Physicochemical transformations of ZnO NPs under aging process in aquatic environment and the toxicity to green algae Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Hong Zhang, Qing Huang* August 24-26, 2015 Philadelphia, USA 4 th Global Summit on Toxicology

Background Nanomaterials and nanotechnology have attracted the attention of the scientific community globally and emerged as a fast developing and fascinating area of research Nanoparticles (NPs) potentially undergo physicochemical transformation in the environment, which may lead to unexpected environmental and health risks ZnO NPs are one of the most abundantly used nanomaterials in materials science and nanotechnology-based industries, and the physicochemical properties can be affected by water chemistry and thus the toxicity of ZnO NPs may be changed in the aquatic environment Microalgae are the primary producers in the aquatic food chain and they are more sensitive to contaminants than fish and invertebrates, a model for evaluating the ecological impact of toxicants and NPs in aquatic environment Lack of quantitative assessment of the neoformation of ZnO NPs and clear understanding of the relationship between the neoformation and the toxicity of ZnO NPs under aging process on the aquatic organisms such as green algae

Outline Morphological and qualitative changes of ZnO NPs under aging process in aquatic environment Interactions of ZnO NPs and algal cell surface Effects of aging process on the toxicity of ZnO NPs to green algae cells Identification and quantification of the neoformations transformed from ZnO NPs under aging process Contribution of the neoformations in aged ZnO NPs to the toxicity on green algae C. vulgaris Identification of the difference in toxicity between fresh and aged ZnO NPs by infrared spectroscopy

(a) (b) (c) (d) Morphological and qualitative changes of ZnO NPs ZnO NPs-freshZnO NPs-aged SEM TEM SEM: spherical and short-rod, no obvious changes TEM: sheet-like neoformations

Interactions of ZnO NPs and algal cell surface(C. vulgaris) ZnO NPs-fresh Control ZnO NPs-aged Time ： 7days ZnO NPs ： 50 μg/ml ZnO NPs attached to the surface of the algal cells: distorted morphological features

Toxicity of aged ZnO NPs to green algae (C. vulgaris) Specific growth rate ZnO suppressed the growth of algea The growth rate and chlorophyll a contents in group ZnO-120 days and ZnO-210 days are higher than that in group ZnO-30 days. chlorophyll a

Changes in physiological status of algal cells The integrality of algal cell membrane Intracellular ROS content Photosynthetic activity Comparisons in membrane damage, photosynthetic activity, intracellar ROS content in five groups: Control without ZnO NPs ZnO NPs fresh ZnO-30 NPs days ZnO-120 NPs days ZnO-210 NPs days

FTIR, Raman and XRD spectroscopic analysis confirmed that the main components were Zn (OH) 2, Hydrozincite and ZnO NPs during aging process Identification of the neoformations transformed from ZnO NPs under aging process Raman FTIR XRD

Quantification of the neoformations transformed from ZnO NPs (two compounds)

Quantification of the neoformations transformed from ZnO NPs (three compounds) P 145 /P 437 P 830 /P 437

Molar ratios of neoformations and zinc ions concentration in ZnO NPs during aging process Molar ratios of neoformations in ZnO NPs during aging process Zinc ions concentration in the different aged ZnO NPs

TPEN ： 10 μM ZnO NPs ： 10 μg/ml TPEN can significantly reduce the toxicity of the fresh or aged ZnO NPs Zinc ion inhibitor (TPEN) influence on the effect of ZnO NPs toxicity on green algae C. vulgaris

Distinguishing the species and components in algal cells by FTIR

Changes of components in algal cells treated by fresh or aged ZnO NPs detected by FTIR Lipid Protein carbohydrate lipid band 3000–2800 cm −1 carbohydrate band 1200–950 cm −1 protein amide I (1724–1585 cm −1 ) amide II (1585–1490 cm −1 ) Bioresource Technology, 2014, 347–354

Discrimination of toxicity of the fresh or aged ZnO NPs by FITR combined with Principal component analysis(PCA)

Conclusions ZnO NPs undergoing aging process in water were transformed into basic zinc carbonate and zinc hydroxide and other substances, and these neoformations can be quantitatively determined by Raman spectroscopy. The toxicity of ZnO NPs aged for 30 days is larger than that for fresh ZnO NPs, but smaller than that of ZnO NPs aged for longer time (e.g. 120, 210 days). The toxicity of aged ZnO NPs on Chlorella vulgaris were decreased during the aging process due to less toxic zinc hydroxide and basic zinc carbonate transformed from ZnO NPs in aquatic environment. Physiological status changes of Chlorella vulgaris treated by fresh or aged ZnO NPs can be discriminated by infrared spectroscopy combined with chemometric methods.

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