower than expected dissolved Ag ions when experiments were performed with AgNO3 used in control experiments. We report that the actual amount of Agþ in the medium is much smaller than the nominal concentration, likely due to precipitation of Ag as insoluble compounds. 5. Conclusions Ag NPs suspended in E3 medium exhibit dramatic reduction of silver dissolution due to formation of insoluble salts of silver when dispersed in E3 medium. As a result, the environment in which Ag NPs are suspended can alter the toxicity behavior. Even though there is a decreased dissolution rate in E3 medium, we find distinct differences in toxicity dependent on NPs size. Only 40 nm Ag NPs demonstrate significant lethality beginning at 100 ppm during the last day of exposure (between 96 and 120 hpf). Embryo mortality on the last day of embryogenesis is consistent with the period when embryos begin eating and ingest Ag NPs, which indicates that the internalization of NPs within the intestinal lumen may be one of the underlying causes of lethality. We suggest that the combination of size and targeted toxicity at the intestinal level in the later stages of exposure to 40 nm Ag NPs contribute to the higher mortality. Exposure to silver ions does not play a significant role in the observed lethality. When embryos are exposed to soluble Ag ions similar to concentrations released by Ag NPs, we observe developmental defects in the intestine, skeletal muscle defects, and nitrosative stress, but no increased mortality. Similarly, the exposure to 10 or 100 nm Ag NPs and silver ions only induces sublethal effects, without mortality. Therefore, we conclude that the dissolution of Ag NPs in E3 medium is contributing along the particulate silver itself to induction of sublethal effects over an extended range of concentrations. Future research should focus on the investigation of developmental changes in organisms exposed to sublethal concentrations of nanomaterials. Funding sources This work was supported by the National Science Foundation: NSF [grant numbers 1336493; 1610281]. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.02.085.

633

References Abdelhalim, M., Jarrar, B., 2012. Histological alterations in the liver of rats induced by different gold nanoparticle sizes, doses and exposure duration. J. Nanobiotechnol. 10, 5. Anderson, J.W., Semprini, L., Radniecki, T.S., 2014. Influence of water hardness on silver ion and silver nanoparticle fate and toxicity toward Nitrosomonas europaea. Environ. Eng. Sci. 31, 403e409. AshaRani, P., Low Kah Mun, G., Hande, M.P., Valiyaveettil, S., 2008. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3, 279e290. Badawy, A.M.E., Luxton, T.P., Silva, R.G., Scheckel, K.G., Suidan, M.T., Tolaymat, T.M., 2010. Impact of environmental conditions (pH, ionic strength, and electrolyte type) on the surface charge and aggregation of silver nanoparticles suspensions. Environ. Sci. Technol. 44, 1260e1266. Bar-Ilan, O., Albrecht, R.M., Fako, V.E., Furgeson, D.Y., 2009. Toxicity assessments of multisized gold and silver nanoparticles in zebrafish embryos. Small 5, 1897e1910. Barker, L.K., Giska, J.R., Radniecki, T.S., Semprini, L., 2018. Effects of short- and longterm exposure of silver nanoparticles and silver ions to Nitrosomonas europaea biofilms and planktonic cells. Chemosphere 206, 606e614. Beer, C., Foldbjerg, R., Hayashi, Y., Sutherland, D.S., Autrup, H., 2012. Toxicity of silver nanoparticlesdnanoparticle or silver ion? Toxicol. Lett. 208, 286e292. Bowman, C.R., Bailey, F.C., Elrod-Erickson, M., Neigh, A.M., Otter, R.R., 2012. Effects of silver nanoparticles on zebrafish (Danio rerio) and Escherichia coli (ATCC 25922): a comparison of toxicity based on total surface area versus mass concentration of particles in a model eukaryotic and prokaryotic system. Environ. Toxicol. Chem. 31, 1793e1800. Browning, L.M., Lee, K.J., Nallathamby, P.D., Xu, X.-H.N., 2013. Silver nanoparticles incite size-and dose-dependent developmental phenotypes and nanotoxicity in zebrafish embryos. Chem. Res. Toxicol. 26, 1503e1513. Carlson, C., Hussain, S.M., Schrand, A.M., Braydich-Stolle, K., L, Hess, K.L., Jones, R.L., Schlager, J.J., 2008. Unique cellular interaction of silver nanoparticles: sizedependent generation of reactive oxygen species. J. Phys. Chem. B 112, 13608e13619. Chithrani, B.D., Ghazani, A.A., Chan, W.C.W., 2006. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6, 662e668. Choi, J.E., Kim, S., Ahn, J.H., Youn, P., Kang, J.S., Park, K., Yi, J., Ryu, D.-Y., 2010. Induction of oxidative stress and apoptosis by silver nanoparticles in the liver of adult zebrafish. Aquat. Toxicol. 100, 151e159. Cunningham, S., Brennan-Fournet, M.E., Ledwith, D., Byrnes, L., Joshi, L., 2013. Effect of nanoparticle stabilization and physicochemical properties on exposure outcome: acute toxicity of silver nanoparticle preparations in zebrafish (Danio rerio). Environ. Sci. Technol. 47, 3883e3892. Diab, N., Schuhmann, W., 2001. Electropolymerized manganese porphyrin/polypyrrole films as catalytic surfaces for the oxidation of nitric oxide. Electrochim. Acta 47, 265e273. Fabrega, J., Luoma, S.N., Tyler, C.R., Galloway, T.S., Lead, J.R., 2011. Silver nanoparticles: behaviour and effects in the aquatic environment. Environ. Int. 37, 517e531. Foldbjerg, R., Olesen, P., Hougaard, M., Dang, D.A., Hoffmann, H.J., Autrup, H., 2009. PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes. Toxicol. Lett. 190, 156e162. George, S., Lin, S., Ji, Z., Thomas, C.R., Li, L., Mecklenburg, M., Meng, H., Wang, X., Zhang, H., Xia, T., Hohman, J.N., Lin, S., Zink, J.I., Weiss, P.S., Nel, A.E., 2012. Surface defects on plate-shaped silver nanoparticles contribute to its hazard potential in a fish gill cell line and zebrafish embryos. ACS Nano 6, 3745e3759. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci. Technol. 43, 9216e9222. Guo, X., Li, Y., Yan, J., Ingle, T., Jones, M.Y., Mei, N., Boudreau, M.D., Cunningham, C.K., Abbas, M., Paredes, A.M., Zhou, T., Moore, M.M., Howard, P.C., Chen, T., 2016. Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays. Nanotoxicology 10, 1373e1384. Gupta, G.S., Dhawan, A., Shanker, R., 2016. Montmorillonite clay alters toxicity of silver nanoparticles in zebrafish (Danio rerio) eleutheroembryo. Chemosphere 163, 242e251. Ispas, C., Andreescu, D., Patel, A., Goia, D.V., Andreescu, S., Wallace, K.N., 2009. Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish. Environ. Sci. Technol. 43, 6349e6356. Jiao, T., Guo, H., Zhang, Q., Peng, Q., Tang, Y., Yan, X., Li, B., 2015. Reduced graphene oxide-based silver nanoparticle-containing composite hydrogel as highly efficient dye catalysts for wastewater treatment. Sci. Rep. 5. Jin, X., Li, M., Wang, J., Marambio-Jones, C., Peng, F., Huang, X., Damoiseaux, R., Hoek, E.M.V., 2010. High-throughput screening of silver nanoparticle stability and bacterial inactivation in aquatic media: influence of specific ions. Environ. Sci. Technol. 44, 7321e7328. Johnston, H.J., Hutchison, G., Christensen, F.M., Peters, S., Hankin, S., Stone, V., 2010. A review of the in vivo and in vitro toxicity of silver and gold particulates: particle attributes and biological mechanisms responsible for the observed toxicity. Crit. Rev. Toxicol. 40, 328e346. Kang, J.S., Bong, J., Choi, J.-S., Henry, T.B., Park, J.-W., 2016. Differentially transcriptional regulation on cell cycle pathway by silver nanoparticles from ionic silver

634

X. Liu et al. / Environmental Pollution 248 (2019) 627e634

in larval zebrafish (Danio rerio). Biochem. Biophys. Res. Commun. 479, 753e758. Ki-Tae, K., Lisa, T., Leah, W., Robert, L.T., 2013. Silver nanoparticle toxicity in the embryonic zebrafish is governed by particle dispersion and ionic environment. Nanotechnology 24, 115101. Kim, S., Choi, J.E., Choi, J., Chung, K.-H., Park, K., Yi, J., Ryu, D.-Y., 2009. Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol. in vitro 23, 1076e1084. €ller, M., Epple, M., 2010. Toxicity of silver Kittler, S., Greulich, C., Diendorf, J., Ko nanoparticles increases during storage because of slow dissolution under release of silver ions. Chem. Mater. 22, 4548e4554. Kumar, A., Aerry, S., Goia, D.V., 2016. Preparation of concentrated stable dispersions of uniform Ag nanoparticles using resorcinol as reductant. J. Colloid Interface Sci. 470, 196e203. Lai, D.Y., 2012. Toward toxicity testing of nanomaterials in the 21st century: a paradigm for moving forward. Wiley Interdiscipl. Rev.: Nanomed. Nanobiotechnol. 4, 1e15. Lai, C., Cheong, C., Mandeep, J., Abdullah, H., Amin, N., Lai, K., 2014. Synthesis and characterization of silver nanoparticles and silver inks: review on the past and recent technology roadmaps. J. Mater. Eng. Perform. 23, 3541e3550. Lem, K.W., Choudhury, A., Lakhani, A.A., Kuyate, P., Haw, J.R., Lee, D.S., Iqbal, Z., Brumlik, C.J., 2012. Use of nanosilver in consumer products. Recent Pat. Nanotechnol. 6, 60e72. Levard, C., Reinsch, B.C., Michel, F.M., Oumahi, C., Lowry, G.V., Brown, G.E., 2011. Sulfidation processes of PVP-coated silver nanoparticles in aqueous solution: impact on dissolution rate. Environ. Sci. Technol. 45, 5260e5266. Levard, C., Hotze, E.M., Lowry, G.V., Brown, G.E., 2012. Environmental transformations of silver nanoparticles: impact on stability and toxicity. Environ. Sci. Technol. 46, 6900e6914. Levard, C., Mitra, S., Yang, T., Jew, A.D., Badireddy, A.R., Lowry, G.V., Brown, G.E., 2013. Effect of chloride on the dissolution rate of silver nanoparticles and toxicity to E. coli. Environ. Sci. Technol. 47, 5738e5745. Liu, J., Hurt, R.H., 2010. Ion release kinetics and particle persistence in aqueous nano-silver colloids. Environ. Sci. Technol. 44, 2169e2175. Lubick, N., 2008. Nanosilver toxicity: ions, nanoparticles or both? Environ. Sci. Technol. 42, 8617. Marambio-Jones, C., Hoek, E.M.V., 2010. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanoparticle Res. 12, 1531e1551. Massarsky, A., Dupuis, L., Taylor, J., Eisa-Beygi, S., Strek, L., Trudeau, V.L., Moon, T.W., 2013. Assessment of nanosilver toxicity during zebrafish (Danio rerio) development. Chemosphere 92, 59e66. Massarsky, A., Trudeau, V.L., Moon, T.W., 2014. Predicting the environmental impact of nanosilver. Environ. Toxicol. Pharmacol. 38, 861e873. McGillicuddy, E., Murray, I., Kavanagh, S., Morrison, L., Fogarty, A., Cormican, M., Dockery, P., Prendergast, M., Rowan, N., Morris, D., 2017. Silver nanoparticles in the environment: sources, detection and ecotoxicology. Sci. Total Environ. 575, 231e246. Moore, M.N., 2006. Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ. Int. 32, 967e976. Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramírez, J.T., Yacaman, M.J., 2005. The bactericidal effect of silver nanoparticles. Nanotechnology 16, 2346. Mukhopadhyay, P., Mishra, R., Rana, D., Kundu, P.P., 2012. Strategies for effective oral insulin delivery with modified chitosan nanoparticles: a review. Prog. Polym. Sci. 37, 1457e1475. Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N., Sigg, L., Behra, R., 2008. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii. Environ. Sci. Technol. 42, 8959e8964. Osaki, F., Kanamori, T., Sando, S., Sera, T., Aoyama, Y., 2004. A quantum dot conjugated sugar ball and its cellular uptake. On the size effects of endocytosis in the subviral region. J. Am. Chem. Soc. 126, 6520e6521. € Ozel, R.E., Alkasir, R.S.J., Ray, K., Wallace, K.N., Andreescu, S., 2013. Comparative evaluation of intestinal nitric oxide in embryonic zebrafish exposed to metal oxide nanoparticles. Small 9, 4250e4261.

Ozel, R.E., Wallace, K.N., Andreescu, S., 2014. Alterations of intestinal serotonin following nanoparticle exposure in embryonic zebrafish. Environ. Sci.: Nano 1, 27e36. € Ozel, R.E., Liu, X., Alkasir, R.S.J., Andreescu, S., 2014. Electrochemical methods for nanotoxicity assessment. Trac. Trends Anal. Chem. 59, 112e120. Park, K., Tuttle, G., Sinche, F., Harper, S.L., 2013. Stability of citrate-capped silver nanoparticles in exposure media and their effects on the development of embryonic zebrafish (Danio rerio). Arch Pharm. Res. (Seoul) 36, 125e133. Peterson, K.I., Lipnick, M.E., Mejia, L.A., Pullman, D.P., 2016. Temperature dependence and mechanism of chloride-induced aggregation of silver nanoparticles. J. Phys. Chem. C 120, 23268e23275. Powers, C.M., Yen, J., Linney, E.A., Seidler, F.J., Slotkin, T.A., 2010. Silver exposure in developing zebrafish (Danio rerio): persistent effects on larval behavior and survival. Neurotoxicol. Teratol. 32, 391e397. Powers, C.M., Slotkin, T.A., Seidler, F.J., Badireddy, A.R., Padilla, S., 2011. Silver nanoparticles alter zebrafish development and larval behavior: distinct roles for particle size, coating and composition. Neurotoxicol. Teratol. 33, 708e714. Rai, M., Yadav, A., Gade, A., 2009. Silver nanoparticles as a new generation of antimicrobials. Biotechnol. Adv. 27, 76e83. Randhawa, M.A., 2009. Calculation of LD50 values from the method of Miller and Tainter, 1944. J. Ayub Med. Coll. Abbottabad 21, 184e185. Riaz Ahmed, K.B., Nagy, A.M., Brown, R.P., Zhang, Q., Malghan, S.G., Goering, P.L., 2017. Silver nanoparticles: significance of physicochemical properties and assay interference on the interpretation of in vitro cytotoxicity studies. Toxicol. in vitro 38, 179e192. €v, M., Taylor, C., Ribeiro, F., Gallego-Urrea, J.A., Jurkschat, K., Crossley, A., Hassello Soares, A.M.V.M., Loureiro, S., 2014. Silver nanoparticles and silver nitrate induce high toxicity to Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio. Sci. Total Environ. 466e467, 232e241. Ribeiro, F., Van Gestel, C.A.M., Pavlaki, M.D., Azevedo, S., Soares, A.M.V.M., Loureiro, S., 2017. Bioaccumulation of silver in Daphnia magna: waterborne and dietary exposure to nanoparticles and dissolved silver. Sci. Total Environ. 574, 1633e1639. Roque, A.C.A., Bicho, A., Batalha, I.L., Cardoso, A.S., Hussain, A., 2009. Biocompatible and bioactive gum Arabic coated iron oxide magnetic nanoparticles. J. Biotechnol. 144, 313e320. Truong, L., Zaikova, T., Richman, E.K., Hutchison, J.E., Tanguay, R.L., 2012. Media ionic strength impacts embryonic responses to engineered nanoparticle exposure. Nanotoxicology 6, 691e699. van Aerle, R., Lange, A., Moorhouse, A., Paszkiewicz, K., Ball, K., Johnston, B.D., deBastos, E., Booth, T., Tyler, C.R., Santos, E.M., 2013. Molecular mechanisms of toxicity of silver nanoparticles in zebrafish embryos. Environ. Sci. Technol. 47, 8005e8014. van der Zande, M., Vandebriel, R.J., Van Doren, E., Kramer, E., Herrera Rivera, Z., Serrano-Rojero, C.S., Gremmer, E.R., Mast, J., Peters, R.J., Hollman, P.C., 2012. Distribution, elimination, and toxicity of silver nanoparticles and silver ions in rats after 28-day oral exposure. ACS Nano 6, 7427e7442. Vance, M.E., Kuiken, T., Vejerano, E.P., McGinnis, S.P., Hochella Jr., M.F., Rejeski, D., Hull, M.S., 2015. Nanotechnology in the real world: redeveloping the nanomaterial consumer products inventory. Beilstein J. Nanotechnol. 6, 1769e1780. Westerfield, M., 1993. The Zebrafish Book : a Guide for the Laboratory Use of Zebrafish (Brachydanio Rerio). M. Westerfield [Eugene, OR]. Wu, Y., Zhou, Q., 2013. Silver nanoparticles cause oxidative damage and histological changes in medaka (Oryzias latipes) after 14 days of exposure. Environ. Toxicol. Chem. 32, 165e173. Xia, T., Kovochich, M., Brant, J., Hotze, M., Sempf, J., Oberley, T., Sioutas, C., Yeh, J.I., Wiesner, M.R., Nel, A.E., 2006. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett. 6, 1794e1807. Yong, K.-T., Law, W.-C., Hu, R., Ye, L., Liu, L., Swihart, M.T., Prasad, P.N., 2013. Nanotoxicity assessment of quantum dots: from cellular to primate studies. Chem. Soc. Rev. 42, 1236e1250. Zhang, H., Oyanedel-Craver, V., 2011. Evaluation of the disinfectant performance of silver nanoparticles in different water chemistry conditions. J. Environ. Eng. 138, 58e66.