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Article

Short-Term Toxicity of ZnO Nanoparticles on Microalgae at Different Initial Nutrient Concentrations

by
Nikolaos Tzanakis
,
Andriana F. Aravantinou
and
Ioannis D. Manariotis
*
Environmental Engineering Laboratory, Department of Civil Engineering, University of Patras, 26504 Patras, Greece
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(10), 7853; https://doi.org/10.3390/su15107853
Submission received: 31 March 2023 / Revised: 6 May 2023 / Accepted: 8 May 2023 / Published: 11 May 2023
(This article belongs to the Special Issue The Application of Nanomaterials for the Removal of Emerging Pollutants from Water)
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Abstract

:
The aim of this work was to investigate the combined short-term toxic effect of zinc oxide (ZnO) nanoparticles (NPs) and nitrate concentration of the medium on freshwater microalgae. For this purpose, freshwater microalgae Chlorococcum sp. was cultivated in modified Blue-Green medium (BG-11) containing nitrate concentrations ranging from 0 to 300 mg/L, and exposed to ZnO NPs in different concentrations (0.081 to 810 mg/L) for a period up to 96 h. The experimental results revealed that algal growth was affected by the exposure time, NPs concentrations, and mainly the initial nitrate concentration. Differences in microalgae growth rates were observed. The toxic effect of ZnO NPs was higher on microalgae cultured in modified BG-11 with low and high nitrate concentrations. During the 4-day exposure, the highest growth rates were observed at 24 h at an initial nitrate concentration of 50 mg/L; 1.94 d−1 and 0.22 d−1 for 0 and 810 mg/L ZnO NPs, respectively. Nitrate uptake by algal biomass reached up to 40.1% after 96 h of operation in the control culture with an initial nitrate concentration of 50 mg/L. Finally, the results of this study showed the need for the investigation of ZnO NPs toxicity on microalgae under optimum and stressful nutrient conditions for microalgae growth.

1. Introduction

Green microalgae are eukaryotic unicellular organisms that are mainly found in water and rarely in soil. They are autotrophic organisms using sunlight, carbon dioxide, and other nutrients to develop [1]. The main elements used for their growth are nitrogen (N), phosphorus (P), and potassium (K). Depending on the environmental conditions and the chemical composition of the culture medium, the intracellular composition of microalgae (colorants, lipids, proteins) can vary significantly both qualitatively and quantitatively [2,3].
Microalgae exhibit quite high growth rates and are found to be quite resilient and capable to adapt to different environmental conditions. The optimal pH for their growth is found to be between 7 and 9 [1], with extreme pH values causing lysis of the cells [4]. Single-cell microalgae dimensions usually range between 0.2 and 50 µm [5].
Nitrates are a vital compound for cell growth since microalgae are not capable of assimilating the atmospheric or dissolved nitrogen and thus rely on inorganic or organic nitrogen, an element that is crucial for biomass production [6]. On the other hand, it has been found that the increase in nitrate concentration is not always analogous to the biomass concentration [7].
Manufactured nanoparticles (NPs) have gained great attention over the past decades. Their small size and large specific area provide them with some particular optical and physicochemical properties that are not found in their bulk materials. Nowadays, nanoparticles are used in a multitude of industrial and household applications [8]. The increase in manufactured NPs quantities that are synthesized and commercialized through plenty of nanomaterials has resulted in an increased quantity ending up in natural bodies and especially in natural waters [9].
Zinc oxide (ZnO) nanoparticles are the second most used nanoparticles after TiO2. They are used in various manufactured products such as cosmetic products, sunscreens, solar cells, antibacterial agents, paints, and coatings [10]. This increase in production and use has raised concerns regarding their end-of-life behavior and their potential toxic effects on fauna and flora.
NPs are found to be able to generate reactive oxygen species (ROS) under solar irradiation and cause oxidative stress to cells. They can also be harmful to photosynthetic microorganisms by the “shading effect”, i.e., by preventing sunlight from reaching the cells through absorption or scattering, thus inhibiting the photosynthesis process that is vital for the development of algal cells [9]. They have also been found to be adsorbed on cells’ surfaces and block some active sites of the cell wall that are necessary for the growth and metabolism of microalgae or for the uptake of nutrients [11]. NPs have also been detected inside the cell, when their size is smaller than the cellular membrane pores, therefore disturbing the cell from properly functioning [12]. Many studies imply that the toxicity of ZnO nanoparticles is mainly due to Zn2+ ions that are dissolved from the particles since ZnO NPs are quite soluble compared to other nanoparticles, and not by the nanoparticles themselves. Zn2+ ions are toxic to many aquatic species, including microalgae [13,14,15]. In natural waters, NPs have been found to tend to coagulate and create larger aggregates as a result of the high ionic strength of water [16]. They also can alter the physicochemical conditions of the extracellular environment (pH, nutrient composition or bioavailability, irradiation) that directly affect the cell’s metabolism and functioning [17].
Microalgae produce great quantities of oxygen and absorb carbon dioxide, making them key species in global ecosystems, especially in an era of climate change and atmospheric heating. Chlorococcum is a genus of green microalgae that belongs to the family of Chlorococcaceae. These single-celled organisms are commonly found in freshwater habitats, such as ponds, lakes, and streams. They are known for their ability to photosynthesize, producing oxygen as a byproduct. Chlorococcum sp. is used in various industries, such as food, aquaculture, pharmaceutical, wastewater treatment, and biofuel production [18,19,20,21,22].
During the last decade, many researchers have been interested in determining the toxic effect of nanoparticles on living organisms and especially on microalgae that form the basis of the food chain on the planet [7,9,13,17]. The results of these studies showed that the type of nanoparticles [13], morphology, concentration, and exposure time [7,9,17], as well as the species of microalgae that were exposed, determined the effect of NPs. In the short-term toxicity of NPs on microalgae, besides the algal species, an important factor is the cultivation medium that is provided to microalgae during their exposure to NPs [17]. The main difference between the media is the concentration of nutrients provided to microalgae, especially nitrogen, and phosphorus. Additionally, when microalgae are exposed to NPs in real environmental conditions (long-term), the first thing that the microalgae will have to deal with is exposure under nutrient-limited or other stressful conditions.
Even though many studies have focused on the toxic effects of ZnO NPs on living organisms, especially in aquatic environments, their action is yet to be completely explained, mainly due to the complexity of the natural compartments and the multitude of possible mechanisms. The purpose of this study was to examine whether ZnO NPs could have toxic effects on Chlorococcum sp. growth and also whether the concentration of nitrates in the medium could affect the ZnO NPs toxicity. The variation in nitrate content of the medium aims to simulate and predict the behavior of nanoparticles under different environmental field conditions. Common toxicity tests use specific concentrations of nitrates, while in natural and engineered ecosystems nutrient concentration can vary significantly both seasonally and locally, mainly due to human activities.

2. Materials and Methods

2.1. Nanoparticles

ZnO nanoparticles were obtained from Sigma-Aldrich, St. Louis, MO, USA (Catalog Number 544906) in the form of ZnO nanopowder. The nominal particle size provided by the manufacturer was smaller than 100 nm. Two stock suspensions of 8.1 g ZnO NPs/L and 81 mg ZnO NPs/L in deionized water were prepared and used in the experiments. ZnO nanoparticles were ultrasonicated for 30 min before their use in an ultrasonic bath (Transsonic TI-H-5, Elma Hans Schmidbauer GmbH & Co., KG, Singen, Germany), to break down potential large aggregates.

2.2. Microalgae and Culture Medium

Chlorococcum sp. SAG 22.83 freshwater microalga was obtained from the Culture Collection of Algae at the University of Göttingen (SAG). Chlorococcum species have been reported in many parts of the world and are generally considered to have a cosmopolitan distribution [23] this was the main reason that these microalgae were selected to investigate.
The culture medium used in this study was BG-11 (BlueGreen-11) with the following composition: Na2CO3 (20 mg/L) (Sigma-Aldrich, Steinheim, Germany); ACS reagent, Na2Mg EDTA (ethylene diamine tetraacetic acid) (1 mg/L) (Sigma-Aldrich, Steinheim, Germany); 99% ferric ammonium citrate (6 mg/L) (Merck, R.G., Darmstadt, Germany); citric acid∙1H2O (6 mg/L) (Fisher Chemicals, Kandel, Germany); ACS reagent, CaCl2∙2H2O (36 mg/L) (Merck, Reag. Ph. Eur., Darmstadt, Germany); MgSO4 7H2O (75 mg/L) (Merck, Darmstadt, Germany); ACS reagent, K2HPO4 (30.5 mg/L) (Merck, Darmstadt, Germany); ACS reagent, H3BO3 (2.86 mg/L) (Penta, A.G., Prague, Czech Republic); MnCl2∙4H2O (1.81 mg/L) (Merck, Darmstadt, Germany); ACS reagent, ZnSO4∙7H2O (0.222 mg/L) (Lachner, G.R., Neratovice, Czech Republic); CuSO4∙5H2O (0.079 mg/L) (Panreac, Reag. Ph. Eur., Barcelona, Spain); CoCl2∙6H2O (0.05 mg/L) (Merck, Darmstadt, Germany); ACS reagent, 98%, NaMoO4∙2H2O (0.391 mg/L) (Fisher Scientific, Kandel, Germany); ACS reagent, NaNO3 variable (Penta, A.G., Prague, Czech Republic). Four different compositions of the culture medium were tested with respect to their NO3− content. The investigated concentrations were 0, 50, 150, and 300 mg NO3−/L.

2.3. ZnO NPs Exposure and Microalgal Growth

Toxicity of ZnO NPs on Chlorococcum sp. was assessed based on the Organisation for Economic Co-operation and Development (OECD) toxicity guideline 201 [24]. Prior to each experiment, a preculture of Chlorococcum sp. was prepared as follows. A 2 L solution of BG-11 medium containing each time one of the four different nitrate concentrations tested was prepared and sterilized in an autoclave (121 °C, 20 min), to which a concentration of algal cells was introduced from the stock.
The preculture was stirred using a magnetic stirrer and continuously provided with air by an air pump (air pump, HP-400, Sunsun, Zhoushan City, China, air flow rate 3 L/min), filtered through a 0.22 µm pore diameter Nylon syringe filter and constantly irradiated by artificial lighting (radiation intensity 100 μmol m−2 s−1).
The toxicity experiments were performed in 250 mL Erlenmeyer flasks. Chlorococcum sp. cells were left to proliferate in the preculture until they reached their exponential growth phase. At that point, an adequate quantity of cells was extracted from the preculture and then added to each flask containing a sterile medium of the same nitrate concentration as the preculture. The initial cell concentration in the test samples was 104 cells/mL, and the working volume was 100 mL [24]. Moreover, an appropriate quantity of ZnO NPs was withdrawn from the stock solutions and transferred to the flasks to obtain five different initial ZnO NPs concentrations (0.081, 0.81, 8.1, 81, and 810 mg ZnO NPs/L). NPs/L corresponds to 1 μΜ, 10 μΜ, 100 μΜ, 1 mM, and 10 mM of ZnO NPs. The Erlenmeyer flasks were then covered with foil to avoid any potential biological contamination and kept statically, without air supply, exposed to constant artificial irradiation (radiation intensity 100 μmol m−2 s−1), in a walk-in incubator room.
In conclusion, for each nitrate concentration of BG-11 medium, six different samples were prepared, a control sample only containing the algal cells without any ZnO nanoparticles and five samples containing each one of the five ZnO NPs concentrations mentioned above. Each sample was prepared in triplicate.

2.4. Analytical Methods and Statistical Analysis

The duration of each toxicity assay was 96 h, and four parameters were measured to assess microalgae growth and nutrient removal: cell number, pH, NO3− concentration, and UV–Vis–NIR absorption. Sampling took place every 24 h. A total of five measurements were made for each parameter, starting from 0 h to 96 h, and flasks were shaken vigorously before every sampling.
Cell number was counted in triplicate in a Neubauer hemocytometer (0.1 mm, 0.0025 mm2, Optic Labor, Görlitz, Germany) under an optical microscope (model DMLB, Leica Microsystems GmbH, Wetzlar, Germany). For the measurement, 1 mL of sample was placed in an Eppendorf tube and mixed with 100 µL Lugol colorant solution. Lugol is used for dead cell identification by penetrating the interior of the cell and making it appear dark colored to black. Cell number was the primary indicator used for biomass determination because of its high accuracy.
pH was measured by a pH meter (pH 300/310 Waterproof Hand-Held pH, Oakton Instruments, Singapore). Nitrate concentration was measured via ion chromatography (Metrohm 850 Professional IC). Samples were filtered before measurement through a syringe filter (0.22 µm) and diluted with deionized water when necessary.
UV–Vis–NIR absorption spectra were obtained using a Specord 210 Plus spectrophotometer (Analytik Jena, Jena, Germany) by placing a volume of 3 mL of the sample in a quartz cuvette. UV–Visible absorption provides interesting information about biomass growth, as well as ZnO NPs presence and stability. Focus was put on two main absorption peaks. For samples exposed to NPs, a peak at around 370 nm appeared, which is attributed to ZnO NPs electron transitions from the valence to the conduction band [25,26,27]. The second important peak was the one attributed to biomass, at around 680 nm, as reported in the literature as well [28,29]. This band was used to assess the cells’ growth, but could not be used as a primary indicator for precise biomass quantification, since its absorbance has contributions from both biomass pigments. In our case, chlorophyll-a absorbing at this particular wavelength, and biomass turbidity, i.e., cells. The increase in the absorbance of this peak indicates the proliferation of the cells and validates the results obtained by cell number counting.

2.5. Data Analysis

The specific growth rate (μ) was determined from the growth phase by the following equation:
μ = (lnXt − lnX0)/(t − t0)
where Xt is the number of cells at time t (days), and X0 the initial number of cells at time t0.
The growth inhibition rate (I%) was calculated according to the OECD 201 guideline [14] [OECD, 2011]:
Inhibition (%) = [(μcontrol − μtoxicity)/μcontrol] × 100
where μcontrol is the mean value of the average specific growth rate (μ) in the control, and μtoxicity is the average specific growth rate for the treatment replicate. After checking for homogeneity of the variance (Levene’s test of equality of error variances), the significant differences among the parameters measured in algae were tested, with the use of the Mann–Whitney U-test (p < 0.05). Significant alterations in the growth rate (μ) observed in each alga treated with each concentration of ZnO NPs for a period of 24, 48, 72, and 96 h were tested with the use of the Friedman test (p < 0.05).
Statistical analysis of data was performed in IBM SPSS Statistics 26 software, using ANOVA or Kruskal–Wallis tests.

3. Results and Discussion

3.1. Effect of ZnO NPs on Algae Growth

Figure 1 shows the cell numbers of Chlorococcum sp. cultured in modified BG-11 media in the presence or absence of ZnO NPs. The first plot (Figure 1a) shows cultures where no nitrates were added to the BG-11 media. Despite the absence of nitrates, biomass growth was observed, and there was a high impact of ZnO NPs, especially at a concentration of 810 mg/L, which was the highest concentration of ZnO tested. The increase in microalgae biomass is likely due to the release of nutrients from the lysis of dead algal cells, which provide nutrients to the culture solution [30,31]. The highest cell number was observed on the fourth day in the control culture and the lowest ZnO NPs concentration (0.081 mg/L), which were at 8.8 × 104 cells/mL and 4.6 × 104 cells/mL, respectively.
The results of microalgae cultivation with an initial nitrate concentration of 50 mg/L are shown in Figure 1b. In this case, the highest biomass production of all cultures was observed. Specifically, the highest cell number was in the control and at the lowest ZnO NPs concentration (0.081 mg/L), which were 47.4 × 104 cells/mL and 18.2 × 104 cells/mL, respectively. These results confirm that the nutrient supply in algal culture can have a significant impact on the growth rate of the algal species cultured. At higher ZnO NPs concentrations, the correlation between the cell number of cultures with different nitrates’ initial concentrations is more obvious due to the highly toxic effects of ZnO NPs. On the other hand, at lower ZnO NPs concentrations (0.081 and 0.81 mg/L) the toxic effect is limited, and cell number is highly affected by nitrates’ initial concentration, therefore, there is less correlation. As shown in Figure 1c, the cell concentration with the initial nitrate concentration of 150 mg/L, is higher at 0.81 mg/L ZnO NPs than in the control and 0.081 mg/L ZnO NPs cultures. Although the experiments were conducted in triplicate and two samples were taken from each flask, when working with living organisms such as microalgae, there is not always perfect linearity or correlation in the observations, and some deviations can be observed [32]. Different algal species have different nutritional requirements, so the nutrient composition of the growth medium must be tailored to meet the specific needs of each microalga [1].
Table 1 shows the growth rates and confirms that the nitrate concentration of 50 mg/L is the optimum concentration among the tested ones for Chlorococcum sp. to reach the highest biomass production for a culture volume of 100 mL. More precisely, at 96 h, for the culture containing 50 mg/L nitrates, the growth rate decreases from 0.96 to 0.45 day−1, while moving from the control culture towards the one exposed at 8.1 mg/L ZnO NPs, or a decrease of approximately 50%. The same motive is observed for the cultures with nitrates of 150 and 300 mg/L, which results in a decrease of 100% and of around 75% for 0 mg/L nitrates. Overall, the culture with 50 mg/L nitrates is quite resistant to ZnO NPs toxicity for concentrations up to 8.1 mg/L. On the other hand, at higher ZnO NPs concentrations (81 and 810 mg/L), the toxic effects become important with a contribution of shading effect due to the high concentration of ZnO NPs, which turns the culture opaque and thus prevents light from reaching the cells. Regardless of the significant inhibition of algal growth rate, following the increased nitrate concentration currently reported, the presence of relevant high sodium levels in the culture media and the concomitant interference with the obtained results should not be neglected. To our knowledge, there are no literature data regarding the sodium modulatory mode of action in freshwater algal species tested, and further investigation should be a challenge.
The results of this case also confirm that the toxic effect of ZnO NPs on algae growth depends on nutrient content and that under favorable nutrient conditions, cells tend to be more resilient to ZnO NPs toxic effects. Hence, it is possible that the toxicity of NPs could be higher when microalgae are grown under sub-optimal nutrient conditions. This is because microalgae that are grown under nutrient stress or other unfavorable conditions may be more susceptible to various stressors, including NPs, which could lead to greater toxicity. An increase in microalgae biomass was observed at an initial nitrate concentration of 50 mg/L, even at the highest ZnO NPs concentration (810 mg/L) according to the growth rate results (Table 1). This implies that the optimum culture conditions of microalgae growth could strengthen microalgae cells to face the effects of ZnO NPs.
Figure 1c,d illustrates the results of microalgae cultivation with initial nitrate concentrations of 150 mg/L and 300 mg/L, respectively. The exposure of microalgae to high ZnO NPs concentrations (81 and 810 mg/L) results in decreased biomass production. This implies that the toxicity of NPs is higher due to the fact that microalgae are grown under sub-optimal nutrient conditions. High nutrient concentrations can actually be sub-optimal and prevent algal growth. This phenomenon is commonly known as nutrient inhibition or nutrient toxicity. Nutrient inhibition occurs when the concentration of a particular nutrient exceeds the optimal range for microalgal growth, leading to reduced or even inhibited growth rates. This is because high nutrient concentrations can interfere with various cellular processes, including photosynthesis, respiration, and cell division, and can lead to the accumulation of toxic byproducts [33]. Excessive nitrogen and phosphorus concentrations in the growth medium can lead to overstimulation of algal growth and an imbalance in the nitrogen-to-phosphorus ratio, which can result in the production of harmful metabolites that can inhibit further growth. Similarly, high concentrations of heavy metals and other trace elements can also be toxic to microalgae and interfere with various cellular functions [17].
For samples exposed to NPs, a peak at around 370 nm appeared, which is attributed to ZnO NPs electron transitions from the valence to the conduction band [25,26,27]. The peak was visible in samples containing 8.1, 81, and 810 mg ZnO NPs/L, but not in samples with 0.081 and 0.81 mg ZnO NPs/L where the nanoparticles’ concentrations were very low (Figure 2). The decrease in this peak over time is due to ZnO nanoparticle dissolution [34]. The latter was also observed conducting UV–Vis–NIR scanning of ZnO NPs samples in BG-11 medium. UV–Vis-NIR raw data were normalized at 1100 nm for better comparison. The second important peak was the one attributed to the biomass, at around 680 nm, as reported in the literature as well [28,29]. This band is used to assess the cells’ growth, but it cannot be used as a primary indicator for precise biomass quantification, since its absorbance has contributions from both biomass pigments, in our case chlorophyll-a absorbing at this particular wavelength, and biomass turbidity, i.e., cells. The increase in the absorbance of this peak indicates the proliferation of the cells and validates the results obtained by cell number counting.

3.2. Effect of ZnO NPs on Nutrient Uptake

The pH values ranged from 7 to 10 (Figure 3). Changes in pH were observed in the control and the 0.081 and 0.81 mg/L ZnO NPs cultures, which showed increased algal biomass. During microalgae culturing, pH values can be increased due to photosynthesis, nutrient uptake, etc. Microalgae use light energy to convert carbon dioxide (CO2) into organic matter and release oxygen (O2) as a byproduct. This process leads to a pH increase in the culture medium, because CO2 is consumed and O2 is released [35,36]. Moreover, microalgae require nutrients such as nitrogen and phosphorus for their growth. When these nutrients are depleted from the culture medium, the microalgae will start to take up carbonates and bicarbonates from the medium, which can also contribute to the increase in pH [17]. pH increase was used as a secondary indicator for algal growth.
Figure 4 shows the results of nitrate concentrations of Chlorococcum sp. cultures in modified BG-11 media in the presence or absence of ZnO NPs. Specifically, it presents the results with initial nitrate concentrations of 0, 50, 150, and 300 mg/L in the plots Figure 4a, 4b, 4c, and 4d, respectively. A general observation was that nitrate concentration decreased with exposure time only when it was associated with biomass production. Nitrogen is an essential nutrient for microalgae growth because it is a key component of many cellular molecules, such as proteins, nucleic acids, and chlorophyll [37]. Proteins are made up of amino acids, which contain nitrogen, and nucleic acids, which are the building blocks of DNA and RNA and also contain nitrogen. Chlorophyll, the pigment that gives green microalgae their color, contains a nitrogen atom in its structure. Without sufficient nitrogen, microalgae cannot synthesize these essential molecules, which are necessary for cell growth and division. Nitrogen is often the limiting nutrient in aquatic ecosystems, and its availability can strongly influence microalgae growth rates. In fact, many studies have shown that nitrogen availability can significantly affect the growth, biomass productivity, and biochemical composition of microalgae [1,38]. However, it is important to note that excessive nitrogen concentration can also be harmful to microalgae, as it can lead to overstimulation of growth, an imbalance in the nitrogen-to-phosphorus ratio, and the production of harmful metabolites that can inhibit further growth. This is easily confirmed by the results of Chlorococcocum sp. growth rates (Table 1). In the case of the highest initial nitrate concentration (300 mg/L), the quick uptake of nitrates by algal cells may also lead to quick consumption of zinc, which gives way for nanoparticles to enter the algal cell. Specifically, the cultures exposed to 8.1 mg/L ZnO NPs with initial nitrate concentrations of 300 mg/L and 150 mg/L from 24 h to 96 h, presented a change from 0.52 to 0.08 d−1 and from 0.22 to 0.58 d−1, respectively. Therefore, a balanced nutrient supply is essential for optimal microalgae growth and productivity.
The control cultures showed the highest removal of nitrates. In the presence of ZnO NPs, the nitrate removal decreased with the increase in NPs concentration (Figure 5). Specifically, the highest nitrate uptake by microalgal biomass was in the control culture after 96 h of operation and was at 40.1% of the initial concentration of 50 mg/L nitrates. The lowest nitrate uptake was 2% in cultures with initial nitrate concentrations of 150 and 300 mg/L and ZnO NPs concentration of 810 mg/L. As mentioned above, these values were expected since nitrate uptake is analogous to biomass production. The high concentration of ZnO NPs in the culture medium can lead to the release of zinc ions, which can bind to and interfere with the uptake of essential nutrients by microalgae. The release of zinc ions from ZnO NPs can lead to an increase in the concentration of zinc in the growth medium, which can compete with other micronutrients like iron and copper for their uptake by microalgae. This can disrupt the balance of essential nutrients in microalgae and inhibit their growth.
In addition to the competition for nutrient uptake, high concentrations of ZnO nanoparticles can also induce oxidative stress in microalgae, leading to the production of reactive oxygen species that can damage cell components and disrupt metabolic processes [12]. This can further reduce nutrient uptake and growth inhibition in microalgae. Overall, the negative effects of high concentrations of ZnO nanoparticles on nutrient uptake in microalgae are primarily due to the release of zinc ions, which can interfere with nutrient uptake and disrupt metabolic processes. The binding or encapsulation of nutrients within nanoparticles is not a common phenomenon and is not typically responsible for the effects of nanoparticles on nutrient uptake.
Finally, the presence of ZnO NPs can have both positive and negative effects on nutrient uptake during algal growth, depending on the concentration and exposure time to NPs. At low concentrations, ZnO NPs can act as a source of zinc. Zinc plays a significant role in chlorophyll formation, which is essential for photosynthesis [39]. Therefore, the presence of ZnO NPs can enhance microalgae growth and improve nutrient uptake, especially under sub-optimal nitrate concentrations (0, 150, and 300 mg/L), as seen in Figure 1. However, high ZnO NPs concentrations can have toxic effects on microalgae.
The mechanisms by which ZnO NPs affect nutrient uptake in microalgae are not yet fully understood, but they are thought to involve a combination of physical and biochemical processes. ZnO NPs can interact with the cell membrane of microalgae, leading to changes in membrane permeability and the release of intracellular components. They can also induce oxidative stress, leading to the production of reactive oxygen species, which can damage cell components and disrupt metabolic processes [12].

3.3. Nitrate Concentration Impact on ZnO NPs Toxicity

The inhibition of Chlororcoccum sp. exposed to different concentrations of ZnO NPs (0.081 to 810 mg/L) is shown in Figure S1. ZnO NPs inhibited algal growth even at low concentrations (0.081 mg/L), in all cultures, for a short-term exposure of 96 h. In some cases, the inhibition of algal growth was up to 100%. Inhibition rates are calculated by comparing the cell numbers of a culture exposed to ZnO NPs with their corresponding control culture for each day. An inhibition rate of almost 100% does not mean that all cells are dead, and two cultures with similar inhibition rates can have significantly different cell numbers. For instance, cultures with 0 and 50 mg/L nitrates exposed to 810 mg/L ZnO NPs both have inhibition rates of around 90% after 96 h of exposure (as can be seen in Figure S1a,b). However, their corresponding cell numbers are 0.5 × 104 and 1.3 × 104 cells/mL, respectively (Figure 1a,b). Additionally, the inhibition values have shown that the cultures with the lower biomass concentrations (150 and 300 mg/L) can be better adapted to ZnO NPs exposure after a period of time when their growth rate is slower. The fast growth of microalgae biomass could lead to the internal impact of metabolism reactions of the cell due to the presence of ZnO NPs. It is possible that if microalgae were exposed for a longer period, the toxicity of ZnO NPs on Chlorococcum sp. would be different. In previous work [9] Scenedesmus rubescens were cultured in two different media and exposed to different ZnO NPs concentrations. With ZnO NPs up to 0.81 mg/L the inhibition initially observed was not sustained after the 4th and 7th days for the ⅓N BG-11 and BBM, respectively. In general, the long-term toxicity of ZnO NPs to S. rubescens demonstrated opposite results when compared to the short-term toxicity [17].
In addition, it should be mentioned that the toxicity of ZnO NPs could be higher when microalgae are grown under nutrient stress or other unfavorable conditions, which makes them more susceptible to various stressors. Miao et al. [40] reported that the toxicity of silver-engineered nanoparticles to the microalga Thalassiosira weissflogii was greater under nutrient-limited conditions than under optimal nutrient conditions. Another study by Das et al. [41] showed that silver nanoparticles were more toxic in natural phytoplankton when they were grown under phosphorus limitations.

4. Conclusions

ZnO NPs can have adverse effects on Chlorococcum sp. growth, depending on their concentration and nitrate content in the medium. The toxicity of ZnO NPs can be modulated by nutrient supply, with low and high nutrient concentrations exacerbating the toxicity and medium nutrient concentrations mitigating the toxicity. These findings highlight the importance of considering the availability of nutrients in the assessment of the environmental risk of ZnO NPs and suggest that nutrient management could be a potential strategy for mitigating the toxicity of ZnO NPs on microalgae. It is important to note that the relationship between nutrient concentration and nanoparticle toxicity may be complex and species-specific. Some microalgae may be more tolerant to NPs under nutrient stress than others, and the type and concentration of NPs could also affect toxicity. Finally, further research is needed to better understand the mechanisms underlying the interaction between nutrient availability and NPs toxicity in microalgae.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su15107853/s1, Figure S1: Growth inhibition rate (%I) of ZnO NPs-treated microalgae species (○ 0, □ 0.081, Δ 0.81, ◊ 8.1, ■ 81, ● 810 mg/L ZnO NPs) with initial concentration of NO3− at (a) 0 mg/L, (b) 50 mg/L, (c) 150 mg/L, and (d) 300 mg/L.

Author Contributions

N.T.: investigation, data curation, writing—original draft preparation; A.F.A.: data curation, writing—reviewing; I.D.M.: conceptualization, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effect of different ZnO NPs concentrations (○ 0, □ 0.081, Δ 0.81, ◊ 8.1, ■ 81, ● 810 mg/L ZnO NPs) on cell numbers of Chlorococcum sp, with an initial concentration of N O 3 − at (a) 0 mg/L, (b) 50 mg/L, (c) 150 mg/L, and (d) 300 mg/L.
Figure 1. Effect of different ZnO NPs concentrations (○ 0, □ 0.081, Δ 0.81, ◊ 8.1, ■ 81, ● 810 mg/L ZnO NPs) on cell numbers of Chlorococcum sp, with an initial concentration of N O 3 − at (a) 0 mg/L, (b) 50 mg/L, (c) 150 mg/L, and (d) 300 mg/L.
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Figure 2. UV–Vis–NIR spectra of Chlorococcum sp. cultures after 96 h of exposure to different ZnO NPs concentrations (0, 0.081, 0.81, 8.1, 81, 810 mg/L ZnO NPs) with 50 mg/L initial concentration of N O 3 − .
Figure 2. UV–Vis–NIR spectra of Chlorococcum sp. cultures after 96 h of exposure to different ZnO NPs concentrations (0, 0.081, 0.81, 8.1, 81, 810 mg/L ZnO NPs) with 50 mg/L initial concentration of N O 3 − .
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Figure 3. pH values of Chlorococcum sp. cultures after exposure to different ZnO NPs concentrations (○ 0, □ 0.081, Δ 0.81, ◊ 8.1, ■ 81, ● 810 mg/L ZnO NPs) with initial concentration of N O 3 − at (a) 0 mg/L, (b) 50 mg/L, (c) 150 mg/L, and (d) 300 mg/L.
Figure 3. pH values of Chlorococcum sp. cultures after exposure to different ZnO NPs concentrations (○ 0, □ 0.081, Δ 0.81, ◊ 8.1, ■ 81, ● 810 mg/L ZnO NPs) with initial concentration of N O 3 − at (a) 0 mg/L, (b) 50 mg/L, (c) 150 mg/L, and (d) 300 mg/L.
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Figure 4. NO3− concentration variation of Chlorococcum sp. cultures exposed in ZnO NPs (○ 0, □ 0.081, Δ 0.81, ◊ 8.1, ■ 81, ● 810 mg/L ZnO NPs) for initial concentration of N O 3 − at (a) 0 mg/L, (b) 50 mg/L, (c) 150 mg/L, and (d) 300 mg/L.
Figure 4. NO3− concentration variation of Chlorococcum sp. cultures exposed in ZnO NPs (○ 0, □ 0.081, Δ 0.81, ◊ 8.1, ■ 81, ● 810 mg/L ZnO NPs) for initial concentration of N O 3 − at (a) 0 mg/L, (b) 50 mg/L, (c) 150 mg/L, and (d) 300 mg/L.
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Figure 5. Nitrate removal (%) in Chlorococcum sp. cultures exposed to ZnO NPs (0.081, 0.81, 8.1, 81, 810 mg/L) at an initial concentration of nitrates (50, 150, and 300 mg/L) after 96 h.
Figure 5. Nitrate removal (%) in Chlorococcum sp. cultures exposed to ZnO NPs (0.081, 0.81, 8.1, 81, 810 mg/L) at an initial concentration of nitrates (50, 150, and 300 mg/L) after 96 h.
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Table
Table 1. ZnO NPs effect on Chlorococcum sp. growth rate at different initial nitrate concentrations.
Table 1. ZnO NPs effect on Chlorococcum sp. growth rate at different initial nitrate concentrations.
ZnO NPs (mg/L)
Initial NO3− ConcentrationTime (h)Control0.0810.818.181810
Growth Rate (day−1)
0 mg/L240.12 ± 0.080.06 ± 0.040.00 ± 0.000.45 ± 0.210.00 ± 0.000.00 ± 0.00
480.42 ± 0.260.00 ± 0.000.00 ± 0.000.09 ± 0.040.00 ± 0.000.00 ± 0.00
720.52 ± 0.000.00 ± 0.000.12 ± 0.060.13 ± 0.120.00 ± 0.000.00 ± 0.00
960.54 ± 0.290.38 ± 0.080.32 ± 0.010.13 ± 0.050.04 ± 0.010.00 ± 0.00
50 mg/L241.94 ± 0.701.42 ± 0.621.43 ± 0.231.14 ± 0.440.32 ± 0.010.22 ± 0.09
480.56 ± 0.190.57 ± 0.110.73 ± 0.010.30 ± 0.100.46 ± 0.070.14 ± 0.07
720.90 ± 0.220.87 ± 0.020.75 ± 0.060.39 ± 0.130.00 ± 0.000.08 ± 0.01
960.96 ± 0.350.72 ± 0.050.74 ± 0.070.45 ± 0.130.18 ± 0.050.0 ± 0.00
150 mg/L240.75 ± 0.230.00 ± 0.000.22 ± 0.140.00 ± 0.000.00 ± 0.000.00 ± 0.00
480.30 ± 0.170.14 ± 0.010.18 ± 0.050.00 ± 0.000.00 ± 0.000.00 ± 0.00
720.52 ± 0.250.45 ± 0.100.51 ± 0.060.00 ± 0.000.00 ± 0.000.00 ± 0.00
960.50 ± 0.100.43 ± 0.010.58 ± 0.090.00 ± 0.000.00 ± 0.000.00 ± 0.00
300 mg/L240.89 ± 0.310.66 ± 0.140.52 ± 0.190.00 ± 0.000.00 ± 0.000.00 ± 0.00
480.34 ± 0.040.30 ± 0.120.61 ± 0.210.26 ± 0.130.00 ± 0.000.00 ± 0.00
720.10 ± 0.010.15 ± 0.010.23 ± 0.090.00 ± 0.000.00 ± 0.000.00 ± 0.00
960.28 ± 0.140.30 ± 0.150.08 ± 0.010.00 ± 0.000.01 ± 0.010.00 ± 0.00
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Tzanakis, N.; Aravantinou, A.F.; Manariotis, I.D. Short-Term Toxicity of ZnO Nanoparticles on Microalgae at Different Initial Nutrient Concentrations. Sustainability 2023, 15, 7853. https://doi.org/10.3390/su15107853

AMA Style

Tzanakis N, Aravantinou AF, Manariotis ID. Short-Term Toxicity of ZnO Nanoparticles on Microalgae at Different Initial Nutrient Concentrations. Sustainability. 2023; 15(10):7853. https://doi.org/10.3390/su15107853

Chicago/Turabian Style

Tzanakis, Nikolaos, Andriana F. Aravantinou, and Ioannis D. Manariotis. 2023. "Short-Term Toxicity of ZnO Nanoparticles on Microalgae at Different Initial Nutrient Concentrations" Sustainability 15, no. 10: 7853. https://doi.org/10.3390/su15107853

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