With the widespread applications of nanomaterials, e.g., in drug delivery, catalysis, and production of personal care products, their discharge into the environment and eco-toxic effects have become persistent causes of concern for researchers (Gottschalk et al. 2009; Li et al. 2015; Lin and Xing 2008). Specifically, nanoscale zinc oxide (nZnO) particles are used in preparing plastics, ceramics, rubber, lubricants, paints, food materials, batteries, fire retardants, personal care products, solar voltaic electronic sensors, medical disinfectants, and so on, because of their unique properties, such as optoelectronic property, UV emission, transparent conductivity, piezoelectricity, and UV absorption and reflection (Ma et al. 2013; Padmavathy and Vijayaraghavan 2008; Wilkie and Morgan 2009). The widespread use of nZnO leads to an increase in the amount discharged into the environment through waste incineration plants, wastewater treatment plants, and landfill, and aquatic ecosystems eventually receive a bulk of the discharged nZnO particles (Keller et al. 2013, 2014). The microalgae are the base of the aquatic ecosystem and suffer directly from the discharged nZnO particles. These particles substantially impact the energy and contaminant transport from microalgae up to higher-trophic-level animals in the food web (Miller et al. 2010; Yung et al. 2015).

The medium of nZnO suspension impacts the behavior of nZnO particles, so they present different chemical effects in fresh water and sea water. However, the main behavioral characteristics, namely, aggregation, dissolution, and sedimentation, occur in both aqueous media. The nZnO behavior impacts the mechanisms of nZnO toxicity in organisms such as microalgae. Dissolution results in the release of Zn2+ into the aqueous medium (Ma et al. 2013; Yamabi and Imai 2002). Free ionic Zn is harmful to cells. The pH, temperature, total organic carbon (TOC) of the exposed medium, and specific surface area of nZnO particles substantially affect the dissolution. In addition, toxic hydroxyl radicals (·OH) and reactive oxygen species (ROS) may be produced by surface interactions of nZnO (Applerot et al. 2009). Further, due to the photo-catalytic property of nZnO, photo-induced toxicity may occur (Hoffmann et al. 1995). However, the interaction between nZnO particles and cells can be ignored in the toxicity assessment. The nZnO particles were observed to adhere on the surface of microalgal cells (Li et al. 2017). Therefore, we present the following hypothesis: swimming microalgal cells may act as nZnO vehicles if exposure dose does not affect the ability of the cells to swim, which results in Zn diffusion. A group of experiments was performed in the present study to analyze the impact of the marine flagellate Tetraselmis helgolandica var. tsingtaoensis on nZnO sedimentation and Zn diffusion.

Materials and Methods

Pure nZnO (code Z713, purity 99.8%, particle size 20–30 nm, surface area 50 m2/g) was purchased from HongwuNewMaterial, Guangzhou, China. Subsamples of clean nZnO powder were analyzed by a scanning electron microscope (HITACHI S-3000 N, Japan) to characterize size distribution (Fig. 1). Further, NaCl, Na2SO4, KCl, NaHCO3, KBr, H3BO3, NaF, MgCl2∙6H2O, CaCl2, SrCl2∙6H2O, NaNO3, NaH2PO4∙H2O, Na2SiO3∙9H2O, CuSO4∙5H2O, ZnSO4∙7H2O, CoCl∙6H2O, MnCl2∙4H2O, Na2MoO4∙2H2O, Na2EDTA, FeCl3∙6H2O, Vitamin B1, B12, Biotin, HNO3, for preparing the artificial sea water (ASW) were purchased from Sinopharm Chemical Reagent Co., Ltd, Shanghai, China. The composition of F/2 ASW enrichment solution was as described by Guillard (1975).

Fig. 1
figure 1

Scanning electron microscopy images show clean nZnO powder (a, b). Top-right inset b is a zoom of part surface of nZnO powder

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The Tetraselmis helgolandica var. tsingtaoensis (Chlorodendraceae: Tetraselmis) cell strain was purchased from Shanghai Guangyu Biological Technology Co., Ltd., China, and was maintained in the ASW medium at 20°C (Li et al. 2017). The inoculant was incubated under cool white fluorescent lights (L:D = 16:8, 2000 lx) at 20°C until the log phase growth to obtain sufficient number of cells for experiments. The cell density was measured using a hemacytometer (MarienFeld, Germany).

Then, 0.124 g of nZnO was added to the ASW to a final volume of 1 L to prepare the stock suspension of nZnO particles (100 mg Zn/L). The nZnO stock suspension was subjected to ultrasonic dispersion at 50 W for 30 min before the test suspension samples were prepared.

Based on the EC50 of 2.17 mg Zn/L calculated using data from a group of preliminary experiments, two concentrations 0.434 and 0.217 mg Zn/L of nZnO suspensions were set in 2 L flasks and microalgal cells were added with a density of 2 × 105 cells/mL. Adequate dispersion was achieved by stirring (120 s at 300 r/min) with a magnetic stirrer at the beginning of suspension preparation. Then, the Zn concentrations and microalgal cell densities were measured every 24 h for 4 days by pipetting 5 mL samples of the suspensions from the three resultant layers of the suspension: surface layer: 0–1 cm from the surface, middle layer: 5–6 cm from the surface, and lower layer: 10–11 cm from the surface but not from the bottom layer. The sampling using a pipette was conducted very carefully to avoid mixing of the layers. Two nZnO suspensions at different concentrations and without microalgal cells were set as control groups. All experimental sets of control groups about nZnO suspension concentrations, volumes, exposure time and sampling way are same as test groups and only without adding algae. The differences between test and control groups are changes brought by algal cells presence.

Two communicating flasks connected by a U-type glass vessel (Φ = 1 cm) were used for the Zn diffusion test (Fig. 2). In the first and second flasks, 0.434 mg Zn/L and 0.217 mg Zn/L nZnO suspensions, respectively, were added, resulting in a concentration imbalance between them. Microalgal cells at 2 × 105 cells/mL were added to the first flask at the beginning, and a light was set from the second flask throughout suspensions to the first flask (Fig. 2). Cells from the first flask would transfer by phototaxis to the second one via the U-type glass vessel, resulting in Zn diffusion. A total of 5 mL suspensions was sampled from each flask every 24 h for 7 days in order to measure the Zn concentrations and microalgal cell densities. Same suspension solutions in similar communicating flasks without microalgae were set as the control.

Fig. 2
figure 2

Two communicating flasks connected by a U-type glass vessel for Zn diffusion experiment design: a front view and b top view

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A microwave digestion system (MARS Xpress, CEM, USA) and an atomic absorption spectrometer (AAS; AA240 Duo, Varian, USA) were used for the Zn concentration measurement. A 1 mL sample of suspensions was digested in 10 mL HNO3 (69%) using the microwave digestion system. The microwave digestion and Zn concentration measurement steps have been described in detail by Li et al. (2018). 10% blank samples and 10% standard samples (GNM-SZN-001-2013) were used to conduct quality control, keeping the error range in ± 10%. The limit of detection of Zn is 0.003 mg/L by this method.

All data are expressed as the mean ± SD (standard deviation) of the different samples specified and were analyzed by one-way analysis of variance (ANOVA) with the SPSS software. The differences were considered statistically significant at p ≤ 0.05. All experiments were set as three replicates (n = 3).

Results and Discussion

The swimming microalgae were exposed to nZnO particles for 4 days. During nZnO sedimentation, the microalgal cell densities and Zn concentrations in the surface layer, middle layer, and lower layer of the ASW column were measured every 24 h; the results are shown in Table 1 and Figs. 3 and 4. The cell densities do not increase obviously in any of the layers of the ASW column during the entire exposure period. In four days, the microalgal population grows by a small extent. No obvious difference is found among the three layers for the two concentration tests. The cells dispersed well in suspensions because T. helgolandica var. tsingtaoensis microalgae swim well by using their four flagella even when the suspensions are not stirred for four days. Air and light made the cells swim up to the surface of the ASW column. In a previous study, the enhanced motility of green flagellate Tetraselmis subcordiformis under illumination had been observed (Ma et al. 2012). In addition, the cells of Tetraselmis suecica gather in the surface layer of a water column via vertical migration under solar irradiation (Richter et al. 2007). In our case, the microalgal cells showed similar swimming ability and migrated up vertically against gravity (which induces sedimentation).

Table 1 Average values of Zn concentrations in three layers and differences between suspensions with and without microalgae (n = 3)
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Fig. 3
figure 3

Microalgal densities in three layers of two suspensions with different nZnO concentrations: a 0.434 mg Zn/L nZnO suspension and b 0.217 mg Zn/L nZnO suspension (n = 3)

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Fig. 4
figure 4

Zn concentrations in three layers of two suspensions with different nZnO concentrations with or without microalgal cells: a 0.434 mg Zn/L nZnO suspension and b 0.217 mg Zn/L nZnO suspension. Red lines separate “microalgae absent” (ma) and “microalgae present” (mp) box plots. White circles indicate the data for each group. Box range is defined by the standard deviation value (n = 3)

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The Zn concentrations in the surface layer exhibited a declining trend during exposure. In the middle layer, the declining trend started later than in the top layer. The lower layer showed gradually increasing Zn concentration. This indicates the typical sedimentation of suspended nZnO particles, as observed in previous studies (Li et al. 2017, 2018). The rate of sedimentation is related to the nZnO concentration that aggregation and further sedimentation occur easier in rich nZnO suspension than low-content counterpart. In this study, the sedimentation rate for the lower concentration (0.217 mg Zn/L) was higher than that in the case of the higher concentration (0.434 mg Zn/L), and the sedimentation rates for both concentrations was lower than that reported by Li et al. (2018) (100 mg Zn/L). This was clear from the data of Zn concentrations in four days (Fig. 4). However, the sedimentation in suspension with microalgal cells seems being promoted than control. In most layers with cells, the Zn concentration is significant lower than that of the control (p < 0.05, see Table 1). The presence of microalgae probably promotes the sedimentation of nZnO particles partly via adhesion with ZnO or Zn ions and subsequent formation of hetero-aggregates that quickly sink (see Fig. 5). Some of the microalgal cells appear to play the role of nZnO vehicles. However, the harmful effect of nZnO on the flagellum adversely affects the swimming ability, leading to the promotion of nZnO sedimentation.

Fig. 5
figure 5

Microalga Tetraselmis helgolandica var. tsingtaoensis and hetero-aggregates of microalgal cells and nZnO particles: a–c cells without nZnO exposure; d–f hetero-aggregates of 0.434 mg Zn/L nZnO suspension at 1 h. Panel d shows nZnO particles adhered to the flagellum, thus affecting the swimming ability of cells. Panel e shows nZnO particles adhered on the surface of cells, and f shows nZnO particles adhered between two flagella

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The toxicity of nZnO toward microalgae was mainly due to the nanoscale size of the particles with a series of hazardous substances and dissolved Zn ions (Aruoja et al. 2009; Franklin et al. 2007; Manzo et al. 2013). Zn diffusion results in toxic effects towards microalgal cells and higher-level organisms in aqueous environments. The Zn concentrations of the nZnO suspension with flagellate organisms and the cell concentrations in the communicating flasks over a 7-day period measured every 24 h are shown in Fig. 6.

Fig. 6
figure 6

Zn concentrations and microalgal cell densities in communicating-flask experiments: a high-nZnO-concentration flask of the control group (initial concentration: 0.434 mg Zn/L); b low-nZnO-concentration flask of the control group (initial concentration: 0.217 mg Zn/L); c high-nZnO-concentration flask of the test group (initial concentration: 0.434 mg Zn/L, initial density: 2.0 × 105 cells/mL); d low-nZnO-concentration flask of the test group (initial concentration: 0.217 mg Zn/L, initial density: 0 cells/mL). White circles indicate the data for each group. Box range is defined by standard deviation value (n = 3)

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Dissolution occurs after nZnO is added to water, and this usually involves the following reactions (Ma et al. 2013; Yamabi and Imai 2002):

$${\text{ZnO}}(s) \, + {\text{ H}}_{{2}} {\text{O}}(l) \rightleftharpoons {\text{Zn}}\left( {{\text{OH}}} \right)_{{2}} (s)$$
$${\text{Zn}}\left( {{\text{OH}}} \right)_{{2}} \left( s \right) \rightleftharpoons {\text{Zn}}\left( {{\text{OH}}} \right)^{ + } \left( {aq} \right) \, + {\text{ OH}}^{ - } \left( {aq} \right)$$
$${\text{Zn}}\left( {{\text{OH}}} \right)^{ + } \left( {aq} \right) \rightleftharpoons {\text{Zn}}^{{{2} + }} \left( {aq} \right) \, + {\text{ OH}}^{ - } \left( {aq} \right)$$

Then, Zn remains in the aqueous medium as Zn(OH)+ and Zn2+, in addition to solid ZnO. Ionic Zn diffuses more easily than ZnO does, so Zn diffusion occurs spontaneously in the first flask and spreads to the second one via the vessel. This is clear from the Zn concentration data in the control group for days 2–3 (Fig. 6a, b): an insignificant increase in the Zn concentration was observed in the second flask. The Zn concentrations in the two flasks gradually decreased during the later part of the exposure period in both groups. According to the Zn concentration changes, sedimentation was the main process occurring in the flasks.

The microalgal density in the first flask remained stable with a slight increase at the end of the exposure period (Fig. 6c). The microalgae bloomed in the second flask from day 3 with an increasing trend seen till the end of the period. Metal ions can be absorbed by microalgal cells (Nakajima et al. 1981; Sandau et al. 1996; Takimura et al. 1992). Most of the absorbed ions corresponded to the intracellular soluble fraction and only a small number was present in the cell wall (Nakajima et al. 1981). The Zn ions could be absorbed by flagellate cells and transported via swimming. Of course, the Zn concentration should not be high enough to damage the swimming ability of the cells. Our hypothesis is that the swimming microalgal cells may transport nZnO particles or Zn ions from the first flask to the second flask via the vessel. Now, the obtained data indicate that a limited number of microalgal cells pass via the vessel, so nZnO or Zn ion detection is not possible. Thus, it is uncertain whether the flagellate cell plays a role in the transport of Zn.

On the basis of the results of the nZnO sedimentation and Zn diffusion experiments, it can be inferred that Zn was not transported by T. helgolandica var. tsingtaoensis cells via ZnO adhesion on the cellular surface and intracellular Zn ion absorption. The hypothesis that the flagella enabled the swimming microalgae to act as a vehicle for Zn transportation with phototaxis-guided direction of Zn transportation, has not been proven in the present study. A more accurate method for the detection of very low concentrations of nZnO should be developed in future, to confirm the role of the microalgae in nZnO transport. In the marine environment, the released nZnO starts to settle by dissolving to form free Zn ions (Li et al. 2017; Merdzan et al. 2014). If the swimming microalgae slowed down the sedimentation of nZnO and promoted the diffusion of Zn, the abundant flagellate cells could retain Zn in the ocean surface water. Most organisms in the marine ecosystem remain near the surface where they can remain exposed to sufficient light. Therefore, Zn can be imported effectively via the food chain from the producer microalgae via the pathway described above. Therefore, the presence of flagellate cells may generate a route of Zn transportation vertically up from nZnO suspension via ZnO adhesion and ion adsorption by flagellate cells, which should be confirmed by conducting further study.

Experiments of sedimentation and Zn diffusion using nZnO were conducted to reveal the role of swimming microalgal cells in transporting nZnO in suspensions. The obtained results indicate that the swimming microalgae could not be vehicles for nZnO particles or Zn ions to transport Zn vertically in the ASW column. In addition, the role of microalgal cells may not be determined arbitrarily based on limited data. Therefore, to confirm the role of the microalgae in nZnO transport, further study using a more accurate method for detecting very low nZnO concentrations is needed. In addition, experiments with a longer exposure time and larger volume of water need to be performed.