Diminishing toxicity of P25 TiO2 NPs during continuous exposure to freshwater algae Chlorella

Nano-titanium dioxide (Nano-TiO 2 ) has been widely used in industrial manufacture and life science eld due to its excellent physical and chemical properties in the past few decades, which makes it inevitably released into the aquatic environment. In freshwater ecosystem, Chlorella is the most commonly used species to study the effect brought by metal oxide nanoparticles. In this work, both the short term and long term effect of P25 TiO 2 on Chlorella were investigated. Here, we found that Nano-TiO 2 would cause serious damage to chlorella in a short period of time, as conrmed by oxidative stress and algal cell morphology. However, different from the short-term condition, the damaging effect was gradually weakened along with the prolongation of exposure time of chlorella to Nano-TiO 2 and the nal observed inhibition rate of biomass was nearly zero. In addition, the other important nding of this work is that extracellular polymeric substance (EPS) has remarkably protective effect on algae cells. Algae cells with EPS removed were more vulnerable to nano titanium dioxide and exhibited more diculties in returning back to normal growth compared with normal algae cells, which might be related to the attachment of nanoparticles to the cell surface.


Introduction
With the high-speed development of nanotechnology, nanomaterial is playing an increasingly important role in our daily life and has commonly been applied into the process of industrial manufacturing.
Engineered nanoparticles (NPs) like Ag NPs, ZnO NPs, carbon nanotube and TiO 2 NPs are widely used (Jahan et al. 2017). Compared with other nanoparticles, large amount of TiO 2 NPs has been utilized in paints, plastics, sunscreens, solar cells, and food additives due to its unique characteristics such as shape, morphology and different crystal structure. ( Microalgae is a crucial part of the whole food chain and aquatic ecosystem since it is one kind of major primary producer (Becker 1995;Ramaraj et al. 2014). They are featured by simple structure, wide distribution, low cost, rapid propagation and stronger adaptability. Hence, the microalgae have been used as bio-indicator to evaluate the risk of toxicity of various pollutants (Rashid N  suspensions that aggregate to varying degrees. Therefore, the stability and ecological toxicology behavior of NPs will exert a unique toxicological effect on the aquatic food web (Christian et al. 2008;Domingos et al. 2009).
In freshwater ecosystem, Chlorella is one kind of the most common algae species. Due to the primary producer status of microalgae in the aquatic environment, any changes at "g" level of the food web will affect organisms with a higher level of nutrition. Therefore, Chlorella vulgaris was selected to evaluate its toxicological feedback towards the exposure to P25 TiO 2 NPs. Cardinale et al. (Cardinale et al. 2012) reported that P25 TiO 2 NPs could inhibit the growth of chlorella through affecting the metabolic process of chlorella. Xia et al. (Xia et al. 2018) used marine microalgae chlorella as the research object, which proved that ocean acidi cation would enhance the toxic effect of TiO 2 NPs on Chlorella. Roy et al. (Roy et al. 2016) emphasized that there is a signi cant interspecies difference in the toxicity of TiO 2 NPs to chlorella cells under visible light compared with other algae, which may be associated with the bioavailability of TiO 2 NPs. Although many relative research on the toxicity of chlorella exposure to TiO 2 NPs have been reported, almost no study focused on the effects of long-term growth of chlorella.
Continuous interactions between nanoparticles and algae might exist in actual aqueous environment, and almost no relative study has been reported on this so far. No studies to date have been conducted to systematically investigated the interaction between P25 TiO 2 NPs and Chlorella vulgaris under continuous exposure, which would better re ect natural conditions. Hence, evaluating the toxicity of P25 TiO 2 NPs under a long-term toxicity examination is required to better understand the effects of nanoparticles in aqueous environmental matrix.
Accumulation of the NPs in the aqueous environment is a serious menace for the producers as well as the organisms at the higher levels of the food chain. An assessment of these aqueous pollutants in producing toxicity is crucial in keeping dynamic balance of water ecosystem. In this study, the behavior of TiO 2 NPs under the culture medium water was analyzed, meanwhile their complex effects on freshwater microalgae Chlorella vulgaris was also studied. This work will be the rst to report the in uence of EPS on the toxicity of nanomaterials with long-term exposure to Chlorella vulgaris. A preliminary characterization of TiO 2 NP was performed. The toxicity of TiO 2 NPs with and without EPS was evaluated by measuring chlorophyll and cell counting techniques. Oxidative stress determination through superoxide dismutase (SOD) and lipid peroxidation (LPO) tests further convinced the toxic aspects of NPs. In addition, electron microscopy analysis was performed to analyze the surface changes of algae after exposure to toxicants.
According to the manufacturer, the average particle size of TiO 2 NPs is 21 nm with over 99.5% purity. The morphology and size of TiO 2 NPs were con rmed by transmission electronic microscopy (TEM; JEM-2100, JEOL, Japan). Initially, TiO 2 NPs dispersed in ultra-pure water were imaged by transmission electron microscopy (TEM) through depositing a 10 µL drop on formvar/carbon-coated copper grids and drying overnight, and micrographs were obtained at 200 keV. BG11 were procured from Haibo (Qingdao, China). All other chemicals used in the experiment were of analytical reagent grade and without further puri cation.

Stock suspensions of TiO 2 nanoparticles
200 mg of the nanoparticle powder were added to 100 mL of ultrapure water and sonicated for 30 min.
The stock solution was stored at 4 °C and sonicated for 30 min before use.

Algal cultures
The algae, Chlorella vulgaris (FACHB-1068), were purchased from Freshwater Algae Culture Collection at the Institute of Hydrobiology (Wuhan, China). Algae in rapid growth phase were transferred into a conical ask containing BG-11 medium (Xiao et al. 2016). The cultures were maintained under 25 ± 0.5 °C (a photoperiod of 12:12 h light/dark cycle with a white uorescent lighting). The asks were shaken three times every day, which will help to kept the samples from precipitating and rearranged randomly in the whole system. The initial cell density was approximately 10 6 cells/mL(Wu et al. 2019).

Aggregation and stability analysis of TiO 2 NPs in freshwater matrix
The varied TiO 2 hydrodynamic diameter (Dh) over time (t) was measured by dynamic light scattering (DLS) (Zetasizer Nano-S90, Malvern Instruments Ltd., UK) performed by a He-Ne laser at the wavelength of 633 nm. The sonicated TiO 2 NPs stock solution was added to the medium, which had been sterilized.

Algal growth assay
Aliquots of the algal suspension after interaction with/without TiO 2 NPs (50 mg/L) visible light and dark conditions were counted at least three times using a hemocytometer under a metallographic microscope (zeiss supra-55, Germany), and the density in each sample was calculated. The initial algal density was 1.6 × 10 6 cells/mL. The normal growth rate(R) of control algae was calculated as follows: R(%)=(C 0 -C t )/C 0 х 100%, where C 0 is the initial density and C t denotes the density at the time t. The algae of test groups had a growth inhibition rate(IR), which was calculated as follows: IR(%)=(C-C t )/C х 100%, where C is the density of the control algal at time t and C t represents the density of the test groups at the time t.

Evaluation parameter
Total protein, superoxide dismutase (SOD) and malondialdehyde (MDA) assays were performed according to previously reported methods (Xiao et al. 2016). The appropriate commercial kits were used to determine the total protein, SOD and MDA of algae, which were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The total protein was determined from the Bradford method (Bradford, 1976). The extent of lipid peroxidation can generally be determined by detecting the amount of MDA, through monitoring the color reaction of thiobarbituric acid (TBA method) (Janero 1990;Navarro et al. 2008). The ability of an organism to scavenge oxygen free radicals itself could be indirectly indicated by the activity of SOD (Wang et al. 2010). A photochemical reduction method of nitroblue tetrazole was applied to determine the activity of SOD (NBT method) (Beyer and Fridovich 1987).
Morphological change on algae due to the exposure to toxicants were observed using SEM. Control samples with only algae, and algae treated with TiO 2 NPs (50 mM) with and without EPS were analyzed under scanning electron microscope (Zeiss supra55). After 72 h interaction, aliquots of algal suspension were placed on a piece of glass, air dried, and sputtered with gold before SEM analysis (Thiagarajan et al. 2019).

Characterization of TiO 2 NPs
SEM image Fig. 1 (a) showed that the TiO 2 nanoparticles are nearly spherical without obvious clumps.
TEM image Fig. 1 (b) indicated the average size is near 20 nm, which is in accordance with the size of 21 nm provided by the manufacturer. The hydrodynamic particle size was found to be 233.6 ± 6.5 nm by DLS, and negative (-3.38 ± 0.11 mV) charge on TiO 2 surface in neutral aqueous solution. The larger hydrodynamic particle size of TiO 2 than the counterpart powder size may attributed to the formation of TiO 2 aggregate.

Aggregation and stability analysis of nanoparticles in freshwater matrix
In order to investigate the short-term toxicity effects of nano TiO 2 on algae cells, the short-term aggregation and sedimentation of P25 TiO 2 NPs were examined. The aggregation of TiO 2 NPs (50 mg/L) in the algal cell culture was studied in 3 hours. In the experiment, the algal culture medium with high salt content and abundant monovalent and divalent counter ions (Na + , Ca 2+ , Mg 2+ ) can partly neutralize the NPs surface charge, the zeta potential was negative (TiO 2 NPs isoelectric point ~ 6.2-6.4) at which the present value of pH (i.e. 7.2). Therefore, NPs dispersed in media colliding had a high attachment e ciency with easily aggregation due to thermal movement. This phenomenon was certainly con rmed by DLS analysis (Fig. 2). The hydrodynamic diameter shows an increasing trend from 484.8 ± 50.8 µm to 2288.7 ± 231.3 µm within two hours. This study demonstrated that nano TiO 2 dispersion was extremely easy to form aggregates that reached micrometric size in the present media within 30 min. It was con rmed from the sedimentation experiment that the sedimentation rate of NPs was up to 80% within 6 hours, as shown in Fig. 3. In these cases, nano TiO 2 can easily forms large clusters and then gradually precipitates in the test vessel under the role of gravity, thus the amount of TiO 2 NPs in the suspension of the sample was reduced. Therefore, the way of agitation was selected to simulate the dynamic process, which helps to enhance the contact of algae cells with particles for a short-term toxicity test under the function of nano TiO 2 .

Morphological variation of Chlorella cell
The effect of nano TiO 2 on the morphology upon the short time exposure of algal cells can be observed in SEM images. The morphological aberrations of vulgaris exposed to TiO 2 NPs were compared between control and treated cells were depicted in Fig. 4. It can be observed that the aggregated TiO 2 NPs adhere to the algal cell surface as the exposure time increased. Compared with the control group, the amount of chlorella attached to the surface of TiO 2 after removing EPS was relatively higher. The adhesion of TiO 2 NPs on the cell surface is one of the signi cant reasons leading to cell growth inhibition and death in a short period of time. Fig.5 demonstrated that the growth inhibition effect was observed in the experimental group under suitable growth conditions for algae. The inhibition rates of the algae cells exposed to nano TiO 2 were detected to be 0.18, 0.36, 0.11 in the rst three days. However, the rate was found to be decreased from 0.38 to -0.02 when the time was more than ten days. The observed growth inhibition of algae cells could remove algae-soluble EPS, and we found that algae cells without EPS were more susceptible to nano-TiO 2 . In the absence of nano-TiO 2 , the growth inhibition rate of the algae-removing algae was 0.36, 0.45, and 0.55 in the rst three days, then a decreased trend was observed in the range of 0.55 to 0.04. A high inhibition rate within 1 to 10 days was maintained with EPS-removed algal cells exposed to nano TiO 2 .

Effect of P25 TiO 2 nanoparticles on Chlorella algae growth
Regularly after the tenth day, the inhibition rate also showed a decreasing trend in the range of 0.52 -0.23. In the terms of biovolume, nano-titanium dioxide exhibits a great in uence on algal cell growth, especially in the early stage. However, as the growth of algae cells matures, the toxicity of nanoparticles to algal cells is gradually reduced, which proved that the nal growth inhibition rate was greatly reduced. Given the negligible dissolution of TiO 2 NPs in water (Dasari et al. 2013), the growth inhibition was mainly due to the attachment of nano TiO 2 to algae cells. It can be concluded that EPS has a protective effect on algae cells simultaneously. Under the same exposure conditions, algae cells without EPS were subjected to higher growth inhibition, which may be relate to the absence of EPS protection, and nanoparticles were more likely to adhere to the surface of algae cells. Fig.6 demonstrated the variation of total protein content of chlorella exposed to nano TiO 2 . Algae cells exposed to nano TiO 2 exhibited a higher total protein content than that without nano TiO 2 . As we expected, the total protein content of the EPS-removed algae was lower than normal condition. Under 1 to 15 days of exposure to nano-titanium dioxide, the total protein content of algae cells was lower than that without exposure to nano TiO 2 , whereas the total protein content of algae cells without nanoparticles were exceeded at 15 to 30 days. The photocatalytic action of nano TiO 2 can lead to cell membrane damage of the organism and eventually cell death by producing oxidative stress against the organism.

Antioxidation and lipid peroxidation
The algae secreted a variety of compounds with antioxidant function that help to resist the survival of toxic stress. This is why the algae cells exposed to TiO 2 NPs have a higher total protein content. The algae cells with EPS removed possibly damaged by TiO 2 NPs, which reduced biomass and resulted in less total protein compared with the algal cells without TiO 2 NPs.
The superoxide dismutase is the rst and primary antioxidant enzyme against the oxidative threats. Figure 7 showed the varied production of SOD during the growth of chlorella as the function of TiO 2 NPs.
We clearly understand that the production of chlorella SOD was reduced under the effect of TiO 2 NPs, thereby lowering the oxidative stress response of organism, which result in reduced biomass. At the same time, algae cells with EPS removed were more easily affected by TiO 2 NPs, so algae cells with EPS removed had minimal biomass when exposed to TiO 2 NPs. However, this effect was gradually weakened.
As the organism continues to grow, the SOD secreted by the algae cells exposed to the TiO 2 NPs eventually reach a normal level.
Malondialdehyde (MDA) was a kind of peroxidation product of lipids and the accumulation of oxygen free radical possibly causes the peroxidation of lipid in algal cells. The change of MDA levels indicates the extent of oxidative damage in cells. As shown in Fig. 8, the level of MDA was signi cantly higher than the control group. This indicated that nano-titanium dioxide exhibits an oxidative damage to cells. In addition, it can be concluded that nano-titanium dioxide had a sustained damage effect to algae cells which was performed by comparison of MDA levels during 30 days.

Conclusion
In conclusion, TiO 2 NPs was highly ecotoxicity mainly due to the attachment of nanoparticles on the surface of algae cells. Meanwhile, a protective role of EPS on the growth of algae was observed. The algae cells with EPS removed were more easily damaged by nanoparticles. Furthermore, the damage of TiO 2 NPs to chlorella was reduced from the perspective of the entire growth cycle. After 25 days, the biomass and SOD activity of chlorella tend to be normal, indicating the negligible effect of nanoparticles on algae cells. This research will be helpful to further understand the biological toxicity of nanoparticles and the response mechanism of organisms. However, the natural water environment conditions are more complicated and more involved factors. It is necessary to further investigate the interaction mechanism of organisms with nanoparticles.