Silver nanoparticles (AgNPs) have physicochemical and biological properties considerably different from those of their bulk or ionic counterparts. This feature has led to the promising utilization of nanosilver in antimicrobial materials, medical devices, photonics, optoelectronics, and catalysis 1. The surface-enhanced Raman scattering effect of AgNPs also encourages their use as a biological probe 2, 3. Silver nanoparticles are currently the most extensively used engineered nanomaterial in the market (http://www.nanotechproject.org/inventories/consumer/analysis_draft/). The increasing production and widespread application of AgNPs raise the chance that these nanomaterials will be discharged into the aquatic environment from commercial products 4. Different research groups have recently demonstrated that AgNPs incorporated into commercial fabrics can be steadily released into the aquatic system 5, 6. As a result, there has been increasing concern about the toxicity of nanosilver to aquatic organisms.
A few reports suggest that AgNPs are acutely toxic to adult fish such as Cyprinus carpio, Carassius carassius, and Perca fluviatilis7, 8. A 48-h toxicity test on adult female zebrafish has indicated that AgNP treatment significantly increases the metal burden in gill tissue 9. Bilberg et al. 10 administered a graded dose of nanosilver to P. fluviatilis for 24 h and found an increased critical oxygen tension (Pcrit). A recent study reveals that AgNPs have cytotoxic and genotoxic effects on the medaka (Oryzias latipes) cell line in a concentration-dependent manner 11. As far as we know, only an initial study demonstrates that AgNPs produce detrimental effects to Cyprinodon variegatus under chronic exposure conditions 12.
Although some studies have focused on the toxicological evaluation and environmental risk assessment of AgNPs to aquatic organisms, most of them have been conducted under in vitro or cell culture conditions and in short-term tests 13. Gaps in our knowledge of the long-term toxicity of AgNPs to freshwater fish still exist. Little is also known of the uptake and toxicological mechanism of AgNPs in adult fish. To evaluate the effects of AgNPs on adult medaka involving 14-d exposure, the metal burdens in fish tissues were measured, and toxicological endpoints such as mortality, oxidative stress, and histopathological changes were determined.
MATERIALS AND METHODS
Silver nitrate (AgNO3), tricaine-methanesulfonate (MS 222), and polyvinylpyrrolidone (PVP) were purchased from Sigma Aldrich. Bovine serum albumin (BSA) and nitric acid (HNO3) were purchased from Roche and Merck, respectively. Biochemical kits for analyzing lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) peroxidase (GPx), reduced GSH, and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Bioengineering Institute.
Synthesis and characterization of AgNPs
Particle synthesis was performed as previously described 14. Briefly, the NPs were prepared in an aqueous solution by reducing silver nitrate with sodium hypophosphite in the presence of PVP as a protective agent. After cleaning and drying, the crystal structure of the AgNP powder was examined by X-ray diffraction analysis using a D8 Advance X-ray powder diffractometer. The Brunauer–Emmett–Teller (BET) method was used to determine the specific surface area. The optical absorption spectra of the AgNPs were recorded in the ultraviolet-visible (UV-vis) region with a spectrophotometer (DU800; Beckman Coulter). The morphology, average diameters, and dispersion of AgNP were determined with a transmission electron microscopy (TEM) system (H-7500; Hitachi). Hydrodynamic diameter and size distribution were monitored by dynamic light scattering (DLS) spectrometry (BI-200SM; Brookhaven). Zeta potential measurements were taken on a Malvern Zetasizer Nano ZS system. The powdered nanosilver was redispersed in deionized water at 10.0 mg/L prior to UV-vis, TEM, and DLS analyses and at 100 mg/L prior to zeta potential measurements.
Fish maintenance and exposure design
A batch of nine-month-old adult medaka fish were chosen for the experiments. The fish were cultured in 10-L aquaria, which were equipped with a flow-through system with dechlorinated tap water at 25 ± 2°C, and fed with Artemia nauplii twice per day. A constant water quality and a controlled photoperiod (16 h light:8 h dark) were maintained.
For the 96-h toxicity test, healthy fish were selected and divided into different AgNP treatment groups (0, 0.3, 0.6, 1.2, 2.4, and 4.8 mg/L). Duplicate experiments were carried out with n = 20 animals or treatments or replicates. All fish were kept in 10-L aquaria and fasted for the duration of exposure. The test water was changed each day. During the 96-h static exposure, dead animals were recorded for the calculation of median lethal concentration (LC50) values.
We selected, based on the LC50 values, AgNP concentrations of 0, 0.05, 0.1, 0.25, and 0.5 mg/L for a subchronic exposure study. Adult fish in each group (32 per group) were equally allocated into two 10-L chambers and were consecutively treated with a graded series of AgNPs for 14 d. Any dead fish were removed as soon as possible. The animals were fed Artemia nauplii once per day, and test solutions were renewed daily. To minimize the decrease in the nominal concentration of AgNPs, which potentially adsorb onto residual food and feces in the test water, each experimental aquarium was supplied with Artemia nauplii for 1 h prior to dosing. After draining, the aquaria were replenished with 10 L of fresh water and redosed immediately. Water samples were collected from each test tank after 24 h, digested with 50% HNO3, and diluted with deionized water. The actual concentrations of silver (means ± standard deviation [SD], n = 6) in the exposures measured by inductively coupled plasma-mass spectrometry (ICP-MS; Agilent 7500CE) were less than the detection limit (detection limit = 0.26 µg/L), 37.2 ± 5.6, 83.3 ± 3.7, 186.8 ± 11.4, and 412.5 ± 42.1 µg/L for the control and low-, middle-, and high-concentration treatments, respectively.
Water quality and AgNP stability in test solutions
Nanoparticles agglomerate at high concentrations or in aqueous solutions with high ionic strengths 15, 16, affecting their bioavailability and toxicological effects on living organisms. The measurements of absorbance spectra with red and blue shifts can be used to detect size changes 17. The oxidation of AgNP (zero-valent silver) in aqueous solutions can occur via a mechanism involving molecular oxygen (O2) 18. We hypothesize a depletion of dissolved oxygen (DO) levels in AgNP-treated water. Thus, the test solutions were sampled from each exposure aquarium at 0, 6, 12, 18, and 24 h, and UV-vis and DO analyses were performed. In the AgNP suspensions, Ag+ ion was also determined by an Ag-ion-selective electrode (ISE).
Distribution of Ag in fish tissues
For Ag distribution analysis, brain, gill, intestine, and liver were dissected from 0.25 mg/L AgNP-dosed medaka after 14 d, and the wet weights were immediately recorded. Tissue samples were digested with a combination of 2.0 ml concentrated HNO3 and 2.0 ml hydrogen peroxide at 60°C for 3 h in a loft dryer. The temperature was subsequently increased to 160°C for 1 h for further digestion. The samples were cooled and diluted in a total volume of 10 g 2% HNO3. Silver contents were quantified via ICP–MS.
At the end of the 14-d exposure, 16 fish in each group were selected. After rinsing, the test species were anesthetized with MS 222, and brain, liver, and gill tissues were collected. The tissues were stored in the refrigerator at −80°C for biochemical analysis. Brains and livers were homogenized using a motor-driven tissue homogenizer, and the gills were homogenized using a Tenbroeck glass homogenizer in iced phosphate buffer solution (PBS) for 2 min. The homogenate was immediately centrifuged at 10,000 g and 4°C for 15 min, and LDH activity, antioxidant enzyme (SOD, CAT, and GPx) activities, GSH content, and MDA levels were measured in the supernatant by spectrometry 19, 20. The activities of the antioxidant enzymes and LDH were defined as international units per gram or milligram of protein, and the reduced GSH contents and MDA products were expressed as the amount of the substances per milligram of protein. Total protein content was measured via the Bradford method with BSA as standard 21. Absorbance values were determined with a microplate reader.
At the end of the 14-d exposure, four individuals were pooled from replicate aquaria per treatment. After rinsing, the test species were anesthetized with MS 222, and brain, liver, and gill tissues were sampled. The samples were immediately fixed with 4% paraformaldehyde (PFA). After rinsing with PBS, the PFA-fixed tissue samples were dehydrated with a graded ethanol series and embedded in paraffin wax. The 4.0-µm sections were stained with hematoxylin and eosin and examined for any sign of histological impairment using a light microscope 14.
Data are given as mean ± SD, and OriginPro 7.5 software was used for statistical analyses. A normality test and a homogeneity of variance test were performed for all data in each group. Valid data were subsequently analyzed by one-way analysis of variance (ANOVA; Tukey's test), with treatment as a factor. The nonparametric data (fish mortality) were analyzed using an unpaired χ2 test. The differences were considered significantly or highly significantly different from the control groups when the F values were higher than those indicated in the statistical tables for p < 0.05 and p < 0.01, respectively.
Characterization of AgNPs
Figure 1A illustrates that the AgNPs had spherical shapes and were well dispersed in water. The TEM images also indicate that the AgNPs had a narrow size distribution, with an average size of 29.9 nm (ranging within 25.9–36.7 nm). The DLS measurements show, in agreement with the TEM analysis, that there was a restricted size distribution of the AgNPs, ranging from 50.4 to 63.1 nm. In the present study, a typical optical spectrum for spherical nanosilver with the maximum absorption (λmax) at 400 nm was characterized using the UV–vis analysis (Fig. 1B). The X-ray diffraction pattern of the AgNP powder is shown in Figure 1C. The crystalline nature of the AgNPs was demonstrated by the diffraction peaks, which matched the face-centered cubic phase of Ag. The AgNPs had negative surface charge (zeta potential –43.4 mV), suggesting good stability in water. The specific AgNP surface area was determined to be 6.84 m2/g, using BET analysis.
Stability of AgNP in test solutions
Under the current exposure conditions, the absorption spectra of the AgNPs were measured during the 24-h exposure period. The results indicated that λmax slightly shifted to 404 nm in the exposure media, compared with 400 nm in deionized water; however, the peak position appeared basically unchanged and fluctuated around 404 to 405 nm. Notably, the absorbance of nanosilver at λmax in the test solutions exhibited a time-dependent increase, but not in the control. As measured by Ag-ISE, Ag+ ion release rate was determined as 1.03% of the total Ag in the AgNP suspensions at 15 min after exposure and increased to 6.4% after 24 h.
Dissolved oxygen levels in test solutions
Silver nanoparticles (zero-valent Ag) can be oxidized in an aquatic environment via a mechanism involving O2 and H2O218, which causes depletion of O2 and negatively affects fish survival. Figure 2 shows a time-dependent decrease in DO concentrations in the lower AgNP-treated groups and the control; however, no dose-related change was observed, and all oxygen levels were above the limit for fish survival. On the other hand, no time-related changes in the DO levels were found at the high AgNP concentration, and a weak increase was detected in the 2.4 mg/L group after 24 h of exposure.
In the 96-h acute toxicity test, no death was observed in the control fish. However, the mortality in the 4.8 mg/L AgNP group was 70% after 6 h of treatment and reached 100% after 12 h of exposure. The time- and dose-related increase in mortality is shown in Figure 3. The LC50 values were calculated as 1.38 (95% confidence interval [CI] 0.81–1.57), 1.12 (95% CI 0.34–1.25), and 0.87 (95% CI, 0.23–1.12) mg/L after 48, 72, and 96 h of exposure, respectively.
In the 14-d subchronic toxicity test, approximately 9.4% (3/32) fish died among the controls, whereas no death occurred in the 0.05 and 0.1 mg/L AgNP groups. However, there was no statistical difference between them (χ2= 1.40, p > 0.05). The lethal effect of the AgNPs was determined from 0.25 mg/L, and a significant increase in mortality was found at 0.5 mg/L (χ2 = 7.05, p < 0.01). Based on these results, the no observed effect concentration (NOEC) of mortality was calculated at 0.1 mg/L.
Metal analysis in fish tissues
Measurements of metal uptake were made in 0.25 mg/L group at day 14 of the exposure experiment. All tissues except for the brain exhibited significantly increased total Ag contents compared with the controls (ANOVA, p < 0.05 or p < 0.01; Fig. 4). The maximal metal concentration was observed in the liver, which was approximately 28-fold higher than that in the control.
A dose-dependent decrease in LDH activity in the liver was induced by AgNP exposure, and a significant difference was observed at high AgNP concentrations (0.1 mg/L, p < 0.05; 0.25–0.5 mg/L, p < 0.01) compared with the control (Fig. 5). However, the LDH content in the gills did not exhibit a significant discrepancy between the control and the AgNP-treated groups.
The GPx activity in all samples and CAT level in the gill were below the sensitivity of the assay method used. Figures 6 and 7 show that the hepatic SOD and CAT activities were highest in the control but were progressively inhibited in the AgNP groups, exhibiting a significant dose-dependent response. In contrast, the SOD activity in the gills did not change under the experimental conditions, except for the 0.1 mg/L AgNP treatment.
The GSH levels in the liver, gills, and brains of the AgNP-treated medaka significantly differed from the control values after 14 d of exposure. A dose-dependent GSH depletion was also observed in all tissues (Fig. 8). However, increased MDA concentrations in the liver and gill were observed, signifying dose-related responses (Fig. 9). Although the MDA values did not change in the brain, the concentrations in liver and gill tissues were statistically different from the control when the AgNP concentration was more than 0.25 mg/L.
After exposure to AgNP for 14 d, histological measurements were performed, and the representative images are illustrated in Figure 10. Normal morphology was found in the control, that is, compact liver parenchyma, clear hepatic cords, and centred nuclei in cells. However, adverse histological changes were observed in the AgNP-exposed groups. The low AgNP induced hemocyte overfilling in blood vessels, hepatocyte enlargement, global basophilia, ballooning degeneration, and loosened liver parenchyma. The hepatic impairments became more extensive following AgNP exposure at higher concentrations. These effects included the disorganization of hepatocytes, focal necrosis, focal lymphocytic infiltration, and diffused vacuolated hepotocytes.
Figures 10D and 11A show that the histological section in the control had intact strips of primary lamellae (PLs) and secondary lamellae (SLs), on which epithelial and chloride cells were present. In contrast, AgNP exposure at all concentrations increased the morphological changes. Such changes included lamellar aneurism in PLs, epithelial hyperplasia, increased mucus generation, hematocyst, lamellar fusion, and SL curling (Fig. 11B–J). Additional alterations such as decreased chloride cells, swollen epithelium, and epithelial lifting in PLs were observed compared with the control fish. In the highest AgNP group, branchial lesions became more severe where desquamation of lamellar epithelium and disruption of cartilaginous rod were prominent (Fig. 11K and L).
Ultraviolet-visible spectroscopy allows the detection of the surface plasmon intensity of the spherical AgNPs with a strong resonance peak centered at 400 to 430 nm 17, 22. The surface plasmon resonance depends on the particle size and shape and can be influenced by dielectrics, surface oxidation, and modification of the particle 23. In the present study, UV-vis measurements showed a slight red shift of AgNPs from 400 nm to 404 nm in the test solutions compared with that in deionized water. Generally, the peak absorbance of AgNPs shifted to a longer wavelength is believed to result from their size increase or agglomeration 24, 25. A high level of ionic Ag release in water can also result in the red shift of the UV-vis peak position 26. However, the very weak change in the plasmon peak position during the 24-h exposure also indicated a stable distribution of the particles in the test water. A recent study has reported that the surface plasmon resonance absorbance of AgNPs exhibits a time-dependent decrease in aqueous solution because of particle aggregation 23. However, our results suggested a time-related increase in AgNP absorbance during the exposure period. This discrepancy warrants further study.
Most publications to date have shown that AgNPs can affect aquatic organisms. In these studies, median effective concentration (EC50) values have been used to quantify the ecotoxicity. Recently, the toxicity of AgNPs to Paramecium caudatum was evaluated, and the EC50 was calculated as 39 mg/L 27. Estuarine sediments were dosed with 1.0 mg/L AgNPs for 20 d, but a negligible effect on prokaryotes abundance in the estuary water was found 28. Similarly, 0.3 mg/L AgNPs did not result in mortality to Eurasian perch after 48 h of exposure 10. The 48-h toxicity of AgNPs, whose sizes were equivalent to the AgNPs used in the present study, to adult zebrafish was investigated, and the results demonstrated that the LC50 and NOEC were more than 7.0 mg/L and 1.0 mg/L, respectively 9, 29. A recent study indicated that AgNPs appeared toxic to Pimephales promelas embryos, and the 96-h LC50 values were determined to be more than 9.0 mg/L 30. In contrast to these results, adult medaka in subchronic toxicity tests can be chosen as a more susceptible model in which to evaluate the environmental toxicity of AgNPs.
The current results indicate that the uptake of silver occurs in the gill and intestinal tissues. One previous report has demonstrated that AgNP exposure can induce increased metal burden in zebrafish gill 9. Several mechanisms exist by which AgNP exposure enhances the gill metal contents. For example, NPs can be trapped in the mucus layer of the gill or taken up by the gill epithelial cells. Nanosilver exposure results in drinking and ingestion of the particles into the gastrointestinal tract, leading to the increased levels of Ag in the fish intestine. Notably, the concentration of Ag in the liver was approximately 10 times higher than that in the gills. A recent publication has also suggested that AgNPs enhance the metal levels in the liver of rainbow trout, in which the total Ag concentration is more than double the levels in the gill tissue 31. This might be explained by the different uptake routes and histological diversity between liver and gill tissues. Scown et al. 31 have demonstrated that uptake of AgNPs in the liver is not principally through the gills but rather via the gut, and the gastrointestinal epithelium may be an important route of AgNPs exposure to fish, resulting in the transportation of Ag within the blood from the sites of uptake and accumulation of Ag in liver tissue. The gill surface is surrounded by aqueous media, and NPs may be trapped and retained in the mucus layer. A previous publication has suggested that the mucus layer covering the gill epithelia may act as a barrier, preventing NP uptake by the gills 32. In the present study, AgNP exposure indeed caused epithelial hyperplasia and increased mucus generation, which in turn affected the uptake of AgNPs by the fish gills. The drastic increase in hepatic metal burden suggests that fish liver may be the target of AgNP toxicity.
Lactate dehydrogenase exists ubiquitously in the cell plasma. This enzyme is generally used as an indicator of cell injury and hypoxia. Lactate dehydrogenase activity in an impaired cell decreases because of its leakage into the bloodstream 33. In the present study, the LDH measurement result combined with the metal analysis suggests that AgNP exposure caused drastic metabolic damage and cellular deterioration in the fish liver.
Many studies have shown the role of oxidative stress in AgNP toxicity 31, 34, 35. To prevent oxidative stress, primary antioxidant defense is provided by enzymes such as SOD, CAT, and GPx. In the present study, enzyme GPx, with reference to GSH metabolism, was very low in the samples; however, the dose-dependent decrease in hepatic SOD and CAT activities suggested an excessive consumption of these antioxidant enzymes in the tissue. The reduced SOD levels combined with limited CAT activity ultimately failed to catalyze the transformation of oxyradicals. These data indicate that the ability of antioxidant defense in the medaka liver was significantly depressed and that a potential enhancement of reactive oxygen species (ROS) was produced.
Recently, two studies have revealed that acute AgNP exposure induces oxidative stress to the liver of medaka and zebrafish, by measuring the lipid peroxidation, total GSH levels, and gene expression related to detoxification and radical scavenging action 34, 35. In the present study, the GSH analysis results showed decreasing trends in the liver, gill, and brain of the AgNP-exposed medaka. Glutathione depletion in cells exposed to nanosilver, accompanied by decreased antioxidant enzymes and increased lipid peroxidation, is reported to be highly correlated with increased ROS levels 36. Again, a dose-dependent enhancement in MDA concentration (a biomarker for lipid peroxidation) in AgNP-treated liver and gill was observed in the present study. Taken together, these findings suggest that AgNP exposure resulted in functional damage to and ROS accumulation in tissues, which was in turn associated with oxidative damage. According to the measurements of metal burden and biochemical endpoints in different tissues, a selective toxic effect of the AgNPs on the fish liver was observed.
Histopathology, as one basic technique in aquatic toxicity, provides helpful information for identifying target tissues and the mechanisms of injuries because of the enhanced sensitivity of histological monitoring compared with other toxicological parameters, such as mortality or behavioral measurements. Among the toxicological endpoints in this subchronic test, the histological changes in medaka liver showed more susceptibility to AgNP exposure than mortality and biochemical analysis. It has been reported that there is a strong relation between histological changes and oxidative stress in fish liver 37–39. Medaka hepatic lesions may be associated with high Ag accumulation and oxidative damage in liver tissue based on the metal analysis and biochemical measurements.
In the present study, large numbers of histological alterations, such as mucus production, epithelial hyperplasia, lamellar fusion, and swollen epithelium, were measured in the gills of AgNP-dosed fish. It has been demonstrated that the increased mucus layer and cell hyperplasia may constitute a barrier for NP uptake by the gills 31. The obtained data may explain the low concentrations of Ag in the gills. Fish gills are vital organs for respiration and osmoregulation; for example, chloride cells distributed on PLs are involved in branchial ion exchange. Additionally, fish gills serve as the primary tissue that makes contact with exogenous toxicants in the aquatic environment; thus, some branchial impairments with reference to toxicity may influence oxygen consumption and disrupt osmoregulation. After 24 h of exposure in Eurasian perch (P. fluviatilis), nanosilver reportedly causes increased critical oxygen tension (Pcrit), indicating disrupted gill respiration and impaired tolerance to hypoxia 10. The histopathological changes in the gill tissue of medaka may induce a hypoxic state under which oxidative stress was concomitantly generated.
Silver nanoparticles induced greater toxicity in medaka under chronic exposure conditions than the toxicity in most previously tested aquatic organisms and caused the highest accumulation of Ag in liver tissue. The present study shows that the activities of LDH and antioxidant enzymes, as well as accompanying histological changes in the liver, were significantly influenced by AgNPs in a dose-related manner. The histopathological alterations could be attributed to the oxidative stress induced by AgNP exposure. The results suggest that medaka liver is the organ most affected by the Ag that might have been released from NPs. The data also indicate that histological analysis is a highly sensitive endpoint for evaluating NP toxicity in fish models. Further studies are underway to elucidate the mechanism of Ag accumulation and determine the involvement of free radicals/ROS in NP toxicity.
The present study was jointly supported by the National Natural Science Foundation of China (grant 20537020) and the Scientific Research of Anhui Medical University (grant XJ201016).