Critical influence of chloride ions on silver ion-mediated acute toxicity of silver nanoparticles to zebrafish embryos

Abstract

The toxicity of silver nanoparticles (AgNP) to aquatic organisms, including zebrafish (Danio rerio), has been demonstrated, but differing opinions exist on the contribution of the physical properties of the particles themselves and the free dissolved silver ions (Ag⁺) to the observed effects. High concentrations of chloride ions (Cl^?) in the routinely used exposure media can cause precipitation of Ag⁺ as AgCl, as well as complexation of silver in diverse soluble chlorocomplexes, thus masking the contribution of dissolved silver to AgNP toxicity. In the present study, we formulated a zebrafish exposure medium with a low chloride content and exposed zebrafish embryos to AgNO₃ or carbonate-coated AgNP. The severity of toxicity caused by both silver forms depended on the time of exposure start, with younger embryos being most sensitive. Toxicity caused by both AgNO₃ and AgNP was of the same order of magnitude when compared based on the total dissolved silver concentration and could be prevented by addition of the Ag⁺ chelator cysteine. Further, we have analyzed the data from several previous studies to evaluate the influence of interactions between Ag⁺ and Cl^? on silver toxicity to zebrafish embryos. Our analysis demonstrates that the acute toxicity of AgNP to zebrafish embryos is largely mediated by Ag⁺. The influence of particle size and coating can at least partially be explained by the differences in Ag⁺ dissolution. High Cl^? levels in the exposure medium indeed have a pivotal influence on the resulting toxicity of AgNP, appearing to significantly attenuate toxicity in several studies. This consideration should influence the choice of exposure medium to be used when evaluating and comparing AgNP toxicity.     

Cellular uptake and toxicity effects of silver nanoparticles in mammalian kidney cells

The rapid progress and early commercial acceptance of silver‐based nanomaterials is owed to their biocidal activity. Besides embracing the antimicrobial potential of silver nanoparticles (AgNPs), it is imperative to give special attention to the potential adverse health effects of nanoparticles owing to prolonged exposure. Here, we report a detailed study on the in vitro interactions of citrate‐coated AgNPs with porcine kidney (Pk15) cells. As uncertainty remains whether biological/cellular responses to AgNPs are solely as a result of the release of silver ions or whether the AgNPs themselves have toxic effects, we investigated the effects of Ag⁺ on Pk15 cells for comparison. Next, we investigated the cellular uptake of both AgNPs and Ag⁺ in Pk15 cells at various concentrations applied. The detected Ag contents in cells exposed to 50 mg l^?1 AgNPs and 50 mg l^?1 Ag⁺ were 209 and 25 µg of Ag per 10⁶ cells, respectively. Transmission electron microscopy (TEM) images indicated that the Pk15 cells internalized AgNPs by endocytosis. Both forms of silver, nano and ionic, decreased the number of viable Pk15 cells after 24 h in a dose‐dependent manner. In spite of a significant uptake into the cells, AgNPs had only insignificant toxicity at concentrations lower than 25 mg l^?1, whereas Ag⁺ exhibited a significant decrease in cell viability at one‐fifth of this concentration. The Comet assay suggested that a rather high concentration of AgNP (above 25 mg l^?1) is able to induce genotoxicity in Pk15 cells. Further studies must seek deeper understanding of AgNP behavior in biological media and their interactions with cellular membranes. Copyright © 2014 John Wiley & Sons, Ltd.     

Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells

Cytotoxicity induced by silver nanoparticles (AgNPs) and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells. Toxicity induced by silver (Ag⁺) ions was studied in parallel using AgNO₃ as the Ag⁺ ion source. Using cation exchange treatment, we confirmed that the AgNP solution contained a negligible amount of free Ag⁺ ions. Metal-responsive metallothionein 1b (MT1b) mRNA expression was not induced in AgNP-treated cells, while it was induced in AgNO₃-treated cells. These results indicate that AgNP-treated cells have limited exposure to Ag⁺ ions, despite the potential release of Ag⁺ ions from AgNPs in cell culture. AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and induced intracellular oxidative stress. AgNPs exhibited cytotoxicity with a potency comparable to that of Ag⁺ ions in in vitro cytotoxicity assays. However, the toxicity of AgNPs was prevented by use of the antioxidant N-acetylcysteine, and AgNP-induced DNA damage was also prevented by N-acetylcysteine. AgNO₃ treatment induced oxidative stress-related glutathione peroxidase 1 (GPx1) and catalase expression to a greater extent than AgNP exposure, but treatment with AgNO₃ and AgNPs induced comparable superoxide dismutase 1 (SOD1) expression levels. Our findings suggest that AgNP cytotoxicity is primarily the result of oxidative stress and is independent of the toxicity of Ag⁺ ions.     

Silver nanoparticles alter zebrafish development and larval behavior: distinct roles for particle size, coating and composition

Silver nanoparticles (AgNPs) act as antibacterials by releasing monovalent silver (Ag⁺) and are increasingly used in consumer products, thus elevating exposures in human and wildlife populations. In vitro models indicate that AgNPs are likely to be developmental neurotoxicants with actions distinct from those of Ag⁺. We exposed developing zebrafish (Danio rerio) to Ag⁺ or AgNPs on days 0-5 post-fertilization and evaluated hatching, morphology, survival and swim bladder inflation. Larval swimming behavior and responses to different lighting conditions were assessed 24 h after the termination of exposure. Comparisons were made with AgNPs of different sizes and coatings: 10 nm citrate-coated AgNP (AgNP-C), and 10 or 50 nm polyvinylpyrrolidone-coated AgNPs (AgNP-PVP). Ag⁺ and AgNP-C delayed hatching to a similar extent but Ag⁺ was more effective in slowing swim bladder inflation, and elicited greater dysmorphology and mortality. In behavioral assessments, Ag⁺ exposed fish were hyperresponsive to light changes, whereas AgNP-C exposed fish showed normal responses. Neither of the AgNP-PVPs affected survival or morphology but both evoked significant changes in swimming responses to light in ways that were distinct from Ag⁺ and each other. The smaller AgNP-PVP caused overall hypoactivity whereas the larger caused hyperactivity. AgNPs are less potent than Ag⁺ with respect to dysmorphology and loss of viability, but nevertheless produce neurobehavioral effects that highly depend on particle coating and size, rather than just reflecting the release of Ag⁺. Different AgNP formulations are thus likely to produce distinct patterns of developmental neurotoxicity.     

Teratogenic hazard of BPEI-coated silver nanoparticles to Xenopus laevis

Silver nanoparticles (AgNPs) are among the most exploited antimicrobial agents and are used in many consumer products. Size and surface reactivity are critical physico-chemical properties responsible for NPs toxicity, and surface coatings, often used to functionalize or stabilize AgNPs, can influence their toxic profile and biocompatibility. In the current study the developmental toxicity of (1) negatively charged citrate-coated AgNPs (Cit-AgNPs), (2) positively charged branched polyethylenimine-coated AgNPs (BPEI-AgNPs), and (3) Ag⁺ (from 0.0625 to 0.75?mg Ag/L) was investigated by the standard Frog Embryo Teratogenesis Assay – Xenopus (FETAX). In order to identify the most sensitive developmental phase, embryos were also exposed during different embryonic stages. Morphological and bio-physical studies were performed to characterize tissue lesions and NP uptake. The results suggest that Ag⁺ was strongly embryo-lethal. Contrary to Cit-AgNPs, the positively charged BPEI-AgNPs exert a concentration-dependent effect on lethality and malformations of embryos. The BPEI-AgNPs showed the highest teratogenic index (TI?=?1.6), pointing out the role of functional coating in determining the developmental hazard. The highest susceptibility to BPEI-AgNPs was during early embryogenesis, when embryos are still enclosed in the fertilization envelope, and the post-stomodeum opening stages, when NPs ingestion occurs. In BPEI-AgNPs treated larvae, the histological examination revealed irregular intestinal diverticula coupled with edematous connective tissue. Small NPs aggregates are mapped throughout the intestinal mucosa and secondary target organs by two-photon excitation microscopy. We conclude that a teratogenic risk may be associated with BPEI-AgNPs exposure, but the modality of NP-tissue interactions and the teratogenic mechanism need further investigations to be better defined.     

Phosphorus fertilization and mycorrhizal colonization change silver nanoparticle impacts on maize

Environmental risks of silver (Ag) nanoparticles (NPs) have aroused considerable concern, however, their ecotoxicity in soil-plant systems has yet not been well elaborated, particularly in agroecosystems with various fertility levels and soil biota. The aims of the present study were to determine AgNPs impacts on maize as influenced by mycorrhizal inoculation and P fertilization. A greenhouse pot experiment was conducted determine the effects of mycorrhizal inoculation with Rhizophagus intraradices and P fertilization (0, 20, and 50 P mg/kg soil, as Ca(H₂PO₄)₂) on plant growth, Ag accumulation and physiological responses of maize exposed to AgNPs (1 mg/kg), or an equivalent Ag⁺. Overall, AgNPs and Ag⁺ did not significantly affect plant biomass and acquisition of mineral nutrients, activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), chlorophyll contents and photosystem (PS) II photochemical efficiency. In most cases, AgNPs and Ag⁺ caused similar Ag accumulation in plant tissues. P fertilization significantly increased Ag bioavailability and plant Ag accumulation, but only promoted the growth and P uptake of nonmycorrhizal plants. AM inoculation produced positive impacts on plant biomass, nutritional and physiological responses, but slightly affected extractable Ag in soil and Ag accumulation in plants. Mycorrhizal responses in plant growth and P uptake were more pronounced in the treatments without P but with Ag. By and large, AgNPs exhibited similar phytoavailability, phytoaccumulation and low phytotoxicity compared to Ag⁺, but higher fungitoxicity (i.e., lower root colonization). In conclusion, both AM inoculation and P fertilization can improve plant performance in the soil exposed to Ag, but P increases environmental risk of Ag. Our results indicate a beneficial role of arbuscular mycorrhizal fungi but a dual role of P in soil-plant systems exposed to AgNPs or Ag⁺.

     

Differentiation of the toxicities of silver nanoparticles and silver ions to the Japanese medaka (Oryzias latipes) and the cladoceran Daphnia magna

Abstract

Silver nanoparticles (AgNPs) are increasingly used in various fields. However, little is known about the environmental effects of widespread use of products containing AgNPs. The objective of this study was to determine the ecotoxicity caused by AgNPs. The 48-h effective concentration 50 (EC₅₀) values for Daphnia magna of suspensions of 60 nm and 300 nm AgNPs were 1.0 (95% confidence interval [CI] = 0.1–1.3) and 1.4 (95% CI = 0.3–2.1) μg Ag/l, respectively. The 96 h LC₅₀ values for Oryzias latipes of 60 nm and 300 nm AgNP suspensions were 28 (95% CI = 23–34) and 67 (95% CI = 45–108) μg Ag/l, respectively. To show that toxicity is caused only by Ag⁺ and not by AgNPs, Ag⁺ was adsorbed onto the synthesized sorbents packed in a column and D. magna was exposed to the column-passed-300 nm AgNP suspensions. There was no acute toxicity with the AgNP suspensions not containing Ag⁺.     

Both released silver ions and particulate Ag contribute to the toxicity of AgNPs to earthworm Eisenia fetida

Abstract

To disentangle the contribution of ionic and nanoparticulate Ag to the overall toxicity to the earthworm Eisenia fetida, a semi-permeable membrane strategy was used to separate Ag⁺ released from silver nanoparticles (AgNPs) in an aqueous exposure. Internal Ag fractionation, activities of antioxidant enzymes and metabolites in E. fetida were determined after 96?h of exposure to two sizes of polyvinylpyrrolidone-coated AgNPs. The response of the antioxidant system combined with the content of malondialdehyde indicated that the Ag⁺ released from AgNPs induced significant oxidative stress to the earthworms. Ag accumulated from AgNPs was predominantly associated with the granules and cell membrane compartments, whereas dissolved Ag was localized in the cytosol-containing fraction. In both Ag⁺ exposures, two intermediates in the Krebs cycle, succinate and fumarate, were significantly elevated and depleted, respectively. A similar alteration pattern was seen in groups exposed to both smaller AgNPs (S AgNP, 10?nm) and larger AgNP (L AgNP, 40?nm), indicating that these effects in E. fetida were induced by exposure to released Ag⁺. In addition, unique metabolic responses including decreased malate and glucose levels in S AgNP-exposed earthworms could be associated with exposure to nanoparticulate silver. Increased leucine and arginine and decreased ATP and inosine levels were observed in L AgNP exposures only, which clearly demonstrated a size-specific effect of AgNPs. Collectively, this study provided strong evidence that nanosilver acts by a different mechanism than ionic silver to cause acute toxicity to E. fetida, but further verification under different environmental conditions is needed.     

Comparison of acute to chronic ratios between silver and gold nanoparticles,using Ceriodaphnia dubia

As integration of nanoparticles (NPs) into products becomes more common, the need to address the paucity of chronic hazard information for aquatic environments required to determine risk potential increases. This study generated acute and chronic toxicity reference values for Ceriodaphnia dubia exposed to 20 and 100?nm silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) to generate and evaluate potential differences in acute-to-chronic ratios (ACR) using two different feeding methods. A modified feeding procedure was employed alongside the standard procedures to investigate the influence of food on organism exposure. An 8-h period before food was added allowed direct organism exposure to NP dispersions (and associated ions) without food-to-NP interactions. The AgNPs [chronic lethal median concentrations (LC50) between 18.7 and 31.9?µg/L] were substantially more toxic than AuNPs (LC50?=?21 507 to >26 384?µg/L). The modified chronic testing method resulted in greater sensitivity in AgNPs exposures. However, the modified feeding ration had less of an effect in exposures to the larger (100?nm) AgNPs compared to smaller particles (20?nm). The ACRs for AgNPs using the standard feeding ration were 1.6 and 3.5 for 20?nm and 100?nm, respectively. The ACRs for AgNPs using the modified feeding ration were 3.4 and 7.6 for 20?nm and 100?nm NPs, respectively. This supports that the addition of the standard feeding ration decreases C. dubia chronic sensitivity to AgNPs, although it must also be recognized organisms may be sensitized due to less access to food. The ACRs for 20?nm and 100?nm AuNPs (standard ration only) were 4.0 and 3.0, respectively. It is important to also consider that dissolved Ag⁺ ions are more toxic than AgNPs, based on both acute toxicity values in the cited literature and chronic toxicity thresholds generated in this study that support existing thresholds that Ag⁺ are likely protective of AgNPs effects.     

Time-resolved toxicity study reveals the dynamic interactions between uncoated silver nanoparticles and bacteria

It is still unclear whether the toxicity of silver nanoparticles (AgNPs) can be attributed solely to the release of Ag⁺ or whether dissolved and nanoparticulate Ag act in parallel; this is due to the difficulty in distinguishing Ag⁺- from AgNP-effects. Also, AgNPs undergo changes during toxicity tests. This is the first study to investigate the influence of AgNP dissolution over time on viable counts at high time resolution and low cell density, avoiding the apparently reduced toxicity at higher cell densities identified in our study. Uncapped AgNPs were synthesized to avoid any interference from surface coatings. The transformations of AgNPs during storage were reduced. Lowering the concentration of AgNPs reduced their aggregation in Davis minimal medium (DMM). Also, AgNPs dissolved more slowly in DMM than in water. The minimum inhibitory concentrations (MICs) of Ag⁺ and AgNPs increased with cell density according to a power law, suggesting that binding to cells decreased effective concentrations. However, AgNPs acted as a reservoir of Ag, releasing new Ag⁺ to maintain the Ag stress. The toxicity of AgNPs was dominated by dissolved Ag. Combining controlled conditions, high time-resolution and low cell density, we could demonstrate different roles of ionic and nano Ag in bacterial death caused by AgNPs.     

Amino acid-dependent transformations of citrate-coated silver nanoparticles: Impact on morphology,stability and toxicity

Humans face the risk of exposure to silver nanoparticles (AgNPs) due to their extensive application in consumer products. AgNPs can interact with many substances in the human body due to their chemically unstable nature and high activity properties, which might result in unknown hazards and even some serious diseases for humans. As the basic constituent element of human bodies, amino acids (AAs) differ in concentration and variety in different cells and tissues. Thus, understanding the transformation of citrate-coated AgNPs in the presence of AAs is crucial for determining their fate and toxicity in the human body. Our study focused on the transformation of the morphology, dissolution behavior and reaction product of AgNPs in different AA-containing systems and then evaluated the effect of these transformations on the cytotoxicity of AgNPs. The obtained results indicated that the addition of glycine with the lowest Ag⁺ binding energy had little effect on the transformations and toxicity of AgNPs. While in the presence of histidine with higher Ag⁺ binding energy, the Ag⁺ release and particle size of AgNPs obviously increased. These transformations resulted in a decrease in the cytotoxicity of AgNPs due to the formation of Ag–His complex and the growth of AgNPs. Furthermore, l-cysteine with the highest Ag⁺ binding energy could easily interact with AgNPs, transforming them completely to form [Ag(Cys)_n]⁺ and Ag₂S precipitates, which induced the largest decrease in AgNP toxicity. In summary, our results may provide useful information to understand the fate, transformation, and toxicity of citrate-coated AgNPs in the human body.     

Silver nanoparticle-specific mitotoxicity in Daphnia magna

Silver nanoparticles (Ag NPs) are gaining popularity as bactericidal agents in commercial products; however, the mechanisms of toxicity (MOT) of Ag NPs to other organisms are not fully understood. It is the goal of this research to determine differences in MOT induced by ionic Ag⁺ and Ag NPs in Daphnia magna, by incorporating a battery of traditional and novel methods. Daphnia embryos were exposed to sublethal concentrations of AgNO₃ and Ag NPs (130–650 ng/L), with uptake of the latter confirmed by confocal reflectance microscopy. Mitochondrial function was non-invasively monitored by measuring proton flux using self-referencing microsensors. Proton flux measurements revealed that while both forms of silver significantly affected proton efflux, the change induced by Ag NPs was greater than that of Ag⁺. This could be correlated with the effects of Ag NPs on mitochondrial dysfunction, as determined by confocal fluorescence microscopy and JC-1, an indicator of mitochondrial permeability. However, Ag⁺ was more efficient than Ag NPs at displacing Na⁺ within embryonic Daphnia, based on inductively coupled plasma-mass spectroscopy (ICP-MS) analysis. The abnormalities in mitochondrial activity for Ag NP-exposed organisms suggest a nanoparticle-specific MOT, distinct from that induced by Ag ions. We propose that the MOT of each form of silver are complementary, and can act in synergy to produce a greater toxic response overall.     

Toxic effects of colloidal nanosilver in zebrafish embryos

A variety of consumer products containing silver nanoparticles (Ag NPs) are currently marketed. However, their safety for humans and for the environment has not yet been established and no standard method to assess their toxicity is currently available. The objective of this work was to develop an effective method to test Ag NP toxicity and to evaluate the effects of ion release and Ag NP size on a vertebrate model. To this aim, the zebrafish animal model was exposed to a solution of commercial nanosilver. While the exposure of embryos still surrounded by the chorion did not allow a definite estimation of the toxic effects exerted by the compound, the exposure for 48 h of 3‐day‐old zebrafish hatched embryos afforded a reliable evaluation of the effects of Ag NPs. The effects of the exposure were detected especially at molecular level; in fact, some selected genes expressed differentially after the exposure. The Ag NP toxic performance was due to the combined effect of Ag⁺ ion release and Ag NP size. However, the effect of NP size was particularly detectable at the lowest concentration of nanosilver tested (0.01 mg l^–1) and depended on the solubilization media. The results obtained indicate that in vivo toxicity studies of nanosilver should be performed with ad hoc methods (in this case using hatched embryos) that might be different depending on the type of nanosilver. Moreover, the addition of this compound to commercial products should take into consideration the Ag NP solubilization media. Copyright © 2014 John Wiley & Sons, Ltd.     

Differential transformation and antibacterial effects of silver nanoparticles in aerobic and anaerobic environment

Silver nanoparticles (AgNP) undergo various transformations into different Ag species in the environment, which determines their toxicity in microorganisms. In aerobic condition, AgNPs release Ag⁺ that causes cell inactivation. Limited information is known about the AgNP-cell interaction in oxygen-free environment. Here we compared the transformation and antibacterial effects of AgNPs in aerobic and anaerobic environment. The bacterium Pseudomonas aeruginosa was relatively not susceptible to Ag⁺ or AgNP in anaerobic environment, indicated by near two orders of magnitude greater of anaerobic minimum inhibitory concentration (MIC) than the aerobic counterpart. In anaerobic environment, the dissolved Ag concentration decreased due to the reduction of Ag⁺. Electron microscopy images showed the formation of new AgNPs and aggregates, preferably on cell surface or associated with extracellular polymer substances (EPS) matrix. Accumulating AgNPs onto the cells could cause membrane damage, cytoplasm release or bacterial death. Meanwhile, EPS and cell lysate were very likely to bind AgNPs, facilitating the extensively assembling of AgNPs into large aggregates. This reduced the effective Ag exposure to cells and might contribute to the detoxification in anaerobic environment. Further, flow cytometry analysis quantified that bacterial membrane was largely intact under the treatment of AgNPs in anaerobic condition compared to the dose–response manner in aerobic condition.     

Acetyl L‐carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment

Verapamil is a Ca²⁺ channel blocker and is highly prescribed as an anti‐anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca²⁺‐permeable N‐methyl‐d ‐aspartate‐type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl l ‐carnitine (ALCAR) reverses ketamine‐induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post‐fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo . In 48 h post‐fertilization embryos, 2 mm ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mm ALCAR neutralized this effect. ALCAR could reverse ketamine's effect, possibly through a compensatory mechanism involving extracellular Ca²⁺ entry through L‐type Ca²⁺ channels that ALCAR is known to activate. Hence, we used verapamil to block the L‐type Ca²⁺ channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)octyl ester, an inhibitor of intracellular Ca²⁺ release suggesting that ALCAR acts via effectors downstream of Ca²⁺. In fact, ALCAR's protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca²⁺ during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine‐ and verapamil‐induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

The acute toxic effects of silver nanoparticles on myocardial transmembrane potential,INa and IK1 channels and heart rhythm in mice

This study focused on the potential toxicity of silver nanoparticles (AgNPs) on cardiac electrophysiology which is rarely investigated. We found that AgNPs (10^?9–10^?6?g/ml) concentration-dependently depolarized the resting potential, diminished the action potential, and finally led to loss of excitability in mice cardiac papillary muscle cells in vitro. In cultured neonatal mice cardiomyocytes, AgNPs (10^?9–10^?7?g/ml) concentration-dependently decreased the Na⁺ currents (I_Na), accelerated the activation, and delayed the inactivation and recovery of Na⁺ channels from inactivation within 5?min. AgNPs at 10^?8?g/ml also rapidly decreased the inwardly rectifying K⁺ currents (I_K1) and delayed the activation of I_K1 channels. Intravenous injection of AgNPs at 3?mg/kg only decreased the heart rate, while at ≥4?mg/kg sequentially induced sinus bradycardia, complete atrio-ventricular conduction block, and cardiac asystole. AgNPs at 10^?10–10^?6?g/ml did not increase reactive oxygen species (ROS) generation and only at 10^?6?g/ml mildly induced lactate dehydrogenase (LDH) release in the cardiomyocytes within 5?min. Endocytosis of AgNPs by cardiomyocytes was not observed within 5?min, but was observed 1?h after exposing to AgNPs. Comparative Ag⁺ (≤0.02% of the AgNPs) could not induce above toxicities. We conclude that AgNPs exert rapid toxic effects on myocardial electrophysiology and induce lethal bradyarrhythmias. These acute toxicities are likely due to direct effects of AgNPs on ion channels at the nano-scale level, but not caused by Ag⁺, ROS, and membrane injury. These findings provide warning to the nanomedical practice using AgNPs.     

Cellular internalization and intracellular biotransformation of silver nanoparticles in Chlamydomonas reinhardtii

It is necessary to elucidate cellular internalization and intracellular biotransformation in order to accurately assess the toxicity and fate of nanoparticles after interaction with organisms. Therefore, this work employed a combination of high resolution imaging and in situ detection spectroscopic techniques to systematically investigate the intracellular localization, morphology and chemical speciation of silver in the cells of Chlamydomonas reinhardtii, a unicellular freshwater green alga, after exposure to AgNPs coated with polyvinylpyrrolidone at a concentration of 2.0?mg/L. High resolution secondary ion mass spectrometry and high-angle annular dark field scanning transmission electron microscopy together with energy dispersive spectroscopy and selected area electron diffraction collectively confirmed that after 48?h of exposure, AgNPs entered the periplasmic space after cellular internalization into the algal cells. Silver was also found to coexist with sulfur inside the cytoplasm in both crystalline and amorphous forms, which were further identified as β-Ag₂S and silver thiolates with synchrotron X-ray absorption spectroscopy. In combination, these analyses demonstrated that silver inside algae could be attributed to the uptake and sequestration of Ag⁺?ion released from AgNPs, which was further sequestrated into cellular compartments. This study provides solid evidence for particle internalization and biotransformation of AgNPs after interaction with algae.     

Developmental toxicity,EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin‐like PCB126

Dioxin‐like PCB126 is a persistent organic pollutant that causes a range of syndromes including developmental toxicity. Dioxins have a high affinity for aryl hydrocarbon receptor (AhR) and induce cytochrome P4501A (CYP1A). However, the role of CYP1A activity in developmental toxicity is less clear. To better understand dioxin induced developmental toxicity, we exposed zebrafish (Danio rerio) embryos to PCB126 at concentrations of 0, 16, 32, 64, and 128 μg L^?1 from 3‐h post‐fertilization (hpf) to 168 hpf. The embryonic survival rate decreased at 144 and 168 hpf. The fry at 96 hpf displayed gross developmental malformations, including pericardial and yolk sac edema, spinal curvature, abnormal lower jaw growth, and non‐inflated swim bladder. The pericardial and yolk sac edema rate significantly increased and the heart rate declined from 96 hpf compared with the controls. PCB126 did not alter the hatching rate. To elucidate the mechanism of PCB126‐induced developmental toxicity, we conducted ethoxyresorufin‐O‐deethylase (EROD) in vivo assay to determine CYP1A enzyme activity, and real‐time PCR to study the induction of CYP1A mRNA gene expression in embryo/larval zebrafish at 24, 72, 96, and 132 hpf. In vivo EROD activity was induced by PCB126 at 16 μg L^?1 concentration as early as 72 hpf but significant increases were observed only in zebrafish exposed to 64 and 128 μg L^?1 doses (p < 0.005) at 72, 96, and 132 hpf. Induction of CYP1A mRNA expression was significantly upregulated in zebrafish exposed to 32 and 64 μg L^?1 at 24, 72, 96, and 132 hpf. Overall, the severe pericardial and yolk sac edema and reduced heart rate suggest that heart defects are a sensitive endpoint, and the general trend of dose‐dependent increase in EROD activity and induction of CYP1A mRNA gene expression provide evidence that the developmental toxicity of PCB126 to zebrafish embryos is mediated by activation of AhR. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 201–210, 2016.     

Silver exposure in developing zebrafish (Danio rerio): Persistent effects on larval behavior and survival

The increased use of silver nanoparticles in consumer and medical products has led to elevated human and environmental exposures. Silver nanoparticles act as antibacterial/antifungal agents by releasing Ag⁺ and recent studies show that Ag⁺ impairs neural cell replication and differentiation in culture, suggesting that in vivo exposures could compromise neurodevelopment. To determine whether Ag⁺ impairs development in vivo, we examined the effects of exposure on survival, morphological, and behavioral parameters in zebrafish embryos and larvae. We exposed zebrafish from 0 to 5 days post-fertilization to concentrations of Ag⁺ ranging from 10 nM to 100 µM in order to assess effects on survival and early embryonic development. We then tested whether concentrations below the threshold for dysmorphology altered larval behavior and subsequent survival. Ag⁺ concentrations ≥ 3 µM significantly reduced embryonic survival, whereas 1 µM delayed hatching with no effect on survival. Reducing the concentration to as low as 0.1 µM delayed the inflation of the swim bladder without causing gross dysmorphology or affecting hatching. At this concentration, swimming activity was impaired, an effect that persisted past the point where swim bladder inflation became normal; in contrast, general motor function was unaffected. The early behavioral impairment was then predictive of subsequent decreases in survival. Ag⁺ is a developmental toxicant at concentrations only slightly above allowable levels. At low concentrations, Ag⁺ acts as a neurobehavioral toxicant even in the absence of dysmorphology.     

Toxic effects of indoxacarb enantiomers on the embryonic development and induction of apoptosis in zebrafish larvae (Danio rerio)

Indoxacarb is a highly potent insecticide widely used to control Lepidoptera insects in vegetable, tea, cotton, and rice fields. It can run off into aquatic environments. It is consisted of two enantiomers. Environmental risks and aquatic toxicity of indoxacarb enantiomers have not been fully investigated. In this study, zebrafish (Danio rerio ) embryos were exposed to varying concentrations of (?)‐R‐indoxacarb and (+)‐S‐indoxacarb until 96‐h post‐fertilization (hpf) to assess the embryonic toxicity. (?)‐R‐indoxacarb was 1.3‐fold more toxic than (+)‐S‐isomer to zebrafish embryos at 96 hpf. (?)‐R‐indoxacarb exhibited reduction in body length and pericardial edema compared with (+)‐S‐indoxacarb. (?)‐R‐indoxacarb decreased the hatching rate sixfold greater than (+)‐S‐indoxacarb. The rate of pericardial edema induced by (?)‐R‐indoxacarb was 2.5 times greater than that by (+)‐S‐indoxacarb. The heart rate of the larvae exposed to (?)‐R‐indoxacarb was 30% lower than that to (+)‐S‐indoxacarb. In addition, exposure to the chiral isomers resulted in significant increases in apoptosis; interestingly (?)‐R‐indoxacarb induced apoptosis in the heart area, whereas (+)‐S‐indoxacarb induced apoptosis in the head area. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 7–16, 2017.