Toxicity Mechanisms of ZnO Nanoparticles in Nanochloropsis Oculata with a Comparative Approach with CuO and Ag Nanoparticles


 The purpose of present work was the investigation of different concentrations of zinc oxide nanoparticles on the marine microalga Nannochloropsis oculata and compare the results of this study with previous studies. Dissolution of ZnO NPs in nanopure water was 0.378-3.12 mg/L and the rate solubility decreased with increasing the concentrations of ZnO NPs. ZnO NPs were toxic to this microalga with EC50 of 153/72 mg/L. The toxicity of 200 mg/L ZnO NPs was 59.36% for the cell number, 61.27% for MTT test, and 57.34% for the chlorophyll content. Increase the content of malondialdehyde and hydrogen peroxide in response to increasing the concentration of ZnO NPs was indicated the induction of oxidative stress in N. oculata. The activity of catalase and lactate dehydrogenase increased in the treated cells, while the activity of ascorbate peroxidase was decreased. Concurrently, an increase in the content of carotenoids and phenolic compounds was observed in the treated cells. SEM and TEM analyses confirmed the aggregation of algal cells, damages in cell membrane and atypical changes in morphology of cell wall after NPs treatments. The FTIR results cofirmed the interaction of ZnO NPs with C-H, C-O and C=O groups on the cell surface. All of these changes were indicated the significant toxic impacts of ZnO NPs on the N. oculata cells. Comparison between the results obtained in previous studies with our results showed that the defensive mechanisms of N. oculata probably was not effective against the oxidative stress by >10 mg/L of ZnO NPs, > 5 mg/L of CuO NPs and > 1 mg/L of Ag NPs. Therefore, N. oculata is sensitive to such concentrations of these NPs.


Introduction
Nanoparticles (NPs) as atomic or molecular aggregates with a diameter of 1 -100 nm possess different physical and chemical properties compared to the bulk materials (Dolatabadi et al. 2015). The usage of engineered metal oxide NPs has increased in recent years in material science and commercial products (Aravantinou et al. 2013). ZnO NPs have wide applications in different products such as paints, coatings, cosmetics and household appliances because of their bactericidal and fungicidal properties (Manzo et al. 2011;Choi et al. 2016;Zafar et al. 2016). According to an estimation, the production of ZnO NPs is around 55-528 tons/year and there is an increase in production and utilization with time (Choudhary et al. 2018). Large-scale production and consumption are resulted in discharge of the nanomaterialscontaining products to aquatic ecosystems and agricultural lands (Lead et al. 2008). Thus, the risk of natural water contamination by synthetic NPs was continuously enhansed (Lead et al. 2008). Nonetheless, the potential toxic effects of NPs on living organisms are poorly studied. This is mainly because of the complexity of factors that in uence the toxicological charactersics of nanomaterials, including size, surface/area ratio, morphology, surface coatings and their other physicochemical properties (Ates et  there are only limited studies on the tocxicity of ZnO NPs on microalgae. The biotoxicity of ZnO NPs and bulk ZnO on Pseudokirchneriella subcapitata was previously con rmed (Franklin et al. 2007;Aruoja et al. 2009). Nano ZnO (EC50 1.94 mg/L) was more toxic than its bulk counterpart (EC50 3.57 mg Zn/L) for Dunaliella salina (Manzo et al. 2013). Also, the evaluation of toxicity of ZnO NPs on Chlorella vulgaris by ow-cytometric and cytotoxicity assays showed a substantial reduction in the viability of cells on a dose dependent manner (Suman et al. 2015).
The unicellular algae Nannochloropsis oculata (Eustigmatophyta) was considered as one of the most promising marine microalgae for eicosapentaenoic acid (20:5) production, an important polyunsaturated fatty acid for human consumption for prevention of several diseases (Forján et al. 2011). Biomass productivity of this microalgae can be 50 times higher than that of the fast growing terrestrial plants (Li et al. 2008

Algal culture
The strain of N. oculata was obtained from Ecological Research Institute of Persian Gulf and was cultivated in f/2 medium. F/2 growing media was prepared ahead of time for algal growth. To prepare this media, seawater was ltered and autoclaved for 20 min at 121 ℃ to sterilize the water. Subsequently, macronutrients, trace metals, and essential vitamins were added to the seawater. 50 mL of a mother culture of N. oculata was inoculated into the 500 ml Erlenmeyer asks containing 350 mL of liquid f/2 medium. The cultures were then uniformly mixed and an initial count of cell number in solution was performed using a Neubauer counting chamber. The cultures were exposed to 26-28 ℃ and a 12:12 lightdark cycle of 5000 lux illumination (Fazelian et al. 2019).

Nannoparticles characterization
ZnO NPs were purchased from the nanomaterials pioneers company (Houston, TX, USA). The ZnO NPs had particle size smaller than 100 nm and a speci c area of 20-60 m 2 /g. The size and shape of ZnO NPs were assessed using the conventional methods for NPs characterization such as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X-ray diffraction (XRD) ( The concentration of chlorophyll a and carotenoids were determined by Jeffrey and Humphrey (1975).
The samples were centrifuged and immersed in acetone (85% V/V) in darkness and dark. After 24 h, the absorption of the solvent was investigated in 664 nm, 647 nm, and 470 nm.

Total phenol content
To determine the total phenol contents, algal samples were homogenized in methanol for 24 h in the dark. The homogenate was centrifuged at 10000 g for 15 min and then 2.5 mL of 10% Folin-Ciocalteu's reagent and 2.5 mL of 7.5% NaHCO 3 were added to the 0.5 ml of methanolic extract. The content of phenolic compounds was estimated using absorbance at 765 nm. The results were expressed as mg of GA/g of extract (Singleton et al. 1999

Microscopic analysis
Field emission scanning electron microscopy (FESEM) was used to examine surface morphology. The samples were freeze-dryed, prepared and analyzed with FESEM (Zeiss-Sigma VP-500, Germany).
For Transmission electron microscopy (TEM) analysis, the primary-xation of algal samples was performed with 4% glutaraldehyde diluted with 0.1 M cacodylate buffer. By 1% osmium tetroxide in the same buffer was carried the sacendary xation. embedding in resine and sectioning was conducted after

Statistical analysis
All experiments were performed in 3 replicates. The data were showed as mean ± standard error (SE), and the means were compared by Duncan's test. Statistical signi cance was considered at P ≤ 0.05.

Dissolution of ZnO NPs
According to Fig

Growth Parameters
The results of this work revealed that ZnO NPs were toxic to this microalga with EC50 of 153/72 mg/L. The MTT test and cell number analysis con rmed the toxic effect of 10-200 mg/L of ZnO NPs on N. oculata. Actually, a decrease of algal cell viability was found after 72 h exposure to ZnO NPs ( Fig. 2.A).
According to the Fig. 2

Photosynthetic Pigments
Treatments of N. oculata with ZnO NPs resulted in signi cant changes in chlorophyll a content (Fig. 3.A). Although 5 mg/L of ZnO NPs did not show remarkable changes in the amount of chlorophyll a, 10-200 mg/L concentrations of ZnO NPs caused a signi cant decrease in the content of chlorophyll a compared to the control. The content of carotenoids was increased in algal cells after exposure to 5-100 mg/L of ZnO NPs, but was not observed the signi cant change in 200 mg/L (Fig. 3.B).

Phenolic Compounds and PPO Activity
The content of phenolic compounds in N. oculata was increased by 10-200 mg/L of ZnO NPs (Fig. 3.C).
In contrast, the PPO activity in N. oculata increased signi canly under 50 mg/L of ZnO NPs treatment and decreased in higher concentrations ( Table 2).

Oxidative Stress
Our results revealed that exposure of N. oculata cells to 50-200 mg/L of ZnO NPs increased MDA content as a marker for oxidative stress (Fig. 4.A). These ndings showed that ZnO NPs established oxidative damages, possibly by generating ROS (Fig. 4.B). The effect of different concentrations ZnO NPs (10-200 mg/L) on H 2 O 2 content of N. oculata was also signi cant at the level of P < 0.05. (Fig. 4.B).
The activity of CAT was considerably promoted by treatment of ZnO NPs (P < 0.05) and the highest CAT activity was observed at the concentration of 200 mg/L of NPs. The enzyme activiy did not show signi cant changes in presence of 5 and 10 mg/L of ZnO NPs ( Table 2). The application of ZnO NPs signi cantly decreased APX activity in the algal cells (P < 0.05), however, 5 mg/L of ZnO NPs did not cause remarkable change in the APX activity (Table 2).

LDH Activity
Enhanced LDH activity in N. oculata cells was observed after the treatment of algal cells with the increasing concentrations (50-200 mg/L) of ZnO NPs. The signi cant changes of LDH activity was not observed in response to 5-10 mg/L of ZnO NPs (Table 2).

SEM and TEM Analysis
EDX graph showed the presence of ZnO NPs in N. oculata cells ( Fig. 1.B). SEM images con rmed that the particle size of ZnO NPs was ranging from 20 to 75 nm (

Discussion
The results of zinc ione solubility showede that the concentration of Zn 2+ was enhanced with increasing ZnO NPs treatment but the percentage of dissolved ions actually was decreased. In other words, the percentage of the total Zn 2+ ion released from the ZnO NPs were actually lower at 200 mg/L (= 1.6%) compared to 5 mg/L (= 7.5%), which is a common phenomenon described by other investigations, e.g. for On the other hand, the reduction of APX activity in response to 50 mg/L of Ag NPs was similare to our study. APX was reported to be involved in H 2 O 2 removal using ascorbate as a speci c electron donor, particularly in chloroplast (Donahue et al. 1997). A reduction in the activity of APX under ZnO NPs treatment established that APX enzyme is not an effective scavenger of ROS in N. oculata and this microalgae is unable to decompose the excess H 2 O 2 radicals by this enzyme. Furthermore, the destruction of N. oculata chloroplast in response to oxidative stress induced by ZnO NPs was probably another reaseon of the reduction of APX activity.
Cell membrane damagr is also known as one of the causes of NP-toxicity and changes in LDH activity is an important parameter for determining cellular toxicity by metal oxide NPs (Zhang et al. 2012;Suman et al. 2015). LDH is known as an important enzyme that is released from damaged cells (Bergmeyer and Bernt 1965). Pathakoti

Conclusion
Our data revealed that concentrations of 10-200 mg/L of ZnO NPs induced oxidative stress in N. oculata cells. As a result, the activity of a number of antioxidant enzymes and the content of some non-enzymatic antioxidant components such as carotenoids and phenols were changes. In addition, the contents of MDA and H 2 O 2 were elevated that may be a consequence of induced oxidative stress. The growth parameters as well as chlorophyll a content were reduced in N. oculata exposed to ZnO NPs. SEM and TEM surveys showed aggregation of algal cells and uncommon changes in their morphology after treatment with NPs. The presence of ZnO NPs on the surface of algal cells was con rmed by the SEM and TEM images. Concordance between our results with previous studies showed that N. oculata as one of the sensitive species to NP-toxicity and the defense mechanisms of this microalgae against oxidative stress induced by ZnO, CuO, and Ag NPs may not have been strong enough (Table.4). Therefore, the entry of these nanoparticles into aquatic ecosystems causes toxicity in N. oculata.

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