In this study, the nominal diameter of commercial nano-TiO₂ ranged from 40 to 50 nm. As shown in the dynamic light scatting (DLS) results and Scanning electron microscope (SEM) images in Figure 1a, the average diameter of the NPs suspended in BG11 culture medium dramatically increased to approximately 450 to 650 nm. The aggregation tendency of the NPs was ascribed to the relatively low zeta potential (−7.8 mV). This rapid formation of nano-TiO₂ aggregates was also observed in previous studies [29,30], indicating that algae and other living organisms were exposed to nano-TiO₂ beyond their original nanoscale particle size in environment systems. At the same time, aggregated nano-TiO₂ were much more toxic than their bulk counterpart [31]. The nano-TiO₂ attached on the surface of the algal cells and the direct contact is demonstrated clearly by the SEM images in Figure 1b. So, the hydrodynamic size and the adsorption of nano-TiO₂ on algae affected their toxicity.

The interactions of Zn²⁺ with nano-TiO₂ were determined by examining the sorption equilibrium. In the equilibrium isotherm experiment, a correlation between Zn²⁺ adsorbed on the nano-TiO₂ (q_e, mg/g) and the non-adsorbed Zn²⁺ concentration (C_e, mg/L) in the culture medium was determined. Figure 2 shows the sorption density of nano-TiO₂ as a function of initial Zn²⁺ concentration up to 10.0 mg/L. Nano-TiO₂ could adsorb Zn²⁺ from the culture medium. With the initial Zn²⁺ concentration increased from 3.0 to 10.0 mg/L, Zn²⁺ adsorption approached the saturation point. The Langmuir isotherm was used to fit these adsorption data using Matlab.

The calculated adsorption capacity q_max was approximately 11.38 mg/g, and the parameter for the b = 6.92. A good correlation is shown in Figure 2 suggesting a monolayer adsorption of Zn²⁺ on nano-TiO₂. Therefore, nano-TiO₂ adsorption had an important impact on the Zn²⁺ concentration in the culture medium.

The toxicity of nano-TiO₂ to algae was reported by other researchers [11,22,23]. However, these results were not significantly comparable because of the different sources and properties of NPs. The inhibition of Anabaena sp. at different nano-TiO₂ concentrations from 1.0 to 50.0 mg/L is shown in Figure 3. After 96 h of exposure, the inhibition was observed at nano-TiO₂ concentrations more than 10.0 mg/L to algae. The changes in the content of chlorophyll-a and the photochemical transformation of energy were observed. The difference in the toxicity of nano-TiO₂ could be related to particle size, crystal form, and test method. Large aggregates of TiO₂ nanoparticles entrapped algal cells (Figure 1b), which reduced the light available to the algal cells and inhibited their growth [32,33]. Moreover, nutrients adsorbed by nano-TiO₂ in culture medium would contribute to the toxicity [34].

Figure 4 shows the inhibition of Anabaena sp. at different Zn²⁺ concentrations after 96 h. No significant inhibition was observed at Zn²⁺ concentrations below 0.3 mg/L, whereas the biomass of Anabaena sp. notably decreased with increasing Zn²⁺ concentration from 0.5 to 1.0 mg/L. The 96 h growth process of Anabaena sp. with increasing Zn²⁺ concentration from 0 to 1.0 mg/L is shown in Figure 5. The exposure of Anabaena sp. to Zn²⁺ resulted in a clear difference in cell number between the control and experimental samples. Higher initial Zn²⁺ concentrations reduced cell density significantly. Growth inhibition was essentially proportional to Zn²⁺ concentration. However, at the lowest Zn²⁺ concentration considered (0.1 mg/L), an increase in the growth of Anabaena sp. was actually observed. The 96 h EC₅₀ value for Anabaena sp. growth was calculated to be 0.38 ± 0.004 mg/L. This finding was in accordance with the results of a previous study on the exposure of Micractinium pusillum to Zn [35]. When Zn²⁺ was in high concentration, Anabaena sp. created physiological stress leading to generation of free radicals. Stress in turn induced the production of reactive oxygen species (ROS). The ROS could rapidly attack all types of biomolecules such as nucleic acids, protein, lipids, and amino acids, leading to irreparable metabolic dysfunction and algae death [36]. Results from H₂DCF-DA dye test using microplate reader showed that the intracellular ROS was raised in the algal cells with different initial Zn²⁺ concentration in Figure 4. When the initial Zn²⁺ concentration was higher than 0.7 mg/L, the intracellular ROS entered in the medium with the algal cells rupture.

The synergistic toxic effect of Zn²⁺ and nano-TiO₂ was examined using a fixed concentration of nanoparticles; the nano-TiO₂ particles alone were not toxic at low concentrations from 1.0 to 10.0 mg/L. Figure 6 shows the effect of Zn²⁺ on the 96 h growth process of Anabaena sp. in the presence of nano-TiO₂. The nanoparticles significantly impacted the toxicity of Zn²⁺. At high concentration such as 10.0 mg/L, the toxicity of Zn²⁺ was reduced and the EC₅₀ value of Zn²⁺ was 0.49 ± 0.001 mg/L. A high nano-TiO₂ concentration could effectively reduce the soluble Zn²⁺ by adsorbing Zn²⁺ on NP_S in Table S1. At same time, as shown in Figure S2, nano-TiO₂ at high concentrations easily settled to the bottom of the reactor, so the soluble Zn²⁺ concentration around algae was low and the toxicity was reduced. At low concentration such as 1.0 mg/L, the toxicity of Zn²⁺ was enhanced and the EC₅₀ value of Zn²⁺ with 1.0 mg/L nano-TiO₂ was about 0.29 ± 0.005 mg/L. The results indicate that Zn²⁺ toxicity was significantly enhanced by 1.0 mg/L nano-TiO₂ in the culture medium. However, the low concentration of nano-TiO₂ reduced the soluble Zn²⁺ concentration, as shown in Table S1. Soluble Zn²⁺ and adsorbed Zn²⁺ were believed to contribute to the overall toxic effect on algae. The direct adherence of nano-TiO₂ resulted in a high localized concentration on the algal surface, which could be due to high levels of free Zn²⁺[37]. Nano-TiO₂ at low concentrations was relatively stable in the culture medium. Nano-TiO₂ easily attached on the surface of the algal cells, which limited their mobility. The adsorbed Zn²⁺ had direct contact with the algae. The synergistic toxic effect of Zn²⁺ and nano-TiO₂ was attributed to the concentration of nano-TiO₂ and the free Zn²⁺.

Although Zn²⁺ at concentrations below 0.3 mg/L showed no significant toxic effects on Anabaena sp., a synergistic effect might occur if nano-TiO₂ was also present in this system. The toxicity of nano-TiO₂ in the presence of constant concentrations of Zn²⁺ was examined. Figure 7 shows the nano-TiO₂ toxicity result after 96 h of exposure at 0.3 mg/L Zn²⁺. It could be seen that the toxicity of nano-TiO₂ in the presence of Zn²⁺ was significantly different from that of nano-TiO₂ alone. With increasing nano-TiO₂ concentration, the inhibition of Anabaena sp. increased at the initial stage and then decreased afterwards. The same trend was observed in the photochemical transformation of energy and in the chlorophyll content of Anabaena sp. When the added nano-TiO₂ was more than 1.0 mg/L, the overall toxicity decreased. This could have been caused by the adsorption of Zn²⁺ onto the nano-TiO₂, which significantly reduced the soluble Zn²⁺ concentration with high concentration of nano-TiO₂. Increased nano-TiO₂ enhanced aggregation, resulting in a lower suspended concentration. Thus, the overall toxicity could also be decreased by reduced uptake of nano-TiO₂ by algae [37]. The addition of nano-TiO₂ enhanced Zn²⁺ toxicity, with the maximum enhancement observed at 1.0 mg/L nano-TiO₂. This result is consistent with the results shown in Figure 6. By contrast, nano-TiO₂ was non-toxic at concentrations less than 1.0 mg/L, and the contribution of bare nano-TiO₂ to algal toxicity was neglected. The soluble Zn²⁺ concentration decreased in the presence of nano-TiO₂, however, the overall toxicity significantly increased. First, the decrease in residual Zn²⁺ concentration reduced the toxic effect. This result was similar to the scenario for both heavy metals and other carriers [38]. Second, the adsorbed Zn²⁺ on nano-TiO₂ contributed to toxicity once nano-TiO₂ was taken up by algae. The addition of nano-TiO₂ increased the total uptake of Zn²⁺-loaded nanoparticles, and the mortality increased accordingly.

NPs in aquatic systems produced potential risks, not only from nano-particles, but also from their ability to accumulate and enhance the toxicity of these background contaminants. Nano-TiO₂ alone at low concentrations (<10.0 mg/L) did not cause significant inhibitory effects. Thus, its fate and potential aquatic effects could be easily overlooked. However, low-concentration nano-TiO₂ served as Zn²⁺ carriers and increased the total Zn²⁺ uptake by algae. Moreover, the concentrations of the NPs in the water body were always at the microgram level. The biomagnifications of NPs from lower trophic aquatic organisms to higher ones strengthened this risk [6]. Therefore, the synergistic effects of the background toxic substances with released NPs could be more serious than the effects of NP alone.

Samples of Anabaena sp. were obtained from the Institute of Wuhan Hydrobiology (China). The composition of BG11 culture medium is listed in Table S2. NaNO₃, K₂HPO₄, MgSO₄·7H₂O, CaCl₂·2H₂O, citric acid, ferric ammonium citrate, EDTANa₂, Na₂CO₃, H₃BO₃, MnCl₂·4H₂O, Na₂MoO₄, CuSO₄·5H₂O, Co(NO₃)₂, and ZnSO₄·7H₂O were purchased from Sinopharm Medicine. The deionized water (DI) used to prepare reagents and culture medium was purified by Millipore reverse osmosis. The initial pH of the medium was adjusted to 7.0 using 0.01 M HCl or NaOH solution. The algae were produced by cultivation in a constant-temperature incubator at 25 ± 1 °C. The illumination intensity in the incubator was 4000 Lux with a light-dark cycle of 12 h:12 h. The stock culture of Anabaena sp. were shaken three to four times a day, and their growth curves were recorded to ensure that the algae used in the test were in the logarithmic growth phase. The concentrations of experimental samples were measured using a spectrophotometer. The optical density (OD) values were in linear relation to algal concentration. The OD at 680 nm of the algal culture was 0.11 to 0.12, which corresponded to an algal concentration of 2.32 × 10⁹ cells/L.

Nano-TiO₂ particles (rutile form) 40 to 50 nm in diameter were purchased from Zhejiang Hongsheng Material Technology Co., China. The suspensions (1000 mg/L) were placed in an ultra-sound water bath (100 W, 40 kHz) for 30 min before being diluted to different exposure concentrations. Zeta potentials and particle sizes of nano-TiO₂ were measured by a dynamic light scatting (DLS) size analyzer (Zetasizer Nano-ZS, Malvern, UK). Scanning electron microscope (SEM) images were taken using a JEOL SM4800 SEM. Suspensions of 1.0 and 10.0 mg/L nano-TiO₂ were prepared by dilution in the culture medium. At 10 min intervals, the absorbance of nano-TiO₂ suspension was measured using a UV-vis spectrophotometer. The settling behavior of the NPs was investigated by the reduction of absorbance over 600 min.

The interactions of Zn²⁺ with nano-TiO₂ were studied by performing the traditional batch sorption experiment. A stock solution of Zn²⁺ was prepared by dissolving ZnSO₄ into DI water. The solution was diluted into 125 mL flasks to serial concentrations of 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 3.0, 5.0, and 10.0 mg/L with 50 mL culture medium. The pH of the Zn²⁺ solutions was adjusted to 7.0 ± 0.1 using 0.01 M HCl or NaOH. The nano-TiO₂ suspensions were diluted to a concentration of 10.0 ± 0.1 mg/L in each flask. The mixed suspensions were then shaken to achieve sorption equilibrium within 10 h. The mixed suspensions were centrifuged at 5000 rpm for 10 min. The supernatants were collected and again centrifuged at 5000 rpm for 10 min [9]. Zinc concentrations in the supernatants were measured by inductively coupled plasma atomic emission spectroscopy (ICP-optima 2001DV, Perkin-Elmer, Waltham, MA, USA).

In the toxicity tests, the algal growth results were obtained by the difference between the final and initial algae densities and chlorophyll fluorescence parameters. Growth of Anabaena sp. density was monitored daily for 96 h and assessed by initial and final OD value at 680 nm. All chlorophyll fluorescence parameters were determined using a Phyto-PAM fluorometer (Pyhto-PAM, Walz, Germany). Phyto-PAM is a four-wavelength chlorophyll fluorometer used to assess the chlorophyll content and photosynthetic activity of planktonic algae. The variables Chl-a fluorescence (F_v) and maximal fluorescence (F_M) were measured. Photosystem II activity was determined using the ΔF mode (F, fluorescence yield = F_M − F_v). The yield (Y, photochemical transformed energy) was calculated as Y = F_v/F_M. In recent years, fluorescence parameters based on fluorescence yield have been proposed to be a useful tool for the toxic evaluation of pollutants [28].

The toxicity experiments were carried out using 50 mL cultures grown in 125 mL flasks. The Anabaena sp. solutions with a series of Zn²⁺ concentrations were cultured and observed in an incubator. The final and initial algae densities and chlorophyll fluorescence parameters were used to examine Zn²⁺ toxicity. The Anabaena sp. solutions with a series of nano-TiO₂ concentrations were tested following the same methods in Zn²⁺ toxicity tests. To investigate the synergistic effects of Zn²⁺ and nano-TiO₂, two sets of experiments were studied. The first set of experiments studied the toxic effects of Zn²⁺ with fixed nano-TiO₂ concentrations. The second set of experiments examined the toxic effects of nano-TiO₂ with fixed Zn²⁺ concentrations. After the toxicity test, the mixed suspensions were centrifuged and the supernatants were collected. Zinc concentrations in the supernatants were measured by ICP.

ROS production was measured by using the cell permeable indicator 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) [39]. The specific method of operation is that 1.0 mL algal cells grown for 72 h were centrifuged at 10,000 rpm for 10 min, after which the supernatant was discarded, washed with phosphate buffer solution twice, followed immediately by the addition of 10 μM H₂DCF-DA to the cell pellet. Next they were incubated in a water bath at 37 °C for 2 h in the dark, and washed with PBS again. The fluorescence intensity of algae cells was measured by a fluorescence microplate reader (Synergy™, Bio-Tek, Richmond, CA, USA) at excitation/emission wavelengths of 488/525 nm. Changes in ROS levels as compared to the control were evaluated using relative ROS level.

The effective concentrations causing 50% inhibition in algal growth (EC₅₀) were calculated and statistical significance was considered at the p < 0.05 level. Differences in growth rates between the control and experimental samples were demonstrated using a comparison of means test for each test concentration. Algal toxicity tests with Anabaena sp. were performed in triplicate. Data were presented as the average values of three parallel detections.