The Occurrence of Oxidative Stress Induced by Silver Nanoparticles in Chlorella vulgaris Depends on the Surface-Stabilizing Agent.

Silver nanoparticles (AgNPs) are of great interest due to their antimicrobial properties, but their reactivity and toxicity pose a significant risk to aquatic ecosystems. In biological systems, AgNPs tend to aggregate and dissolve, so they are often stabilized by agents that affect their physicochemical properties. In this study, microalga Chlorella vulgaris was used as a model organism to evaluate the effects of AgNPs in aquatic habitats. Algae were exposed to AgNPs stabilized with citrate and cetyltrimethylammonium bromide (CTAB) agents and to AgNO3 at concentrations that allowed 75% cell survival after 72 h. To investigate algal response, silver accumulation, ROS content, damage to biomolecules (lipids, proteins, and DNA), activity of antioxidant enzymes (APX, PPX, CAT, SOD), content of non-enzymatic antioxidants (proline and GSH), and changes in ultrastructure were analyzed. The results showed that all treatments induced oxidative stress and adversely affected algal cells. AgNO3 resulted in the fastest death of algae compared to both AgNPs, but the extent of oxidative damage and antioxidant enzymatic defense was similar to AgNP-citrate. Furthermore, AgNP-CTAB showed the least toxic effect and caused the least oxidative damage. These results highlight the importance of surface-stabilizing agents in determining the phytotoxicity of AgNPs and the underlying mechanisms affecting aquatic organisms.

Particle Size Modulates Silver Nanoparticle Toxicity during Embryogenesis of Urchins Arbacia lixula and Paracentrotus lividus.

Silver nanoparticles represent a threat to biota and have been shown to cause harm through a number of mechanisms, using a wide range of bioassay endpoints. While nanoparticle concentration has been primarily considered, comparison of studies that have used differently sized nanoparticles indicate that nanoparticle diameter may be an important factor that impacts negative outcomes. In considering this, the aim of the present study was to determine if different sizes of silver nanoparticles (AgNPs; 10, 20, 40, 60 and 100 nm) give rise to similar effects during embryogenesis of Mediterranean sea urchins Arbacia lixula and Paracentrotus lividus, or if nanoparticle size is a parameter that can modulate embryotoxicity and spermiotoxicity in these species. Fertilised embryos were exposed to a range of AgNP concentrations (1−1000 µg L−1) and after 48 h larvae were scored. Embryos exposed to 1 and 10 µg L−1 AgNPs (for all tested sizes) showed no negative effect in both sea urchins. The smaller AgNPs (size 10 and 20 nm) caused a decrease in the percentage of normally developed A. lixula larvae at concentrations ≥50 µg L−1 (EC50: 49 and 75 µg L−1, respectively) and at ≥100 µg L−1 (EC50: 67 and 91 µg L−1, respectively) for P. lividus. AgNPs of 40 nm diameter was less harmful in both species ((EC50: 322 and 486 µg L−1, for P. lividus and A. lixula, respectively)). The largest AgNPs (60 and 100 nm) showed a dose-dependent response, with little effect at lower concentrations, while more than 50% of larvae were developmentally delayed at the highest tested concentrations of 500 and 1000 µg L−1 (EC50(100 nm); 662 and 529 µg L−1, for P. lividus and A. lixula, respectively. While AgNPs showed no effect on the fertilisation success of treated sperm, an increase in offspring developmental defects and arrested development was observed in A. lixula larvae for 10 nm AgNPs at concentrations ≥50 µg L−1, and for 20 and 40 nm AgNPs at concentrations >100 µg L−1. Overall, toxicity was mostly ascribed to more rapid oxidative dissolution of smaller nanoparticles, although, in cases, Ag+ ion concentrations alone could not explain high toxicity, indicating a nanoparticle-size effect.

Simultaneous Influence of Gradients in Natural Organic Matter and Abiotic Parameters on the Behavior of Silver Nanoparticles in the Transition Zone from Freshwater to Saltwater Environments.

As nanoparticles have been found to cause a range of harmful impacts in biota, understanding processes and transformations which may stabilize and increase their persistence time in the environment are of great importance. As nanoparticles carried in riverine or wastewaters will eventually reach estuaries, understanding their behavior and transport potential in this transition zone from fresh to marine waters is essential, particularly as estuaries are sensitive ecological zones, oftentimes encompassing ornithologically important areas. In this direction, we report on the influence of combined gradients of riverine and marine natural organic matter (NOM) on the temporal stability of biocorona-encapsulated silver nanoparticles in terms of ion release kinetics. In parallel, salinity, pH and oxygen saturation were simultaneously varied to create a model to mimic the complex estuarine environment. While humic acid (HA) and alginate (Alg) disrupted the stabilizing ability of the nanoparticle protein corona to a greater and lesser degree, respectively, they slowed the rate of ion release in freshwater at pH 6.6 and in saltwater at pH 8, respectively, while oxygen saturation was also found to be an important factor. Thus, as the type of NOM changes with pH along a salinity gradient in an estuary, conditions required to increase the persistence time of nanoparticles are serendipitously met, with greater colloidal stability achieved in cases where there is more rapid replacement of HA with Alg. Despite the strong gradients in ionic strength, pH and oxygen saturation, the protein corona was not sufficiently disrupted at the nanoparticle surface to be substituted by NOM indicating the greater adsorption energy of the protein's hydrophobic domains. Ultimately, it is the specific NOM profile of individual estuaries that may provide the best indicator for predicting the stability and persistence of silver nanoparticles as they transition from fresh to salt water environments.

Phytotoxicity of Silver Nanoparticles on Tobacco Plants: Evaluation of Coating Effects on Photosynthetic Performance and Chloroplast Ultrastructure.

Silver nanoparticles (AgNPs) are the most exploited nanomaterial in agriculture and food production, and their release into the environment raises concern about their impact on plants. Since AgNPs are prone to biotransformation, various surface coatings are used to enhance their stability, which may modulate AgNP-imposed toxic effects. In this study, the impact of AgNPs stabilized with different coatings (citrate, polyvinylpyrrolidone (PVP), and cetyltrimethylammonium bromide (CTAB)) and AgNO3 on photosynthesis of tobacco plants as well as AgNP stability in exposure medium have been investigated. Obtained results revealed that AgNP-citrate induced the least effects on chlorophyll a fluorescence parameters and pigment content, which could be ascribed to their fast agglomeration in the exposure medium and consequently weak uptake. The impact of AgNP-PVP and AgNP-CTAB was more severe, inducing a deterioration of photosynthetic activity along with reduced pigment content and alterations in chloroplast ultrastructure, which could be correlated to their higher stability, elevated Ag accumulation, and surface charge. In conclusion, intrinsic properties of AgNP coatings affect their stability and bioavailability in the biological medium, thereby indirectly contributing changes in the photosynthetic apparatus. Moreover, AgNP treatments exhibited more severe inhibitory effects compared to AgNO3, which indicates that the impact on photosynthesis is dependent on the form of Ag.

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Coating-Dependent Effects of Silver Nanoparticles on Tobacco Seed Germination and Early Growth.

Silver nanoparticles (AgNPs) are used in a wide range of consumer products because of their excellent antimicrobial properties. AgNPs released into the environment are prone to transformations such as aggregation, oxidation, or dissolution so they are often stabilised by coatings that affect their physico-chemical properties and change their effect on living organisms. In this study we investigated the stability of polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB) coated AgNPs in an exposure medium, as well as their effect on tobacco germination and early growth. AgNP-CTAB was found to be more stable in the solid Murashige and Skoog (MS) medium compared to AgNP-PVP. The uptake and accumulation of silver in seedlings was equally efficient after exposure to both types of AgNPs. However, AgNP-PVP induced only mild toxicity on seedlings growth, while AgNP-CTAB caused severe negative effects on all parameters, even compared to AgNO3. Moreover, CTAB coating itself exerted negative effects on growth. Cysteine addition generally alleviated AgNP-PVP-induced negative effects, while it failed to improve germination and growth parameters after exposure to AgNP-CTAB. These results suggest that the toxic effects of AgNP-PVP are mainly a consequence of release of Ag+ ions, while phytotoxicity of AgNP-CTAB can rather be ascribed to surface coating itself.

Effect of silver nanoparticles on Mediterranean sea urchin embryonal development is species specific and depends on moment of first exposure.

With the ever growing use of nanoparticles in a broad range of industrial and consumer applications there is increasing likelihood that such nanoparticles will enter the aquatic environment and be transported through freshwater systems, eventually reaching estuarine or marine waters. Due to silver's known antimicrobial properties and widespread use of silver nanoparticles (AgNP), their environmental fate and impact is therefore of particular concern. In this context we have investigated the species-specific effects of low concentrations of 60 nm AgNP on embryonal development in Mediterranean sea urchins Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. The sensitivity of urchin embryos was tested by exposing embryos to nanoparticle concentrations in the 1-100 µg L(-1) range, with times of exposure varying from 30 min to 24 h (1 h-48 h for S. granularis) post-fertilisation which corresponded with fertilized egg, 4 cell, blastula and gastrula development phases. The most sensitive species to AgNP was A. lixula with significant modulation of embryonal development at the lowest AgNP concentrations of 1-10 µg L(-1) with high numbers of malformed embryos or arrested development. The greatest impact on development was noted for those embryos first exposed to nanoparticles at 6 and 24 h post fertilisation. For P. lividus, similar effects were noted at higher concentrations of 50 µg L(-1) and 100 µg L(-1) for all times of first exposure. The S. granularis embryos indicated a moderate AgNP impact, and significant developmental abnormalities were recorded in the concentration range of 10-50 µg L(-1). As later post-fertilisation exposure times to AgNP caused greater developmental changes in spite of a shorter total exposure time led us to postulate on additional mechanisms of AgNP toxicity. The results herein indicate that toxic effects of AgNP are species-specific. The moment at which embryos first encounter AgNP is also shown to be an important factor in the development of abnormalities, and future applications of the sea urchin embryo development test for nanoparticle toxicity testing should carefully address the specific phase of development of embryos when nanoparticles are first introduced.