INTRODUCTION

Pollution with heavy metals is constantly increasing due to the increase in agricultural land and industrial production. These toxicants are also present in the environment due to natural processes, such as volcanism, weathering, and soil erosion [1].

Most heavy metals, for example, nickel (Ni), are necessary in certain quantities for the normal life of plants. However, those such as cadmium (Cd) and lead (Pb) are nonessential elements that cause negative changes in plants, primarily due to the production of ROS and a negative effect on enzyme activity [1, 2].

Nickel plays an important role in the functioning of the Ni-containing enzyme urease and Ni-superoxide dismutase. Most marine phytoplankton use urease to hydrolyze urea to ammonium and carbon dioxide. For this reason, with a lack of nickel, the growth of microalgae is inhibited [3].

Unicellular algae, as the basis of trophic chains and the main source of oxygen in aquatic ecosystems, are of natural interest in ecotoxicological terms among researchers [2–5]. Most works on the action of heavy metals in ionic form on microalgae are limited to representatives of the Chlorophyta division [4, 6–9]. At the same time, representatives of raphidophyte algae remain poorly studied [10, 11]. One of the most common types of raphidophytes is Heterosigma akashiwo, which causes harmful “blooms” that lead to the mass death of fish and invertebrates [12, 13].

Traditionally, the assessment of the effect of toxic substances on microalgae is carried out by changing the number of cells and their size and shape since these are integral indicators that reflect the processes occurring in microalgae organisms [9, 14]. The close attention of researchers to the effect of heavy metals on the photosynthetic apparatus is due to the fact that chloroplasts are responsible for many processes in the plant cell [15]. As parameters for assessing the state of the photosynthetic apparatus, the content of pigments (chlorophyll a and carotenoids) and fluorescence of chlorophyll a are used [14, 16]. Under adverse effects on a living organism, the content of ROS increases, and, therefore, this indicator is often controlled in ecotoxicological studies [17]. The content of neutral lipids (NL) in microalgae is also studied to assess the stress impact during adverse effects on the body of algae [18, 19].

The goal of the work was to assess the effect of cadmium, lead, and nickel on the number of cells and their size and internal structure, the photosynthetic apparatus, and the content of ROS and NL in microalgae H. akashiwo.

MATERIALS AND METHODS

Plant material. The object of the study was a culture of unicellular algae Heterosigma akashiwo MBRU_HAK-SR11 (Y. Hada) Y. Hada ex Y. Hara, M. Chihara (Raphidophyceae), provided by the Marine Biobank Shared Use Center of the Zhirmunsky National Research Center for Marine Biology, Far Eastern Branch, Russian Academy of Sciences (http://marbank.dvo.ru).

Experimental conditions. Algae were grown on the f medium [20] prepared on the basis of filtered and sterilized sea water with a salinity of 32‰ in 250 mL Erlenmeyer flasks with a culture medium volume of 100 mL, at a temperature of 18°C, and an illumination intensity of 70 µmol/(m2 s) in the visible light region and 14-h daylight hours. A culture at the exponential growth stage was used as an inoculum. The duration of the experiments was 7 days. Samples for the analysis of indicators were taken on days 3 and 7.

Cadmium was added in the form of 3CdSO4·8H2O, Ni–NiSO4·7H2O, and Pb–PbCl2 salts with conversion to metal ions on the day of the experiment. The studied concentrations of Cd, Ni, and Pb were 10 and 20 µg/L. The choice of concentrations is based on the content of these metals in the coastal waters of Russia and their maximum allowable concentrations: the studied concentrations correspond to MAC and 2MAC.

The term growth in this article refers to the growth of the population: an increase in growth is stimulation of the number of microalgae cells, and inhibition of growth is a decrease in the population.

Measurement methods. The parameters (cell number, forward and side scatter) were measured on a CytoFLEX flow cytometer (Beckman Coulter, United States). For analysis, 10 000 events (registered in the particle sample) were recorded during each measurement. The selection of algae cells from the total number of events recorded by the cytometer was carried out by fluorescence of chlorophyll a [21]. Forward light scattering (FS), which indirectly characterizes the size of algal cells, was recorded on the FSC channel. Side light scattering (SS), which characterizes the internal structure (granularity), was recorded on the SSC channel. Chlorophyll a fluorescence intensity was recorded at a wavelength of 690 nm, the excitation wavelength was 488 nm, channel PC 5.5 [21]. ROS production was assessed using a 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye, and staining was carried out for 1 h at room temperature in the dark. The fluorescence index of its oxidized and diacetylated product was determined at a wavelength of 525 nm, the excitation wavelength was 488 nm, channel FITC [17]. The NL content was determined by the fluorescence of Nile Red fluorochrome at a concentration of 1 µg/mL; staining was carried out for 15 min at room temperature in the dark; excitation wavelength 488 nm; emission 580 nm, channel PE [22].

To analyze the content of photosynthetic pigments (chlorophyll a and total carotenoid content), the algae suspension was collected on MFAS-OS-2 membrane filters. The pigments were extracted with 90% acetone, and the resulting extract was centrifuged for 15 min at 7000 rpm in an Allegra X-22R centrifuge (Beckman-Coulter, United States). The supernatant was collected and its optical density was determined using a Shimagzu-UV 2550 spectrophotometer (Shimagzu, Japan) at the following wavelengths: 480, 630, 647, 664, and 750 nm. Pigment concentrations were calculated using standard formulas [23].

Statistical analysis. The experiments were carried out in three biological replicates. Statistical processing was performed using the Excel program. Data on cell number, forward and side scatter, fluorescence of chlorophyll a, chlorophyll a content, carotenoids, ROS, and NL are presented as percentages of the control. The graphs show the average values of nine measurements. The bars on the graphs represent the standard deviation of the measured values. The significance of differences between the samples was assessed by the Mann–Whitney U-test at a significance level R < 0.05.

RESULTS

Cadmium in the environment at all concentrations stimulated population growth of H. akashiwo throughout the experiment (Fig. 1a). The FS indicator significantly differed from the control on day 7 of the experiment at 10 μg/L Cd (Fig. 1b). The SS index increased on day 3 and significantly decreased by the end of the experiment at both metal concentrations (Fig. 1c). Fluorescence of chlorophyll a decreased by day 7 in all experimental variants (Fig. 1d). Chlorophyll a content and carotenoids was higher than the control when 10 µg/L Cd was added throughout the experiment, and the content of chlorophyll a did not differ from the control at 20 µg/L, while the content of carotenoids increased (Figs. 1e, 1f). The ROS content was also higher than the control by day 7 (Fig. 1g). NL content increased by day 3 of the experiment and decreased by day 7 (Fig. 1f).

Fig. 1.
figure 1

Population growth and physiological state of Heterosigma akashiwo under the influence of cadmium: (a) number of cells, % of control; (b) forward light scattering, % of control; (c) side light scattering, % of control; (d) fluorescence of chlorophyll a, % of control; (e) chlorophyll a content, % of control; (f) content of carotenoids, % of control; (g) content of reactive oxygen species, % of control; (h) content of neutral lipids, % of control. (1) Cadmium content in the medium 10 µg/L, (2) cadmium content in the medium 20 µg/L. * Differences with control are significant at P ≤ 0.05.

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Lead in both concentrations caused a slight stimulation of microalgae growth on day 3 and a slight inhibition on day 7 (Fig. 2a). The indices of FS and SS increased at 10 μg/L of metal on day 7 (Figs. 2b, 3c). Fluorescence of chlorophyll a decreased in the presence of 20 μg/L of the toxicant on day 7 (Fig. 2d). Chlorophyll a content and carotenoids increased on day 3 and significantly decreased on day 7, especially at 20 µg/L of lead (Figs. 2e, 2f). The ROS content exceeded the control by 3.5 and 4.6 times at lead concentrations in the medium of 10 and 20 μg/L, respectively, on day 3 of the experiment and was higher than the control on day 7 (Fig. 2g). The NL content was reliably at the control level during the experiment, except for exceeding that level at a lead concentration in the medium of 10 μg/L on day 7 (Fig. 2h).

Fig. 2.
figure 2

Population growth and physiological state of Heterosigma akashiwo when exposed to lead: (a) number of cells, % of control; (b) forward light scattering, % of control; (c) side light scattering, % of control; (d) fluorescence of chlorophyll a, % of control; (e) chlorophyll a content, % of control; (f) content of carotenoids, % of control; (g) content of reactive oxygen species, % of control; (h) content of neutral lipids, % of control. (1) Lead content in the medium 10 µg/L, (2) lead content in the medium 20 µg/L. * Differences with control are significant at P ≤ 0.05.

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Fig. 3.
figure 3

Population growth and physiological state of Heterosigma akashiwo under the influence of nickel: (a) number of cells, % of control; (b) forward light scattering, % of control; (c) side light scattering, % of control; (d) fluorescence of chlorophyll a, % of control; (e) chlorophyll a content, % of control; (f) content of carotenoids, % of control; (g) content of reactive oxygen species, % of control; (h) content of neutral lipids, % of control. (1) Nickel content in the medium 10 µg/L, (2) nickel content in the medium 20 μg/L. * Differences with control are significant at P ≤ 0.05.

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The addition of nickel at concentrations of 10 and 20 μg/L led to an increase in the number of H. akashiwo cells compared with the control on day 3 (Fig. 3a). The FS indicator remained at the control level during the entire experiment (Fig. 3b). The SS indicator increased compared to that in the control by day 7 (Fig. 3c). Fluorescence of chlorophyll a did not differ from the control in all experimental cases (Fig. 3d). Chlorophyll a content exceeded the control only at a nickel concentration in the medium of 20 μg/L on day 7 of the experiment (Fig. 3e). The content of carotenoids significantly increased on day 3 at a nickel concentration of 10 μg/L and on day 7 at 20 μg/L (Fig. 3f). The ROS content was expressed lower than in the control during exposure (Fig. 3g). The content of NL, on the contrary, exceeded the control, especially on day 7 (Fig. 3h).

DISCUSSION

The experiments performed showed that Cd, Pb, and Ni had a negative impact on the physiological state of H. akashiwo, even when simultaneously stimulating cell growth.

Cadmium, in comparison with other studied metals, caused a particularly pronounced increase in the population of H. akashiwo. Previously, an increase in the number of cells under the influence of 100 μg/L cadmium was also noted in Dunaliella salina [24]. In H. akashiwo, the indicator of BS significantly changed throughout the experiment. The same phenomenon was shown in Phaeocystis antarctica [25]. Fluorescence of chlorophyll a decreased, while the content of photosynthetic pigments, on the contrary, increased, which may indicate a decrease in the efficiency of the photosynthetic apparatus. It is known that Cd inhibits PSII as a result of damage to thylakoids and reaction centers [14, 15]. However, there is evidence that cadmium affects both photosystems [26]. This metal, in addition to inducing physiological disorders, causes changes in the chloroplast structure [5, 26, 27], disruption of membrane transport and permeability, synthesis of plastoquinone and carotenoids, and inactivation of a number of enzymes [27]. The increase in the content of carotenoids recorded by us is probably due to the fact that they protect the photosynthetic apparatus from oxidative stress [24]. Carotenoids quench the triplet states of chlorophylls, which cause the formation of atomic oxygen, and help to release energy from damaged chlorophylls [2]. There is evidence that carotenoids are less exposed to cadmium than chlorophyll a [26]. Heavy metals cause oxidative stress as a result of an increase in the number of free radicals [1, 5, 8]. In our experiment, the ROS content decreased on day 3 and significantly increased on day 7. An increase in the level of ROS under the influence of heavy metals leads to a decrease in photosynthetic productivity as a result of damage to biomolecules, disturbances in mitochondrial metabolism and metabolism between the cytosol and chloroplast [2]. Cadmium leads to an increase in lipid content [7, 15]. In H. akashiwo, there was first an increase and then a sharp drop in the content of NL. Neutral lipids play a protective role in adapting to the negative influence of the environment [7]; thus, their decrease may signal the inhibition of microalgae cells.

Lead affects the enzymatic activity of plants; however, the main reason for the inhibition of plant cell growth is the oxidation of IAA, which is actively involved in their growth processes. In addition, the metal causes a violation of membrane permeability and changes the processes involved in mineral nutrition [1]. We noted growth inhibition of H. akashiwo by the end of the experiment, after its stimulation on day 3. In Isochrysis galbana, an increase in the growth rate occurred at 50–100 µg/L, and the content of chlorophyll a increased already at 5 µg/L [28]. In our experiment, the content of chlorophyll a also increased but subsequently decreased. Evidence of damage to chloroplasts and mitochondria in green algae when this metal is introduced into the medium is an increase in starch deposits in the pyrenoid to store the energy necessary to restore damaged organelles [29, 30]. Chloroplast damage has been noted, for example, in D. salina [31]. Lead leads to a decrease in chloroplast fluorescence [32, 33], which, among other changes, was noted in our experiment only at 20 μg/L after 7 days of incubation. Lead can replace magnesium in the chlorophyll molecule, but such chlorophylls are not strongly bound to the ligands of the pigment-protein complex. Thus, algae synthesize more chlorophyll molecules per reaction center to compensate for nonfunctional chlorophylls and maintain the productivity of the photosynthesis process [16, 32]. In addition, inhibition of chlorophyll a biosynthesis by metals is through its effect on the production of protochlorophyllide, and activation of the enzymatic degradation of chlorophylls by chlorophyllase plays a critical role in the loss of photosynthetic pigment [26]. It is perhaps for these reasons that the content of chlorophyll a in H. akashiwo increased and then decreased. An increase in the content of carotenoids protects the cell from the effects of ROS, which, for example, is shown in Nannochloropsis oculata. At particularly toxic concentrations of metals, the content of carotenoids, on the contrary, decreases, which indicates a strong stress for algae [16]. The same phenomenon occurred in our experiments. In Chlorella elipsoides under the influence of lead, the content of MDA increased, while the content of superoxide dismutase and glutathione reductase decreased [33]. Such changes indicate an increase in the content of ROS in the algae. In our experiment, a pronounced increase in the content of ROS was noted upon the introduction of a toxicant. However, the lipid content did not decrease below the control level, which may be due to the adaptation of the algae to adverse conditions.

Nickel binds to proteins and, to a lesser extent, to microalgae lipids [34]. This metal causes damage to the membrane and disruption of mineral metabolism, especially potassium metabolism, leading to an increase in the concentration of MDA [1]. In our experiment, the growth of H. akashiwo was stimulated at nickel concentrations in the medium of 10–20 µg/L, and the chlorophyll a content and carotenoids at a concentration of 20 μg/L also increased on day 7 of the experiment. Previously, it was shown that the number of cells in green microalgae Ankistrodesmus falcatus decreased at nickel concentrations of 15–30 µg/L and the content of chlorophyll a and carotenoids decreased at 1 μg/L nickel [9]. At the same time, the rate of population growth Phaeocystis antarctica was inhibited by 10% at only a nickel concentration of 260 µg/L [25]. Despite the fact that this metal inactivates PSII, which, among other things, is expressed in a decrease in fluorescence, this indicator did not change in our experiment. Nickel was also the only one among the studied metals at whose introduction the ROS content in H. akashiwo did not increase but, on the contrary, decreased. This toxicant is known to affect the genes responsible for the metabolism of nitrogen, fatty acids, and DNA [35]. Perhaps this is due to the increase in NL, especially towards the end of the experiment, when it affects H. akashiwo.

Thus, our studies showed that the addition of 10 and 20 μg/L Cd to the medium stimulated the growth of the H. akashiwo population and an increase in the content of photosynthetic pigments throughout the experiment. Under the influence of Pb, inhibition of algae growth and a decrease in the content of photosynthetic pigments by the end of the experiment were noted. At the same time, indicators of direct and side light scattering and fluorescence of chlorophyll a in most experimental cases did not differ from the control ones. The content of ROS, in general, increased under the influence of heavy metals. A sharp drop in the content of NL was noted only under the influence of cadmium, while lead either led to a slight increase in the content of NL or had no effect on this indicator.