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Toxicity, Bioaccumulation and Biotransformation of Glucose-Capped Silver Nanoparticles in Green Microalgae Chlorella vulgaris.
Mariano S
,
Panzarini E
,
Inverno MD
,
Voulvoulis N
,
Dini L
.
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Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials in consumer products. When discharged into the aquatic environment AgNPs can cause toxicity to aquatic biota, through mechanisms that are still under debate, thus rendering the nanoparticles (NPs) effects evaluation a necessary step. Different aquatic organism models, i.e., microalgae, mussels, Daphnia magna, sea urchins and Danio rerio, etc. have been largely exploited for NPs toxicity assessment. On the other hand, alternative biological microorganisms abundantly present in nature, i.e., microalgae, are nowadays exploited as a potential sink for removal of toxic substances from the environment. Indeed, the green microalgae Chlorella vulgaris is one of the most used microorganisms for waste treatment. With the aim to verify the possible involvement of C. vulgaris not only as a model microorganism of NPs toxicity but also for the protection toward NPs pollution, we used these microalgae to measure the AgNPs biotoxicity and bioaccumulation. In particular, to exclude any toxicity derived by Ag+ ions release, green chemistry-synthesised and glucose-coated AgNPs (AgNPs-G) were used. C. vulgaris actively internalised AgNPs-G whose amount increases in a time- and dose-dependent manner. The internalised NPs, found inside large vacuoles, were not released back into the medium, even after 1 week, and did not undergo biotransformation since AgNPs-G maintained their crystalline nature. Biotoxicity of AgNPs-G causes an exposure time and AgNPs-G dose-dependent growth reduction and a decrease in chlorophyll-a amount. These results confirm C. vulgaris as a bioaccumulating microalgae for possible use in environmental protection.
Figure 1. (A) Ultraviolet (UV)–visible spectra of glucose-capped silver nanoparticles (AgNPs-G)/mL in Bold’s basal medium (BBM) culture medium reported as absorbance in arbitrary unit (a.u., y axis) vs. wavelength (nm, x axis). (B,C) Size distribution and transmission electron microscopy (TEM) micrograph of AgNPs-G. Size distribution is reported as arbitrary unit (a.u., y axis) vs. longitudinal diameter (nm, x axis). Bars = 20 nm. (D) Kinetic of Ag+ dissolution. The dissolution of AgNPs-G in complete BBM culture medium was evaluated by atomic absorption spectroscopy. Data were analysed by performing one-way analysis of variance (ANOVA) at the 95% confidence level. Each value represents the mean ± standard deviation (SD) of 3 technical replicates in each of the 3 independent experiments. Ag+ dissolution degree is expressed as percentage (%) of total AgNO3 used to obtain the highest concentration of NPs solution during treatment.
Figure 2. (A) X-ray diffraction (XRD) spectrum of AgNPs-G before and after the interaction with algae. A culture of C. vulgaris is used as negative control. Numbers refer to diffraction peaks of Ag in its crystalline form. (B) Inductively coupled plasma–optical emission spectrometry (ICP–OES) to determine Ag internalisation by algal cells treated with five concentrations of AgNPs-G. The absorbed Ag was calculated by the total Ag (TAg, also determined by ICP–OES by using stocks at five concentrations) minus the Ag in filtrates (FAg). Therefore, the percentage of absorbed Ag = (TAg − FAg)/TAg × 100. Data were analysed by performing one-way ANOVA at the 95% confidence level. Each value represents the mean ± SD of 3 technical replicates in each of the 3 independent experiments. Asterisks indicate significant differences from respective values at 24 h at the same concentration (p < 0.05).
Figure 3. (A) Analysis of inhibitory rate. Algae were incubated for 24 h and a week with Ag ions and with five concentrations of AgNPs-G. Data were analysed by performing one-way ANOVA at the 95% confidence level. Each value represents the mean ± SD of 3 technical replicates in each of the 3 independent experiments. Asterisks indicate significant differences from the control values (p < 0.05); (B) Analysis of chlorophyll-a content by spectrophotometric analysis of centrifuged samples. Quantitative determination was done according to Arnon et al. (1949). The experiments were conducted in triplicate and results are the mean with standard deviation. Asterisks indicate significant differences from the respective untreated samples (p < 0.05) (C) TEM micrographs of algal cells and elemental X-ray spectrum (lower panel) of the square area of micrograph (e) containing black spots. (a) control cell; (b) algal cells treated with Ag ions. (c) algal cells treated with AgNPs-G for 24 h. Plasma membrane detaches from the cell wall, as indicated in the magnification; (d–g) Algal cells treated with AgNPs-G for a week. AgNPs-G were observed inside large vacuoles (d, white triangle), inside algae (d–e) or crossing the cell wall (f–g). Bar = 500 nm.
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