The results of zinc ione solubility showede that the concentration of Zn2+ was enhanced with increasing ZnO NPs treatment but the percentage of dissolved ions actually was decreased. In other words, the percentage of the total Zn2+ 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 CuO NPs (Sǿrensen et al. 2016). ZnO Particles and Zn2+ ions are observed in algal suspensions, and the toxicity of ZnO NPs can be related to the particles, ionic, or the total effect of both. The toxicity of Zn2+ ions confirmed by available studies (Chen et al. 2012b; Suman et al. 2015; Wang et al. 2016a). The released Zn2+ ion from ZnO NPs can easily penetrate into the algal cells by small pores in algal cell wall (Melegari et al. 2013). Lee (2018) reported that Zn2+ can not permeate across the cellular membrane and the membranes of intracellular structures because it is a hydrophilic ion. Zn2+ transporters (ZnTs) family facilitates the mobilization of Zn2+ in the opposite direction of Zn2+ importers (ZIPs) family (Lee 2018). Further, the other membrane transporter proteins are involved in mobilization of Zn2+ across the cell membrane (Lee 2018). Most researchers have found that there is the significant links between the dissolution rate of ZnO NPs and their toxic effects (Suman et al. 2015; Skjolding et al. 2017). It is well-estabilished that the toxicity of ZnO NPs partly caused by the dissolved zinc ions which can increase the ROS production and induce oxidative damage in cells (Sharifi et al. 2012; Suman et al. 2015). Furthermore, the regulatory role of zinc in mitochondrial homeostasis is imported becuase mitochondria are the main source of ROS production (Lee 2018). On the other hand, zinc is known as a redox-inert metal which play as an antioxidant by the catalytic action of copper/zinc-superoxide dismutase, conservation of the protein sulfhydryl groups, fixation of membrane structure, and upregulation of the expression of metallothionein, which has a metal-connection capacity as well as antioxidant functions (Lee 2018).
The decreased growth of N. oculata was observed in response to 10-200 mg/L of ZnO NPs. Study of the growth of Clorella vulgaris after exposure to 50-300 mg/L of ZnO NPs using MTT test shows that the cell viability was significantly decreased (Suman et al. 2015). Likewise, the investigation of long-term toxic effect of ZnO NPs (0.81-810 mg/L) on the growth of S. rubescens in BG11 and BBM media were showed that algal growth is affected by the exposure time, NPs concentrations, and mainly type of culture medium (Aravantinou et al. 2017). Intereguingly, 0.081-0.810 mg/L concentrations of ZnO NPs had toxic effects on P. subcapita, while 0.81 mg/L of ZnO NPs did not restrict the viability of Chlorella sp. after 24 h in Algo-Gro medium (Aruoja et al. 2009). Taken all together, the effects of ZnO NPs on microalgae were dependent on the algal species, the culture medium, concentration of NPs, solubility of NPs and time of exposure (Arouja et al. 2009; Suman et al. 2015; Aravantinou et al. 2017; Fazelian et al. 2020a). It should be noted that the different NPs cause different toxicity in N. oculata. Fazelian et al. (2020a) reported the order of toxicity of metal oxide NPs in N. oculata as CuO-NPs > ZnO-NPs > Fe2O3-NPs. On the other hand, the EC50 of Ag NPs for N. oculata was 20.88 mg/L, while the EC50 of CuO-, ZnO-, and Fe2O3-NPs was 116.98, 153.72, and 202.92 respectively (Fazelian et al. 2019, 2020a, 2020b). The growth inhibition by ZnO NPs could be associated with the inhibition of photosynthetic processes. In any case, decrease of chlorophyll content might be responsible for the reduction of N. oculata growth. In agreement with of our results, the negative effects of CuO NPs, Ag NPs and TiO2 NPs on the chlorophyll content and growth of N. oculata, Chlorella sp. and Chlamydomonas reinhardtii were reported (Chen et al. 2012a; Wang et al. 2013; Fazelian et al. 2019, 2020b). In the presence of ZnO NPs, a decrease in chlorophyll content might induce oxidative stress or inversely an increase in oxidative stress may cause a decrease in chlorophyll content.
Oxidative damage is a common mechanism responsible for the NPs-induced cell disorders (Sharifi et al. 2012). Lipid peroxidation leads to the destruction of biomembranes and has been used extensively as a sign for in vivo oxidative stress (Sayeed et al. 2003). The produced free radicales by cellular exposure to NPs are extremely reactive and rapidly disrupt normal cell metabolism and increase lipid peroixidation (Dawes 2000). Significant effects of different NPs on generation of MDA in a number of microalgal species were formerly reported (Chen et al. 2012a; Fazelian et al. 2019, 2020b). At high concentrations of ZnO NPs occurs a decrease in the level of some antioxidant structures, which results in rapid accumulation of ROS in algal cells (Sayeed et al. 2003; Suman et al. 2015). The uncontrolled production of H2O2 (as a ROS), may destroy cellular components and oxidative stress can be caused by excess H2O2 accumulation (Sayeed et al. 2003). Accordingly, high lipid peroxidation coupled with high H2O2 content might cause damage to chloroplast, decreased algae biomass and inhibited chlorophyll synthesis, leading to lower chlorophyll concentration in N. oculata.
Many attemps have been made to enhance the tolerance to oxidative stress by modifying the antioxidant defense system (Liu et al. 2008). Carotenoids (canthaxanthin and β-carotene) play an important role in defense against oxidative stress by scavenges of single oxygen and suppressing lipid peroxidation in all photosynthetic organismsand (Salguero et al. 2003). Also, these compounds protect the photosynthetic apparatus from excess photons through xantophylls cycle (El-Bakey et al. 2007). Similarly, the increase of carotenoids was observed in response to TiO2 NPs in the alga Phaeodactylum tricornutum and Ag NPs in N. oculata (Wang et al. 2016a; Fazelian et al. 2020b). It has been also reported that the increase of zeaxanthin and β-carotene in Nannochloropsis is the antioxidant response against UV-A radiation (Forján et al. 2011). Contrary to our results, 5-200 mg/L of CuO NPs significantly decreased the content of carotenoids in N. oculata (Fazelian et al. 2019).
Phenolic compounds have also been studied due to their secondary ecological functions (e.g., reproductive role in algal reproduction and protective mechanism against biotic factors) (Machu et al. 2015). These compounds possess antioxidant activity through inhibition of lipid peroxidation, scavenging molecular species of active oxygen, and chelation of metal ions (Michalak 2006). The similar to our findings, CuO NPs (100-200 mg/L) significantly increased the content of phenolic compounds in N. oculata (Fazelian et al. 2019), while 5-50 mg/L of Ag NPs decreased these compounds (Fazelian et al. 2020b). It has also been reported that photosynthetic microorganisms can produce phenolic compounds in order to prevent cell damages in response to TiO2 NPs stress (Comotto et al. 2014). It seems that changes in PPO activity are directly related to the amount of phenolic compounds, because the enhanced activity of PPO showed a direct correlation with the contents of phenolic compounds of N. oculata. Increase of phenolic compounds and PPO activity was also observed in N. oculata exposed to 50-200 mg/L of CuO NPs (Fazelian et al. 2019). Ruiz et al. (2003) also reported that induction of PPO activity is possibly due to its role in phenolic compound synthesis, which has a function in detoxification in microalgae.
It is well known that antioxidant enzymes are the most sensitive indices in response of algae to environmental stress (Fazelian et al. 2019). In fact, the increased activity of antioxidant enzymes in living cells is as an early warning indicator of pollution in the environment (Song et al. 2008). The potential of ZnO NPs to induce oxidative stress was assessed by measuring the activities of a number of antioxidant enzymes including CAT, APX and PPO (Suman et al. 2015). The activity of CAT was considerably promoted by treatment of ZnO NPs, while APX activity decreased in response to ZnO NPs. Therefore, CAT plays an important role in protection of cells against oxidative damage caused by ZnO NPs. The increase of CAT activity was reported for C. vulgaris by exposure of algal cells to ZnO NPs (Chen et al. 2012b) and for N. oculata in response to 50-200 mg/L of CuO NPs (Fazelian et al. 2019) and 5-50 mg/L of Ag NPs (Fazelian et al. 2020b). Contrary to present results, 5-200 mg/L of CuO NPs and 10-25 mg/L of Ag NPs increased the activity of APX enzyme in N. oculata (Fazelian et al. 2019). 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 H2O2 removal using ascorbate as a specific 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 H2O2 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 et al. (2013) reported that the ROS production results in the degradation of cell membrane by LDH release. These findingss were similar to the previous reports showing that the treatment of ZnO NPs, CuO NPs and Ag NPs increased LDH activity in C. vulgaris and N. oculata respectively (Suman et al. 2015; Fazelian et al. 2019, 2020b). Interestingly, 200 mg/L of ZnO NPs and CuO NPs increased the activity of this enzyme by 2.38 and 5.7 fold respectively, as compared to the control (P < 0.05). Haung et al. (2008) reported that the released Zn2+ ions from ZnO NPs could be the reason for cell toxicity and cell membrane-damages by NPs.
Our data for viability, lipid peroxidation, LDH activity and SEM images showed that ZnO NPs was associated with algal cell surfaces and induced the cell membrane damages. These results are in accordance with the previous results on C. vulgaris, P. subcapitata, and N. oculata showing that ZnO NPs, TiO2 NPs, CuO NPs and Ag NPs caused membrane damages (Metzler et al. 2011; Suman et al. 2015; Fazelian et al. 2019, 2020b). The aggregation of ZnO NPs also observed in SEM images and confirmed the shadding effect as well as decreasing the light availibility which ultimately reduce the photosynthesis rate and the cell growth. Similar to our results, the aggregation of TiO2 NPs on the cell wall of Chlorella sp. and Scenedesmus sp. were reported by SEM images (Mohammed Sadiq et al. 2011). The algal cell aggregation after treatment with ZnO NPs is probably a defensive mechanism of N. oculata against the physical interaction with NPs. The other researchers reported that the aggregation of algal cells acts as a barrier against CuO NPs (Zhao et al. 2016; Fazelian et al. 2019).
TEM images of N. oculata were identical with the results obtained by Lee and An (2013) on P. subcapitata. and Fazelian et al. (2019) on N. oculat. Unlike CuO NPs, the aggregation of ZnO NPs was not observed in TEM images of N. oculata and the wrinkling of N. oculata cell wall in response to ZnO NPs was more than CuO NPs. Likewise, it was reported that the cell wall damages play an important role in the toxicity of TiO2 NPs to the marine microalga Phaeodactylum tricornutum (Wang et al. 2016b). The cell wall of algae acts as a selective and semipermeable barrier with pores ranging from 5-20 nm (Melegari et al. 2013). The interaction of cell wall with ZnO NPs can change the size of pores and/or can create new pores with higher size rather than typical pores which allow the entry of ZnO NPs through the cell wall structure (Navarro et al. 2008). The change of cell wall structure and internalization of NPs into microalgae has been observed by other researchers (Perreault et al. 2012; Melegari et al. 2013). Similarly, the TiO2 NPs were localized around the cell membrane of C. reihardtii while the entering the NPs into the cells was limited even at high concentrations of TiO2 NPs (Al-awady et al. 2015). The entry of ZnO NPs into the N. oculata cell can also be demonstrated according to the XRD image. On the other hand, the entry of ZnO NPs into N. oculata can release Zn2+ ions into the cell. Zinc is a divalent cation and does not directly undergo redox reactions and interact with ROS (Haas and Franz 2009). This property of zinc is unlike other bioactive metals such as iron and copper, and thereby Zn2+ can act as an efficient Lewis acid and it is often interacted with side chains of some amino acids (Haas and Franz 2009; Lee 2018). Also, zinc known as an antioxidant or prooxidant, which its antioxidant function was investigated previously (Lee 2018). Numerous studies have linked the induction of oxidative stress in response to high levels of Zn2+, which owing to its role as a prooxidant (Suman et al. 2015; Lee 2018).
In addition to the TEM image, the FTIR analysis is also a useful tool for the study of NPs interaction with algal cell wall (Liu et al. 2014; Mohammed Sadiq et al. 2011). FTIR results indicated strong interactions between the C-O, C=O and C-H groups on the surface of the algal cells with ZnO NPs (Table 3). Similarly, the interaction of CuO NPs with C-O, C=O and C-H groups as well as Ag NPs with C=O, C-H and O-H groups of N. oculata was observed (Fazelian et al. 2019, 2020b). In actual fact, cellulose, polysaccharides and glycoporteins of the algal cell walls act as binding sites for NPs (Chen et al. 2012b). The uptake of NPs by cells through crossing the cell wall and plasmamembrane were facilitated by the high spicific area and tiny size of NPs (Chen et al. 2012b). On the other hand, a number of functional groups with a net negative charge and high affinity for metal ions located on the surfaces of algal cells. These functional groups interact with the ions and help them in entering into the algal cells (Crist et al. 1994).