| Silver nanoparticles (AgNPs) have taken on wonderful and unique chemo-physical virtues and biological properties that differ from those of their macro scaled or ionic counterparts. These differences have led to ubiquitous applications of silver nanoparticles (AgNPs) in optoelectronics, catalysis system, medical fields and personal healthcare. Presently, nano-silver becomes the most common group of metal engineered nanomaterials in commercial products. The increasing uses of AgNPs in consumer products have increased their release to the environment and, meanwhile, resulted in their exposure to living tissues. This would thus promote special concerns of environmental risks and human health associated with these particles.Some evaluations have been carried out to demonstrate the plausible deleterious effects of nanoscale silver on microorganisms, algae, freshwater fish and rodent animals, however, the information of the AgNPs toxicity and environmental risks remains unclear. Silver nanoparticles that show considerably changed physical, chemical and biological properties increase these complex influences caused by exposure to AgNPs in the environment. In addition, the impossibility of release of ionic silver (Ag+) also should be taken into account to understand the AgNPs toxicity. Present work was performed to decipher in vivo biological activity of Ag nanoparticles on adult medaka fish and embryonic larvae as well as to explain the mechanisms for their toxicity. With a comparative study, we also described the discrepancy between nanoscale silver and ionic silver.The colloidal silver nanoparticles were prepared by reducing a high molar concentration of silver nitrate solution (up to 1 M) with sodium hypophosphite in the presense of polyvinylpyrrolidone 30 (PVP 30). Purified powder of AgNPs was subsequently produced through solvent cleaning, centrifugation and drying in vacuum oven to remove the residual ingredients. We have utilized X-ray diffraction (XRD), UV-vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS) spectrometry to characterize the silver nanoparticles obstained. These data showed spherical shape and face-centered cubic (fcc) phase of AgNPs with shallow size distribution ranged from 20-35 nm, and no agglomeration and oxidation of silver nanoparticles were observed. The experimental AgNPs exhibited a homogeneous dispersion in aqueous solutions. The stable state of these silver nanoparticles should ensure that biological or toxicological activity in medaka model may specially attribute to nanoparticles rather other components and chemical mixtures.In the acute toxicity tests, we found that the UV-vis spectrum pattern of AgNPs showed unchanged and the dissolved oxygen (DO) displayed consistency during 24 h AgNPs exposure. The caculated 96-h LC50 was at 0.87 mg/L, based on the mortality of adult medaka exposed to different concentration of nanoscale silver. In sublethal toxicity tests, adult medaka was treated with 0-0.5 mg/L AgNPs for consecutive 14 days to investigate organ distribution. Meanwhile, biochemical assessment and histopathological analysis were performed to evaluate possible harmful effects in terms of antioxidant enzymes, lipid peroxidation and histopathology. Highest concentration of AgNPs significantly increased mortality of medaka to 37.5% during the exposure (X2=7.05, P＜0.01), though no statistic difference was found between low dose groups and control group. It's showed that AgNPs exposure enhanced the metal burden in gill, intestine and, specially, in liver. Dose-dependent decreases of Lactate dehydrogenase (LDH), Catalase (CAT) and superoxide dismutase (SOD) acitivities and total reduced GSH content were observed in liver, gill or brain of medaka exposed to Ag nanoparticles, however, increase of malondialdehyde (MDA) was induced in the AgNPs treated medaka-, suggesting that oxidative damage was induced by nano-Ag. Histopathological assay also revealed severe damages in the gill and liver. The results indicated liver of medaka could be the target organ of AgNPs toxicity.Using Japanese medaka (Oryzias latipes) at early-life stages as experimental models, the developmental toxicity of silver nanoparticles was investigated following exposure to 100-1000μg/L homogeneously dispersed AgNPs for 70 days, and developmental endpoints were evaluated by microscopy during embryonic, larval and juvenile stages of development in medaka. Meanwhile, histopathological changes in the larval eye were evaluated. Retarded development and reduced pigmentation were observed in the treated embryos by AgNPs at high concentrations (≥400 ug/L). Maximum width of the optic tectum, as an indicator of midbrain development, decreased significantly in a dose-related manner. Furthermore, silver nanoparticles exposure at all concentrations induced a variety of morphological malformations such as edema, spinal abnormalities, finfold abnormalities, heart malformations and eye defects. Histopathological observations also confirmed the occurrence of abnormal eye development induced by AgNPs. The data showed non-linear or U-shaped dose-response patterns for growth retardation at 5 days of postfertilization, as well as the incidence of abnormalities. Preliminary results suggested that the developmental process of medaka may be affected by exposure to silver nanoparticles. Morphological abnormalities in early-life stages of medaka showed the potential developmental toxicities of silver nanoparticles. Further research should be focused on the mechanisms of developmental toxicity in fish exposed to silver nanoparticles.Using light microscopy and scanning electron microscopy (SEM), the results indicated that AgNPs treatments at higher concentration (≥125μg/L) may severely destroy the surface ornamentation and egg envelope. Meanwhile, significant increase in lactate dehydrogenase (LDH) activity, an indicator of anaerobic metabolism, indicated hypoxia in treated groups. Biochemical changes in SOD activity, reduced GSH level, TRARS concentration showed oxidative stress caused by silver nanoparticles in early-stage medaka embryos. Unexpectedly, a weak dose-dependent reduction in levels of ROS and singlet oxygen by high AgNPs exposure (≥250μg/L) as observed. We also noted that responses by antioxidant defenses in well-developed embryos were elevated, but the developmental damages caused by silver nanoparticles showed no recovery. Overall, it suggested that the disturbed egg chorion, hypoxia and oxidative stress were mechanistically associated to AgNPs toxicity in embryonic fish.Ionic silver may be easily released from silver nanoparticles whose surface oxidation occurred in water, medium, and specially, under biological conditions. Toxic effects of AgNPs may be related to interactions of Ag+ ions with proteins and enzymes. As measured by Ag-ISE (Ion-selective electrode), maximum free Ag concentrations present in the fresh AgNPs suspensions were 0.94%-1.01%, and the dissolved silver was time-dependent increased with non-linear pattern for 24 h. We have examined the sub-chronic toxicity of silver nanoparticles and Ag+ to adult medaka by evaluating the distribution, mortality of tested fish, oxidative stress endpoints and histopathological changes. When compared as a function of the free ionic silver levels, Ag+(AgNP3) resulted in much higher toxicity such as mortality and oxidative damages than that of AgNPs.
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