Silver nanoparticles or free silver ions work? An ...

Science of the Total Environment 610–611 (2018) 77–83

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Silver nanoparticles or free silver ions work? An enantioselective phytotoxicity study with a chiral tool Zunwei Chen, Xiaolin Sheng, Jia Wang, Yuezhong Wen ⁎ MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Chiral Ag-cysteine complex was made to elucidate role of Ag+ in AgNP toxicity. • Ag+ was partly photo-reduced to AgNP by cysteine and toxicity was assayed. • Ag-L/D-Cys differ in Ag+/AgNP quantification and show different toxic effects. • Ag-D-Cys was more toxic to S. obliquus and Ag-L-Cys was more toxic to A. thaliana. • AgNP toxicity to S. obliquus and A. thaliana was due to Ag+ and AgNP, respectively.

a r t i c l e

i n f o

Article history: Received 28 June 2017 Received in revised form 1 August 2017 Accepted 3 August 2017 Available online xxxx Editor: Jay Gan Keywords: Silver nanoparticle Cysteine Enantioselectivity Phytotoxicity

a b s t r a c t Nowadays, silver nanoparticles (AgNP) have been widely used and there are raising concerns about their potential adverse effects on organism. As for the exact toxicity mechanism of AgNP, opinions still vary and whether the released silver ions (Ag+) or AgNP themselves are responsible for the toxicity remains debatable. In the present study, we have designed two exposure systems where Ag+ and AgNP coexisted but differed in quantification by using photo-reduced method with cysteine enantiomers, and their toxicities to freshwater microalgae Scenedesmus obliquus and model plant Arabidopsis thaliana were determined. In the results, Ag+ was in suit photo-reduced by cysteine enantiomers, and the UV–Vis and circular dichroism spectrum evidence confirmed the quantification difference between Ag-L-cysteine (Ag-L-Cys) and Ag-D-cysteine (Ag-D-Cys), where there was more AgNP and less Ag+ in Ag-L-Cys. Furthermore, the toxicity assay data revealed that Ag-D-Cys was more toxic to S. obliquus but A. thaliana was more susceptible to Ag-L-Cys. The metal element distribution in Arabidopsis leaves was also influenced in an enantioselective manner, which was related to the oxidative stress. Considering the quantification difference between Ag-L-Cys and Ag-D-Cys, it can be concluded that AgNP exhibited their toxicity to S. obliquus by the action of Ag+, but toxicity brought to A. thaliana was attributed to AgNP themselves rather than Ag+. The results of the present study help to better clarify the role of Ag+ in AgNP toxicity and offer a chiral tool and a new sight to investigate the toxicity mechanism of AgNP. © 2017 Published by Elsevier B.V.

1. Introduction

⁎ Corresponding author. E-mail address: [email protected] (Y. Wen).

http://dx.doi.org/10.1016/j.scitotenv.2017.08.037 0048-9697/© 2017 Published by Elsevier B.V.

The high pace of nanotechnology developments have led to increasing production and utilization of engineering nanoparticles (ENPs). Even though the values in material fields and medical applications

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have long been recognized (Lanone and Boczkowski, 2006; Xu et al., 2014; Li et al., 2015; Liu et al., 2016), ENPs still pose risk to the ecosystem and raising concerns over their potential adverse impacts have also been reported (Colvin, 2003). As one of the most widely commercialized ENPs, silver nanoparticles (AgNP) have received special attentions due to their potential harmful effects on bacteria (Beddow et al., 2017), aquatic and terrestrial animals (Conine and Frost, 2017), and plants (Cox et al., 2016; Wang et al., 2016). In particular, adverse influences to plants may finally threaten our human beings along the food chains (Wijnhoven et al., 2009; Ahamed et al., 2010; Marambio-Jones and Hoek, 2010). Even though AgNP have been extensively investigated, there still remain hot debates about the exact action mechanism. Because AgNP can release Ag+, whether Ag+ or AgNP cause the toxic effects remain controversial. On the one hand, pioneering researches have compared the toxicity of Ag+ and AgNP at same concentrations and found that Ag+ was more toxic than AgNP, and revealed that the toxicity of AgNP was mainly attributed to the release of Ag+ (Xiu et al., 2011; Priester et al., 2014). Therefore, the toxicity of AgNP was controlled primarily by the extent of nanoparticle dissolution (Gunsolus et al., 2015). On the other hand, some investigations also claimed that Ag+ did not account for all of the observed toxicity (Kwok et al., 2016). Evidence was also provided when compared as a function of the Ag+ concentration, toxicity of AgNP appeared to be much higher than that of Ag+, indicating that the culprits of AgNP toxicity can be tracked back to more than the released Ag+ but the nanoparticles themselves (Navarro et al., 2008; Yin et al., 2011). To compare the toxicity of Ag+ and AgNP, some investigations were performed by using the commercial coated AgNP and AgNO3, making sure about the same Ag concentrations before the exposure experiments (Navarro et al., 2015). However, there standing an unavoidable defect is that the effects of anionic such as NO− 3 cannot be ignored, because NO− 3 is one of the most important nitrogen nutrients, which may cause bias in the toxic effects assessments. In addition, to better and more efficiently distinguish the source of the toxicity, AgNP coated with natural organism matters (NOMs) or the Ag+ binding agents were further used in the toxicity assays (Gunsolus et al., 2015). Among the various Ag+ binding agents, cysteine has been frequently applied as a strong Ag+ ligand, which can abolish the inhibitory effects caused by Ag+ (Navarro et al., 2008). However, it also takes a risk to conclude that AgNP exhibited its toxicity by releasing Ag+ even though cysteine alleviates the toxicity. Because the effects of cysteine should be taken into consideration and more importantly, due to the binding ability, the complex Ag-cysteine may also exhibit its own influences.

Unfortunately, no investigation has been performed to explore the effects of Ag-cysteine complex so far. As for the Ag-cysteine complex, former research has found that enantiomers of cysteine, L- and D-cysteine can interact with Ag+ in an enantioselective manner due to the chiral carbon center (Liu et al., 2012), and the chiral recognition of cysteine by Ag+ has also been observed (Zhang and Ye, 2011). Furthermore, it has been reported that Ag+ can be photo-reduced into AgNP by cysteine without any other reducing agents (Liu et al., 2012) and the enantioselectivity was also observed between the AgNP, which enlightens us that AgNP reduced from Ag+ by cysteine enantiomers may be a good tool to investigate the toxic effects of AgNP. Therefore, as shown in Scheme 1, the aims of the present study is to establish two exposure systems containing AgNP and Ag+ at the same time but differing in quantification, by photo-reducing Ag+ with cysteine enantiomers. Furthermore, freshwater microalgae Scenedesmus obliquus and model plant Arabidopsis thaliana were selected as model organisms for their representative role in aquatic toxicology study and short life cycle, respectively. We aimed to (1) compare the toxic effects of Ag-cysteine complexes in an enantiomeric level and (2) to determine the effect of Ag+/AgNP quantification on the toxicity to both type plants. In addition, the phenotypic effects and potential mechanism such as oxidative stress were also investigated. By comparing the toxicity of Agcysteine complexes, we believe the results obtained in the present study may help to better distinguish the role of AgNP and Ag+ in AgNP toxicity and offer a useful tool as well as new sight to investigate the toxic mechanism of AgNP. 2. Materials and methods 2.1. Chemicals and materials AgNO3 (analytically pure, 99.85%) and cysteine (analytically pure, ≥ 97%) with L- and D-enantiomers were purchased from SigmaAldridge (St. Louis, MO, USA). Freshwater microalga Scenedesmus obliquus was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Doubly distilled water (ddH2O) was used throughout the experiments. All other chemical reagents were analytical grade and all glassware was sterilized in an autoclave. 2.2. Preparation and characterization of Ag-L/D-cysteine complex The mixed reaction between Ag+ and L/D-cysteine was performed according to previous study (Liu et al., 2012) with slight modification.

Scheme 1. Synthesis and toxicity assay of Ag-cysteine complex.


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In brief, aqueous AgNO3 (50 mM) and L/D-cysteine (55 mM) were exposed to stable N2 for 30 min to remove the O2 and stored under darkness at 4 °C. Then 50 μL AgNO3 and 50 μL L/D-cysteine were added into 9.9 mL ddH2O and the mixtures were further stirred under N2 for 30 min. The reduction reaction was initiated by the ultraviolet light (λ = 254 nm, 5000 μW/cm2) and shut down by the darkness. The solutions were centrifuged and dried under vacuum in a desiccator to obtain the Ag-cysteine complex (Ag-Cys). Then Ag-Cys were characterized by ultraviolet-visible spectroscopy (Cary 100, Agilent Technology), transmission electron microscopy (JEM-1230, JEOL) and circular dichroism spectroscopy (JASCO-815, JASCO Technology). Ag-Cys was also added into ddH2O, tap water, green alga medium, Daphnia magna OCED M4 medium and Hoagland medium, making the final concentration at 10 μM and stirred for 24 h. Then Ag-Cys in these solutions were analyzed by transmission electron microscopy (TEM). 2.3. Ag-Cys toxicity to Scenedesmus obliquus Scenedesmus obliquus was cultivated in HB-4 medium (0.2 g/L (NH4)2SO4, 0.03 g/L Ca(H2PO4)2·H2O, 0.08 g/L MgSO4·H2O, 0.1 g/L NaHCO3, 0.025 g/L KCl, 1.5 mg/L FeCl3) at 24 ± 1 °C until to logarithmic phase. The acute toxicity (24 h) was determined at the concentrations ranging from 0.05 to 1 μM. The absorbance at 680 nm was measured. The subcellular structures of S. obliquus were obtained by transmission electron microscopy according to previously reported method (Wen et al., 2016). 2.4. Ag-Cys toxicity to Arabidopsis thaliana Seeds of Arabidopsis thaliana were first sterilized with sodium hypochlorite and 75% ethanol solution for 2 min. Then the seeds were placed on the gauze floating on the Hoagland nutrient solution. A. thaliana were cultivated in an incubator at 23 ± 2 °C with 16 h light (4000 lx) and 8 h dark cycles for two weeks until analysis. The exposure concentration was set at 27.5 μM. Distribution of metal elements (K, Ca, Mn, Fe, Cu and Zn) in the leaves of Arabidopsis was measured according previous study (Chen et al., 2015). The experiments were performed based on synchrotron X-ray micro-fluorescence (μ-XRF) with a beamline BL15U1 at Shanghai Institute of Applied Physics, Chinese Academy of Science. At first, the leaves were harvested after two-week cultivation and washed by ddH2O for three times. The samples were fixed on the tape and the beam size of 2 × 2 μm2 was used. The majority of maps were focused on the entire leaf blade. 2.5. Statistical analysis The data were present as the mean standard deviation and analyzed by Origin 9.0 software (OriginLab, Northampton, MA, USA). The growth inhibition rates of S. obliquus were calculated according to the change percentage comparing to the control. The color-coded composite chemical maps of metal elements were obtained by Igor Pro 6.0 software (IGOR). 3. Results 3.1. Formation and characterization of Ag-cysteine complex First of all, the effects of Ag+ vs cysteine ratios (denoted by r) on the formation of AgNP were investigated. We kept the concentration of cysteine to be the same (0.5 μM), but changed concentrations of Ag+ ranging from 0.25 to 1 μM. As shown in Fig. 1, when r was b1, i.e., Ag+ was less than cysteine, small particles (b 20 nm) formed in Ag-L-Cys, but with the presence of D-cysteine, AgNP were much bigger and aggregated together. When r equaled to 1.1, both L- and D-cysteine efficiently promoted the formation of AgNP in similar sizes. When r raised, the formation of AgNP became much clearer (Fig. 1I & J).

Fig. 1. TEM images of Ag-Cys after 6-h reaction with different cysteine enantiomers and Ag+/cysteine ratio. (A) 0.25 μM Ag+ vs 0.5 μM L-cysteine; (B) 0.25 μM Ag+ vs 0.5 μM Dcysteine; (C) 0.40 μM Ag+ vs 0.5 μM L-cysteine; (D) 0.40 μM Ag+ vs 0.5 μM D-cysteine; (E) 0.50 μM Ag+ vs 0.5 μM L-cysteine; (F) 0.50 μM Ag+ vs 0.5 μM D-cysteine; (G) 0.55 μM Ag+ vs 0.5 μM L-cysteine; (H) 0.55 μM Ag+ vs 0.5 μM D-cysteine; (I) 1.00 μM Ag+ vs 0.5 μM L-cysteine; (J) 1.00 μM Ag+ vs 0.5 μM D-cysteine (black bars in each group (A–J) from left to right represent to 2 μm, 0.5 μm, 200 nm and 100 nm, respectively).

Except for unveiling the effect of Ag+ concentrations, we further investigated the influence of reaction time on the formation of AgNP (r = 1.1). The inserted photos in Fig. 2(A) exhibited the change of colors, where the color of Ag-L-Cys became significantly darker than Ag-D-Cys and AgNO3 alone after a specific reaction time. Furthermore, as the UV–Vis spectrum data depicted, the peaks at around 400 nm increased

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as the reaction continued. On the whole, the absorptions of Ag-L-Cys were all greater than those of Ag-D-Cys. The circular dichroism (CD) data also revealed the enantioselective difference between cysteine enantiomers at the photo-reduction of Ag+ to AgNP. To be exact, peak at 260 nm was disappeared in Ag-L-Cys but a new peak occurred at around 350 nm. The spectrum of Ag-D-Cys was similar to D-cysteine except the weaker peak at 260 nm (Fig. 2B). As the TEM images shown in Fig. 3, after 2-h reaction, both L- and Dcysteine promoted the formation of AgNP. However, AgNP in L-cysteine group were like rod shape and in a bigger size (Fig. 3A), but Ag+ and Dcysteine led to smaller and particle shape AgNP (Fig. 3D). As the reaction continued, AgNP grew quickly and gathered in both cysteine enantiomer groups (Fig. 3B, C, E and F).

as the concentration raised, toxicities of Ag+ were much greater than both forms of AgNP. Interestingly, different toxicities of AgNP were found at 0.4 and 0.5 μM, at which concentration the Ag-D-Cys was 4.95- and 1.99-fold more toxic than Ag-L-Cys, respectively. At a relative high concentration (1 μM), both Ag+ and AgNP led to a thorough death of S. obliquus. In addition, TEM images exhibited that the morphologies of S. obliquus exposed to 0.4 μM AgNP were significantly different (Fig. 4 Right). Therein, images in the control (a, b) and Ag-L-Cys group (e, f) were similar and no obvious damages were found. However, when exposed to Ag+, S. obliquus suffered from severe plasmolysis and the cell nucleus was separated (c, d). Comparatively, the damage of Ag-DCys was weaker than Ag+ but greater than Ag-L-Cys, where the cell nucleus was also separated (g, h).

3.2. Toxicity of Ag-Cys to Scenedesmus obliquus

3.3. Toxicity of Ag-Cys to Arabidopsis thaliana

Toxicity of Ag-Cys complex to microalgae Scenedesmus obliquus was investigated. As depicted in Fig. 4 Left, there was no significant difference between Ag+ and AgNP when concentration was b 1 μM. However,

Toxicity of Ag-Cys to terrestrial plant Arabidopsis thaliana was further investigated. As shown in Fig. 5, morphology images revealed that Ag+ were toxic comparing to the control. And Ag-L-Cys resulted

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Fig. 3. TEM images of Ag-cysteine complexes after 2-, 4- and 6-h reaction, ratio of Ag+ vs cysteine equals to 1.1. (A) Ag-L-Cys after 2 h; (B) Ag-L-Cys after 4 h; (C) Ag-L-Cys after 6 h; (D) AgD-Cys

after 2 h; (E) Ag-D-Cys after 4 h; (F) Ag-D-Cys after 6 h (each black bar equals to 0.5 μm).

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Fig. 4. Effects of Ag+ and Ag-Cys on the growth of Scenedesmus obliquus (Exposed for 72 h): inhibition rate (left, error bars represent the standard error of the mean and some of which are not visible when they are smaller than the symbol) and TEM image (right, (a, b) control; (c, d) 0.4 μM Ag+; (e, f) 0.4 μM Ag-L-Cys; (g, h) 0.4 μM Ag-D-Cys). (The amplifications of (a), (c), (e) and (g) are 12,000 X (black bar = 2 μm) and the red rectangle images are showed on their left at 50,000 X (black bar = 0.5 μm)). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Ag+ was more than cysteine, the formation of AgNP in uniform can be expected (Fig. 1). This interesting phenomenon indicated that a little excess of Ag+ over cysteine accelerated the photo-reduced process, which was consistent with previous study (Liu et al., 2012). On the other hand, results obtained above also confirmed that cysteine could simultaneously act as protecting and reducing agent. Then we performed further experiments at r = 1.1, where both cysteine enantiomers started showing its promotion roles in AgNP formation. Except for the effects of r values, the influence of reaction time on AgNP formation was additionally investigated. As shown in Fig. 2(A), the inserted image revealed the change of solution color, which was due to colorimetric chiral recognition of cysteine enantiomers reacted with Ag+ as previously reported (Zhang and Ye, 2011). In addition, the UV–Vis spectrum data revealed the formation difference of AgNP. To be exact, the peak at around 400 nm, which is the characteristic peak of AgNP, got stronger as reaction continued, indicating the formation of AgNP photo-reduced from Ag+ by cysteine was time-dependent. On the other hand, the difference in the peaks strength of Ag-L-Cys and Ag-D-Cys demonstrated the both types of complex differed in AgNP quantification, where there were more AgNP in Ag-L-Cys than Ag-DCys. Their differences were also confirmed by the change of circular dichroism (CD) spectrum data, where Ag-L-Cys had a peak at around 300 nm but Ag-D-Cys didn't (Fig. 2B). And the peak intensity of Ag-DCys at 260 nm was weaker than that of D-cysteine, indicating that D-

in a more severe inhibitory effect on A. thaliana growth than Ag-D-Cys. The metal elements mapping also revealed that metal elements were aggregated in the leaves of A. thaliana exposed to Ag+, especially K and Ca. Under the treatment with AgNP, most metal elements were distributed uniformly in the leaves. However, their distribution patterns differed. Some metal elements such as K and Ca were aggregated at the end edge of Arabidopsis leaves exposed to Ag-L-Cys, but they were more likely to aggregate in the veins when treated with Ag-D-Cys. Furthermore, element mapping image also revealed the difference of quantification of elements. On the whole, uptake of elements K, Ca, Cu and Zn were obviously increased in treated plants comparing to the control. In the comparison between AgNP, their effects varied in Cu, where Ag-LCys promoted the uptake of Cu to more extent than Ag-D-Cys did. 4. Discussion To compare the toxic effects of Ag+ and silver nanoparticles (AgNP) at the same time, we established an exposure system where Ag+ and AgNP were coexisted, but they were different in quantification with the help of chiral cysteine. First of all, as previously reported, Ag+ can be in suit photo-reduced to form AgNP by cysteine without any assistance of other reducing agents, due to the functional group -SH (Liu et al., 2012). In the preparation experiment, we set different ratio of Ag+ vs cysteine in a wide range. The TEM images revealed that once

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Fig. 6. TEM images of Ag-Cys in different solutions. (A) and (F) are Ag-L-Cys and Ag-D-Cys in deionized water; (B) and (G) are Ag-L-Cys and Ag-D-Cys in tap water; (C) and (H) are Ag-L-Cys and Ag-D-Cys in algal HB-4 medium; (D) and (I) are Ag-L-Cys and Ag-D-Cys in Daphnia magna OCED M4 medium; (E) and (J) are Ag-L-Cys and Ag-D-Cys in Hoagland medium. All the images are amplified at 50,000 X and the length of black bars in each image equal to 100 nm.

cysteine was consumed by the reaction with Ag+. At this point, we have finished the preparation of exposure system consisted of Ag+ and AgNP, but differed in the quantification of both types Ag. Thereafter we applied the exposure system to two types of plants, microalgae Scenedesmus obliquus and terrestrial plant Arabidopsis thaliana, to investigate and compare the toxicity of Ag+ and AgNP at the same time, and to further unveil the puzzle that whether AgNP themselves or the released Ag + should be more responsible for the toxic effects. On the one hand, microalgae are important tools for monitoring water quality and aquatic toxicity, because they are often more sensitive to contaminants than are the fishes and invertebrates (Wen et al., 2009, Jin et al., 2010 and Wen et al., 2011). In the toxicity assays, Ag+ and Ag-cysteine complex exhibited no significantly difference in the inhibition rate at low concentration. However, as the concentration raised, toxicities of Ag+ were much greater than those with the presence of L- and D-cysteine, indicating that cysteine played an alleviated role in the toxicity of Ag+ due to the combine ability, which has been confirmed by similar investigations (Xiu et al., 2011; Pokhrel et al., 2013). On the other hand, the toxicities of Ag- L -Cys and Ag- D-Cys were compared for the first time, and Ag-D -Cys caused more severe damage than Ag-L -Cys as depicted in inhibition rate and cell morphology influence (Fig. 4). Considering that there were more Ag+ and less AgNP in Ag-D-Cys than Ag-L-Cys, it could be concluded that Ag+ should take more responsibility for the toxic effects on S. obliquus in the exposure system where Ag+ and AgNP coexisted. On the other hand, for the toxicity to model plant A. thaliana, both exposure systems also exhibited different inhibitory effects. However, the disturbance manner was exactly opposite to that of S. obliquus. As it shown in Fig. 5, Ag-L-Cys caused more toxicity than Ag-D-Cys in the phenotype influences. Similarly, due to composition difference that there were more AgNP and less Ag+ in Ag-L-Cys than Ag-D-Cys, we could also conclude that in the toxicity of AgNP to A. thaliana was attributed to AgNP themselves rather than the released Ag+. Furthermore, to detect possible mechanism involved, metal element distribution in Arabidopsis leaves was also observed. Previous investigations have revealed the cross talk between metal element behaviors and oxidative stress such as the production of reactive oxygen species (ROS) (Chen et al., 2013; Chen et al., 2015), which has been recognized as the action mechanism of AgNP (Navarro et al., 2008; Miao et al., 2009). In the present study, the distribution of metal elements especially K and Ca were disturbed, which became aggregated after being exposed to Ag+. In the comparison between Ag-L-Cys and Ag-D-Cys, different influence pattern was also observed, where K and Ca were aggregated in the edge of leaves and in the veins, respectively. The abnormal behavior of metal elements such as K and Ca may contribute to evaluating the enantioselective effect of AgNP reduced from Ag+ by chiral cysteine. In addition, both types AgNP also differed in Fe and Cu uptake in Arabidopsis. As it known to all, Fe2 + is the important composition of

Fenton reaction, whose products contains a series of ROS like hydroxyl radicals (•OH). However, Fe2+ in the organism is unstable and can be easily oxidized to Fe3 +. On the other hand, Cu2 + toxicity has been well recognized (Lequeux et al., 2010; Wang et al., 2016) and has also been confirmed to take part in Fenton-like reaction to generate ROS (Aguiar and Ferraz, 2007; Peng et al., 2016). Therefore, the mechanism of the toxicity difference between Ag-L-Cys and Ag-D-Cys can be tracked to the over uptake of Cu and then the production of ROS. Questions about whether AgNP or released Ag + should be responsible for the toxicity have been discussed above. In addition, another potential misinterpretation about nanotoxicology study is that once AgNP are released into exposure system such as nutrient solution, they may quickly aggregate to become larger than the nanosize, thus it is inaccurate to claim that the toxic effects are caused by nanoparticles and similar concern has been expressed by pioneering perspective (Alkilany et al., 2016). Therefore, we further investigated the status of AgNP released to different solutions including deionized water, tap water, algal HB-4 medium, Daphnia magna OCED M4 medium and Hoagland medium. As the TEM images depicted in Fig. 6, sizes of Ag-D-Cys were all larger than those of Ag-LCys in all the solutions studied except the algal HB-4 medium, indicating that Ag-D-Cys was more likely to aggregate. Therefore, cysteine enantiomers can be helpful to investigate the effect of sizes on AgNP behaviors. In conclusion, the present study has established two exposure systems containing AgNP and Ag+ by using in suit photo-reduced Ag+ by chiral cysteine. The spectrum data revealed that there were more AgNP but less Ag + in Ag-L -Cys complex than in Ag-D -Cys. Then we selected two types of plants, microalgae Scenedesmus obliquus and terrestrial plant Arabidopsis thaliana and exposed them to Ag-Cys complex. The toxicology data demonstrated that Ag-D-Cys caused more damage to S. obliquus than Ag-L-Cys according to the phenotype and inhibitory rates evidence. However, A. thaliana was more susceptible to Ag-L-Cys, and metal elements behaviors in Arabidopsis leaves, which were correlated with ROS production and oxidative stress, was disturbed in different patterns by Ag-L -Cys and Ag-D-Cys. Therefore, considering the quantification differences of Ag+ and AgNP in the both types of Ag-Cys complexes, we can conclude that the damage to S. obliquus caused by AgNP was due to the released Ag+, but AgNP themselves should take more responsibility for the toxicity to A. thaliana. In summary, it may take risk to conclude absolutely the toxicity of AgNP was attributed to Ag+ or nanoparticle itself. The present study enlightens us that species of the test organism should be taken into consideration when detecting the exact toxicity mechanism of AgNP. In addition, the enantioselective differences between cysteine enantiomers provided useful tools to clarify the role of Ag+ in AgNP toxicity and more importantly, to further investigate the exact behavior and toxicity mechanism of AgNP (Qiu et al., 2011 and Qiu et al., 2012).


Abbreviations AgNP Silver nanoparticles Ag-L-Cys Ag-L-Cysteine Complex Ag-D-Cys Ag-D-Cysteine Complex ROS Reactive Oxygen Species

Acknowledgment This work was supported by the National Natural Science Foundation of China (NSFC, Nos. 21377111 and 21677124). References + Aguiar, A., Ferraz, A., 2007. Fe+ 3 - and Cu2 -reduction by phenol derivatives associated with Azure B degradation in Fenton-like reactions. Chemosphere 66, 947–954. Ahamed, M., AlSalhi, M.S., Siddiqui, M.K.J., 2010. Silver nanoparticle applications and human health. Clin. Chim. Acta 411, 1841–1848. Alkilany, A.M., Mahmoud, N.N., Hashemi, F., Hajipour, M.J., Farvadi, F., Mahmoudi, M., 2016. Misinterpretation in nanotoxicology: a personal perspective. Chem. Res. Toxicol. 29, 943–948. Beddow, J., Stolpe, B., Cole, P.A., Lead, J.R., M. Sapp, M., Lyons, B.P., Colbeck, I., Whitby, C., 2017. Nanosilver inhibits nitrification and reduces ammonia-oxidising bacterial but not archaeal amoA gene abundance in estuarine sediments. Environ. Microbiol. 19 (2), 500–510. Chen, H., Sheng, X., Wen, Y., Zhang, L., Bao, H., Li, L., et al., 2013. New insights into the effects of the herbicide imazethapyr on Cu(II) ecotoxicity to the aquatic unicellular alga Scenedesmus obliquus. Aquat. Toxicol. 140, 407–414. Chen, Z., Chen, H., Zou, Y., Qju, J., Wen, Y., Xu, D., 2015. Are nutrient stresses associated with enantioselectivity of the chiral herbicide imazethapyr in Arabidopsis thaliana? J. Agric. Food Chem. 63 (47), 10209–10217. Colvin, V.L., 2003. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 21, 1166–1170. Conine, A.L., Frost, P.C., 2017. Variable toxicity of silver nanoparticles to Daphnia magna: effects of algal particles and animal nutrition. Ecotoxicology 26 (1), 118–126. Cox, A.P., Venkatachalam, S.S., Sharma, N., 2016. Silver and titanium dioxide nanoparticle toxicity in plants: a review of current research. Plant Physiol. Biochem. 107, 147–163. Gunsolus, I.L., Mousavi, M.P.S., Hussein, K., Buehlmann, P., Haynes, C.L., 2015. Effects of humic and fulvic acids on silver nanoparticle stability, dissolution, and toxicity. Environ. Sci. Technol. 49, 8078–8086. Jin, M.C., Li, L., Xu, C., Wen, Y.Z., Zhao, M.R., 2010. Estrogenic activities of two synthetic pyrethroids and their metabolites. J. Environ. Sci. 22, 290–296. Kwok, K.W.H., Dong, W., Marinakos, S.M., Liu, J., Chilkoti, A., Wiesner, M.R., et al., 2016. Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation. Nanotoxicology 10, 1306–1317. Lanone, S., Boczkowski, J., 2006. Biomedical applications and potential health risks of nanomaterials: molecular mechanisms. Curr. Mol. Med. 6, 651–663. Lequeux, H., Hermans, C., Lutts, S., Verbruggen, N., 2010. Response to copper excess in Arabidopsis thaliana: impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile. Plant Physiol. Biochem. 48, 673–682. Li, X.F., Liu, W.P., Ma, J.Q., Wen, Y.Z., Wu, Z.C., 2015. High catalytic activity of magnetic FeOx/NiOx/SBA-15: the role of Ni in the bimetallic oxides at the nanometer level. Appl. Catal. B Environ. 179, 239–248. Liu, H., Ye, Y., Chen, J., Lin, D., Jiang, Z., Liu, Z., et al., 2012. In situ photoreduced silver nanoparticles on cysteine: an insight into the origin of chirality. Chem. Eur. J. 18, 8037–8041.

83

Liu, W.P., Ma, J.Q., Shen, C.S., Wen, Y.Z., Liu, W.P., 2016. A pH-responsive and magnetically separable dynamic system for efficient removal of highly dilute antibiotics in water. Water Res. 90, 24–33. 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. Nanopart. Res. 12, 1531–1551. Miao, A.-J., Schwehr, K.A., Xu, C., Zhang, S.-J., Luo, Z., Quigg, A., et al., 2009. The algal toxicity of silver engineered nanoparticles and detoxification by exopolymeric substances. Environ. Pollut. 157, 3034–3041. Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N., et al., 2008. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii. Environ. Sci. Technol. 42, 8959–8964. Navarro, E., Wagner, B., Odzak, N., Sigg, L., Behra, R., 2015. Effects of differently coated silver nanoparticles on the photosynthesis of Chlamydomonas reinhardtii. Environ. Sci. Technol. 49, 8041–8047. Peng, J., Li, J., Shi, H., Wang, Z., Gao, S., 2016. Oxidation of disinfectants with Cl-substituted structure by a Fenton-like system Cu2 +/H2O2 and analysis on their structurereactivity relationship. Environ. Sci. Pollut. Res. 23, 1898–1904. Pokhrel, L.R., Dubey, B., Scheuerman, P.R., 2013. Impacts of select organic ligands on the colloidal stability, dissolution dynamics, and toxicity of silver nanoparticles. Environ. Sci. Technol. 47, 12877–12885. Priester, J.H., Singhal, A., Wu, B., Stucky, G.D., Holden, P.A., 2014. Integrated approach to evaluating the toxicity of novel cysteine-capped silver nanoparticles to Escherichia coli and Pseudomonas aeruginosa. Analyst 139, 954–963. Qiu, J.G., Ma, Y., Chen, L.S., Wu, L.F., Wen, Y.Z., Liu, WP., 2011. sirA-like gene, sirA2, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading Pseudomonas sp. HZN6 strain. Appl. Microbiol. Biotechnol. 92 (5), 1023–1032. Qiu, J.G., Ma, Y., Wen, Y.Z., Chen, L.S., Wu, L.F., Liu, W.P., 2012. Functional identification of two novel genes from pseudomonas sp strain HZN6 involved in the gatabolism of nicotine. Appl. Environ. Microbiol. 78, 2154–2160. Wang, Z., Xu, L., Zhao, J., Wang, X., White, J.C., Xing, B., 2016. CuO nanoparticle interaction with Arabidopsis thaliana: toxicity, parent-progeny transfer, and gene expression. Environ. Sci. Technol. 50 (11), 6008–6016. Wen, Y.Z., Yuan, Y.L., Shen, C.S., Liu, H.J., Liu, W.P., 2009. Spectroscopic investigations of the chiral interactions between lipase and the herbicide dichlorprop. Chirality 21, 396–401. Wen, Y.Z., Chen, H., Shen, C.S., Zhao, M.R., Liu, W.P., 2011. Enantioselectivity tuning of chiral herbicide dichlorprop by copper: roles of reactive oxygen species. Environ. Sci. Technol. 45, 4778–4784. Wen, Y.Z., Zhang, L.J., Chen, Z.W., Sheng, X.L., Qiu, J.G., Xu, D.M., 2016. Co-exposure of silver nanoparticles and chiral herbicide imazethapyr to Arabidopsis thaliana: enantioselective effects. Chemosphere 145, 207–214. Wijnhoven, S.W.P., Peijnenburg, W.J.G.M., Herberts, C.A., Hagens, W.I., Oomen, A.G., Heugens, E.H.W., Roszek, B., Bisschops, J., Gosens, I., Meent, D., Dekkers, S., Jong, W.H., Zijverden, M., Sips, A.J.A.M., Geertsma, R.E., 2009. Nano-silver - a review of available data and knowledge gaps in human and environmental risk assessment. Nanotoxicology 3 (2), 109–138. Xiu, Z.M., Ma, J., Avarez, P.J.J., 2011. Differential effect of common ligands and molecular oxygen on antimicrobial activity of silver nanoparticles versus silver ions. Environ. Sci. Technol. 45 (20), 9003–9008. Xu, L.L., Li, X.F., Ma, J.Q., Wen, Y.Z., Liu, W.P., 2014. Nano-MnOx on activated carbon prepared by hydrothermal process for fast and highly efficient degradation of azo dyes. Appl. Catal. A Gen. 485, 91–98 (xu). Yin, L., Cheng, Y., Espinasse, B., Colman, B.P., Auffan, M., Wiesner, M., Rose, J., Liu, J., Bernhardt, E.S., 2011. More than the ions: the effects of silver nanoparticles on Lolium multiflorum. Environ. Sci. Technol. 45 (6), 2360–2367. Zhang, M., Ye, B.C., 2011. Colorimetric chiral recognition of enantiomers using the nucleotide-capped silver nanoparticles. Anal. Chem. 83 (5), 1504–1509.