INTRODUCTION

The exceptional properties of nanomaterials determine the prospects for their wide application in industry [1]. Metal oxide nanoparticles (NPs) are widely used in the production of structural materials, catalysts, energy storage devices, paints, phosphors, cosmetic, and medical preparations [2, 3]. One of the important directions in the development of nanotechnology is the production of nanopowders, 80% of which are powders of metal oxides; the most popular and in demand for production are NPs of aluminum oxide (Al2O3) [4]. Aluminum oxide is used for the manufacture of optically transparent and structural ceramics, heat-protective coatings and paints and varnishes, acts as a catalyst in a number of organic synthesis processes [5, 6]. Due to the large production of Al2O3 and the open nature of many technological cycles in which it is used, this nanomaterial can be a significant pollutant and pose a serious threat to the environment [7]. It should be noted that the toxic effect from particles of the nanorange is much greater than from particles of similar composition of micron sizes [8, 9].

Despite its prevalence, aluminum and its compounds are toxic elements [10]. Al2O3 NPs are easily absorbed by various cell cultures [11, 12] while exerting a cytotoxic effect [12, 13] and possess the ability to catalytically generate free radicals [14]. Alumina dust (~33 g/m3 five hs a day) causes severe damage to the epithelium of the respiratory tract of rats [15].

Aluminum and its compounds enter natural waters during the partial dissolution of clays and aluminosilicates, as well as a result of harmful emissions from industrial enterprises and with wastewater [15]. Every year there are more such emissions into the environment; control over the degree of pollution by them is getting lower. Since Al2O3 NPs are insoluble in water and virtually incapable of biological degradation, they can accumulate in the components of natural ecosystems and have a detrimental effect on most living organisms that inhabit natural water bodies [16]. The harmful effect of aqueous dispersions of Al2O3 on daphnia [17], freshwater snails [18], fish [19], soil nematodes [20], and insects [21] has been shown.

The main methods for monitoring the ecological state of water bodies continue to be physicochemical methods. However, along with analytical methods, biological testing methods are increasingly being used, which make it possible to assess the entire set of properties of the studied environment by the responses of living organisms. Freshwater ciliates are such organisms; they are widespread in water bodies and play a significant role in the self-purification of water. Being unicellular organisms, ciliates simultaneously demonstrate reactions at the organism and cellular levels, thereby expanding the range of criteria for assessing toxicity. Unfortunately, the issues of the reaction of ciliates to Al2O3 has been insufficiently studied.

The main goal of this study is to determine the toxic effect of aluminum oxide nanoparticles in an experiment on the infusoria Paramecium caudatum (P. caudatum).

EXPERIMENTAL

To assess the toxicity of Al2O3 NPs, we used a commercial preparation from Sigma-Aldrich, which is a white nanodispersed powder with a particle diameter of 16.4 ± 10.0 nm and a ζ potential of 44.3 ± 1.8. The ζ potential and hydrodynamic particle diameter were measured using a Zetasizer Nano ZS instrument (Malvern).

Al2O3 NPs were characterized using dark-field electron microscopy (TEM) and atomic force microscopy (AFM). AFM images were obtained with a Dimension Icon microscope (Bruker) operating in PeakForce Tapping mode, using a ScanAsyst-Air probe (Bruker) (nominal length 115 μm, tip radius 2 nm, spring rate 0.4 N m–1). The obtained data were processed using the Nanoscope Analysis v.1.7 software. (Bruker). Al2O3 NPs were visualized using high-contrast TEM CytoViva®. TEM images were obtained with a condenser CytoViva®attached to an Olympus BX51 microscope equipped with a fluorite objective (×100) and CCD-camera.

For biotesting, an aqueous suspension of Al NPs2O3 was prepared just before the study. To eliminate aggregation, the suspension was sonicated (for 2 min at 44 kHz and 40 W). The acute toxicity of Al2O3 NPs was investigated at concentrations of 100, 50, 40, 30, 25, 20, 15, and 10 μg/mL at various exposures (0.16, 0.5, 1, 3, 5, and 24 h).

We used equalciliary ciliates P. caudatum as model organisms. Assessment of the resistance of ciliates to Al2O3 NPs was carried out according to the method [22] based on determining the survival rate of ciliates. P. caudatum were cultivated in a ten-fold dilution of the Lozin-Lozinsky medium prepared by dissolving the following weighed portions of salts in 1 L of distilled water: NaCl (1.0 g), KCl (0.1 g), NaHCO3 (0.2 g), MgSOfour (0.1 g), and CaCl2 (0.1 g) with the addition of an aqueous yeast suspension of Saccharomyces cerevisiae (3 ml) at a temperature of 22–24°С. For the experiments, ciliates were selected manually using a micropipette. A Carl Zeiss Stemi 2000C stereoscopic microscope was used to observe the paramecium. A culture plate with wells was used for biotesting. Using a micropipette, 10–12 individuals were selected in a minimal amount of medium and transferred to the wells of the plate. After placing the ciliates in the plates, 0.2 ml of the culture medium was poured into the control wells and 0.2 ml of the test sample were added to the experimental wells. The start time of the biotesting was noted. During the exposure, the ciliates were not fed either in the control or in the experimental wells. The criterion of toxicity was the death of ciliates. Immobile and reshaped cells were considered dead. In addition, change in the nature of movement of ciliates was assessed.

Survival of the ciliates (N, %) was determined by the formula

$$N = {{{{N}_{2}}} \mathord{\left/ {\vphantom {{{{N}_{2}}} {{{N}_{1}}}}} \right. \kern-0em} {{{N}_{1}}}} \times \;100,$$

where N2, N1 is the arithmetic mean of the number of ciliates at the end and beginning of the experiment, pcs.

The average lethal dose (LD50) is defined graphically as the concentration of the test solution at which the toxicity is 50%.

All experiments were performed in triplicate. Moreover, each series of experiments was performed at least three times. We used the t-Student’s test for the statistical processing of the results. Differences were considered significant for p ≤ 0.05.

RESULTS AND DISCUSSION

This study aimed to investigate the potentially toxic effects caused by Al2O3 NPs, which are now widely used in industry. Typical AFM images show the geometry and size of Al2O3 particles, which are ellipsoidal and 13–16 nm in diameter. These images represent the topography of the surface as much as possible. For visualization of Al2O3 particles, TEM was used (Fig. 1).

Fig. 1.
figure 1

(Color online) Aluminum oxide nanoparticles: (a) dark field microscopy; (b) atomic force microscopy.

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To assess the toxicity of Al2O3 P. caudatum, which are motile unicellular microscopic organisms that feed on yeast and capture other particles suspended in an aqueous medium, were used as a model in vivo. Paramecia have a typical ellipsoid shape, the cells themselves are transparent, which makes it possible to visualize organelles, for example, digestive vacuoles filled with yeast cells (Fig. 2a). Enhanced TEM can be used to observe the absorption of Al2O3 particles. From Fig. 2b it can be seen that particles of Al2O3 fall into the cell of P. caudatum from the aquatic environment. Aluminum oxide inhibits phagocytic activity in P. caudatum, in this case, the process of formation of digestive vacuoles is disrupted in the entire range of the studied concentrations. At lower concentrations of Al2O3 NPs were diffusely distributed in the cytoplasm (Fig. 2b). As an example, silicon oxide at a concentration of 5 mg/mL has a slightly toxic effect [23], but it does not prevent the formation of digestive vacuoles even at high concentrations. After entry, silicon oxide NPs are transferred to the digestive vacuoles and visualized by TEM (Fig. 2c).

Fig. 2.
figure 2

(Color online) TEM images of ciliates P. caudatum: (a) control (the arrow indicates the digestive vacuoles filled with yeast cells), (b) absorption of nanoparticles of aluminum oxide P. caudatum (indicated by an arrow), NP distribution visualization of Al2O3 in the cytoplasm of P. caudatum (indicated by arrows); (c) visualization of the digestive vacuoles filled with silica particles (indicated by arrows).

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High-contrast TEM images allow rapid, simple, and efficient observation of Al2O3 NPs inside the transparent bodies of paramecium, as in previous studies with the microscopic worms Caenorhabditis elegans [24] and ciliates P. caudatum [23].

In a typical experiment, the acute toxicity of Al2O3 NPs was assessed in the concentration range from 10 to 100 μg/mL. At the same time, the survival rate of ciliates was investigated at various exposures (Table 1).

Table 1. The survival of ciliates P. caudatum (%) depending on the concentration of aluminum oxide
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To assess the survival rate, the number of dead individuals was taken into account. Deformation of the body, rupture of the membrane, lysis of the cell, as well as preservation of immobility, are indicators of the death of ciliates. After 0.16 h of the experiment at concentrations of 100 and 50 μg/mL the absence of motor activity in 50% of the cells was noted. After 0.5 h at a concentration of 100 μg/mL, rupture of the cell membrane and cell lysis were observed, while at a concentration of 50 μg/mL, approximately 30% of the cells retained locomotor activity. In other concentrations, most of the cells retained normal locomotor activity. The survival rates of ciliates after 24 h of incubation with Al2O3 are presented in Fig. 3a. The greatest decrease in survival was observed at concentrations of 20–100 μg/mL, at which Al2O3 has an acute toxic effect on ciliates. Concentrations of 10 and 15 μg/mL have a slightly toxic effect on Paramecium.

Fig. 3.
figure 3

(Color online) The toxicity of aluminum oxide on ciliates P. caudatum: (a) survival of ciliates after 24 h of incubation with aluminum oxide, (b) LD50 the lethal dose of aluminum oxide that causes the death of 50% of the ciliates in 24 h.

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The LD50 is the lethal dose of Al2O3 that causes the death of half (50%) of the organisms within a certain period of time (24 h). As a result of the studies, the toxicity parameter of Al2O3 under acute exposure was LD50 = 23 μg/mL (Fig. 3b).

In studies by other authors, Al2O3 NPs were less toxic. It was found in [25] that the concentration of Al2O3 (particle size 83 nm), at which 50% mortality occurs in the ciliates Paramecium multimicronucleatum in a 48-h exposure was 9269 mg/L. The toxicity of NP Al2O3 (with a particle size Δ50 of 7 and 70 nm) was studied by the chemotoxic response of ciliates P. caudatum [26]. According to the conducted studies Al2O3 NPs (Δ50 = 70 nm) turned out to be more toxic (LD50 = 1.22 mg/L) than (Δ50 = 7 nm) NPs, which had no toxic effect.

Inconsistency of the data on the dependence of the toxicity of Al 2O3 NPs on particle size can be caused by the authors’ use of various toxicity assessment methods.

The toxicity of Al2O3 depends on the interfacial interaction of NPs with the cell surface, as well as on the physicochemical properties of NPs (size and surface charge) [25]. With a decrease in NP size, the surface area increases, which causes a dose-dependent increase in oxidative stress [27]. Oxidative stress is one of the main mechanisms of the toxic effect of Al2O3 NPs under the influence of NPs on aquatic organisms [26]. Particle charge is also a significant factor. Positively charged particles with high affinity for DNA macromolecules which, therefore, carry a genotoxic potential, are the most dangerous [27].

The toxicity of Al2O3 NPs is different for different test organisms. The inhibitory effect of Al2O3 with a particle size of 70 nm for the growth of microalgae Chlorella vulgaris (LD50 = 15 mg/L), and for Daphnia magna Al2O3 turned out to be less; the LD50 is more than 100 mg/L [26]. Aluminum oxide (with a particle size of 16 nm) at a concentration of 4 mg/L induced irreversible histopathological lesions of the branchial, liver, and brain tissues of freshwater fish Oreochromis mossambicus after 96 h of exposure [19]. When comparing the median lethal dose of Al2O3 in different species it was found that P. caudatum ciliates are more sensitive organisms for assessing the toxicity of Al2O3 in aqueous media (LD50 = 23 μg/mL).

When analyzing the literature data, it can be concluded that the toxic effect of Al2O3 NPs is specific and depends on the size and surface charge of the particles, as well as on the concentration and exposure time.

Thus, the high toxicity of Al2O3 NPs can be caused by the small size of particles (13–16 nm) and their high penetrating ability, which facilitates their redistribution within the cell. The aluminium oxide, used in this study has a positive surface potential (44 mV), which can also contribute to increased toxicity. Al2O3 NPs are capable of generating reactive oxygen species, damaging DNA, disrupting protein expression, depolarizing the cell membrane, and causing morphological changes and cell death [12, 28]. Aluminum oxide has a harmful effect on lower aquatic organisms involved in the self-purification of water bodies that are food resources for fish. Consequently, the contamination of the aquatic environment with Al2O3 NPs can have a negative impact on living organisms and pose a threat to aquatic ecosystems.

Despite the fact that ciliates are widely used as a model object to assess toxicity, the mechanisms of toxic effects of Al2O3 NPs on the P. caudatum have practically not been studied. This indicates the need for additional toxicological studies.

CONCLUSIONS

The danger of nanoparticles of aluminum oxide was assessed by the survival rate of ciliates P. caudatum. Al2O3 NPs have an acute toxic effect on paramecium. At a concentration of 100 μg/mL (exposure 0.5 h), 100% death of ciliates is observed. The LD50 is 23 μg/mL at a 24-h exposure. The relatively high toxicity Al2O3 NPs can be caused by the small size (13–16 nm) and positive charge of the particles, as well as their high penetrating ability, which facilitates their redistribution within the cell.