Aquatic Toxicity of Photocatalyst Nanoparticles to Green Microalgae Chlorella vulgaris

: In the last years, nanoparticles such as TiO 2 , ZnO, NiO, CuO and Fe 2 O 3 were mainly used in wastewater applications. In addition to the positive aspects concerning using nanoparticles in the advanced oxidation process of wastewater containing pollutants, the impact of these nanoparticles on the environment must also be investigated. The toxicity of nanoparticles is generally investigated by the nanomaterials’ effect on green algae, especially on Chlorella vulgaris . In this review, several aspects are reviewed: the Chlorella vulgaris culture monitoring and growth parameters, the effect of different nanoparticles on Chlorella vulgaris , the toxicity of photocatalyst nanoparticles, and the mechanism of photocatalyst during oxidative stress on the photosynthetic mechanism of Chlorella vulgaris . The Bold basal medium (BBM) is generally recognized as an excellent standard cultivation medium for Chlorella vulgaris in the known environmental conditions such as temperature in the range 20–30 ◦ C and light intensity of around 150 µ E · m 2 · s − 1 under a 16/8 h light/dark cycle. The nanoparticles synthesis methods inﬂuence the particle size, morphology, density, surface area to generate growth inhibition and further algal deaths at the nanoparticle-dependent concentration. Moreover, the results revealed that nanoparticles caused a more potent inhibitory effect on microalgal growth and severely disrupted algal cells’ membranes. of wastewater through efﬁcient and inexpensive methods to maintain the environment in normal parameters.The use of nanoparticles is causing a substantial environmental impact due to the aquatic environment’s toxic effect. This work provides an overview of the key parameters of growing Chlorella vulgaris green algae. The current status of photocatalytic nanomaterials and their toxic effect on Chlorella vulgaris ’s growth provide a direction for future research in this area and scaling-up and industrial-scale applications.


Introduction
Conventional wastewater treatment technologies such as coagulation, precipitation, flocculation, adsorption, and natural aerobic treatment [1] have been sufficient for years. However, the complexity of pollutants discharged into the water has constrained new technologies to removing various emerging pollutants. Technologies based on nanoparticles, such as advanced oxidation processes, are efficient for emerging pollutants in low concentrations, such as pesticides, pharmaceuticals, endocrine disruptors, and personal care products [2]. Nanoparticles are particles obtained by natural or synthetic methods with less than 100 nm [3]. Nanoparticles have applications in various industrial [4,5] and medicine [6,7] fields, including wastewater treatment [8]. The most significant exposures from engineering material applications avenues for assessing environmental risks are water and soil [4,9]. The activity of nanoparticles in the environment depends on several characteristics, such as morphology, porosity, grain size, composition and crystallinity, groups attached on the surface, and charge of the surface. An area of interest for research is the decontamination of wastewater through efficient and inexpensive methods to maintain the environment in normal parameters.The use of nanoparticles is causing a substantial environmental impact due to the aquatic environment's toxic effect. This work provides an overview of the key parameters of growing Chlorella vulgaris green algae. The current status of photocatalytic nanomaterials and their toxic effect on Chlorella vulgaris's growth provide a direction for future research in this area and scaling-up and industrial-scale applications.

Chlorella vulgaris-Culture, Monitoring and Growth Parameters
Microalgae respond rapidly to environmental changes, and they are essential because of their role as primary producers in aquatic systems due to their short cell-doubling time. In aquatic systems, microalgae Chlorella vulgaris have been used for experiments to determine their crucial role in oxygen production and as the producers of food chains/food webs [10]. Destruction of activity would lead to an imbalance in the aquatic environment [11]. Because of their unicellular structure, microalgae suffer abnormal damage that may affect the whole aquatic system after exposure to pollutants. Simultaneously, in multicellular organisms, there are barriers skin and respiration that protect them [10,12].
Chlorella vulgaris, an essential green eukaryotic photosynthetic microorganism capable of rapid adaptation to new environments, with a structure similar to plants in an aquatic system, is a unicellular organism [13] with a spherical shape 2.5-10 µm in diameter [13,14], with a lipid production capacity range 14-56 dw% (dw-dry weight), protein content 10-58 dw%, and carbohydrate content 10-17 dw% [15]. Chlorella vulgaris reproduces asexually most commonly by auto-sporulation (i.e., four daughter cells with their own and dependent cell wall) [16]. In optimum conditions, Chlorella vulgaris can rapidly multiply asexually, and due to this, it is widely used as a model aquatic organism for toxic studies [17].

Chlorella vulgaris Maintenance Medium and Culturing Parameters
According to the Organisation for Economic Cooperation and Development (OECD) criteria, the concentrations of the Chlorella vulgaris growth nutrients, OECD Test no. 201: freshwater alga and cyanobacteria, growth inhibition test [18], are presented in Table 1. Chlorella vulgaris has a rapid growth rate, and it is ideal for production because it is remarkably resistant to conditions and invaders. For growth of this algae, there are three types of method: autotrophic, mixotrophic, and heterotrophic. Microalgae growth depends on pH, temperature, intensity and duration of illumination, and mineral nutrients' concentration [19]. Chlorella vulgaris grows better at a temperature between 22 and 30 • C [20]; a temperature higher than 30 • C leads to slow growth and cell death [21]. The pH is essential for optimal growth, and optimum pH is neutral, but microalgae can survive at pH 3.0 [22,23]. A high concentration of iron, an essential micronutrient, induced a lipid accumulation while increasing Chlorella vulgaris's growth [24].
Intensity and wavelength of light are significant for the growth of Chlorella vulgaris. For algae, light helps cell proliferation and photosynthesis. In order to keep the cell growth rate high and the costs low, it is recommended to alternate light with darkness under 8 h: 16 [28,29]. Chlorella vulgaris has registered more cell division after stopping the light [30]. Light intensity is also important from the point of view of economic and operating efficiency and varied from 2800 to 8000 lux m −2 s −1 [28,31,32].
In terms of wavelength, microalgae usually use wavelengths between 400 nm and 700 nm (red light λ = 630-665 nm [33] and blue light λ = 430-465 nm [34]) for photosynthesis. Chlorella vulgaris cell size measurement demonstrated that microalgae cells' growth had an approximate increase of 60-70% in diameter under blue light, compared to red light [30].
Aeration is ensured through aeration pumps because in the absence of any aeration or mixing, the unicellular form of algae dies, and the growth slowly decreases [35]. After five days of aeration Chlorella vulgaris growth is two times than without aeration [36].

Chlorella vulgaris Growth Monitoring
Algae growth modeling has been attracting attention over the past century regarding the effect of process parameters such as temperature, pH, light, nutrients, and growth rate. A few essential parameters are usually monitored ( Table 2).

The cell Concentration
The cell concentration is needed to estimate the algae growth; for cell density quantification, a few techniques are presented: (1) UV-VIS spectrophotometric optical density (OD), estimating chromophore formation in liquid culture and quantitative evaluation using Beer-Lambert low [46] at different wavelengths of 480, 649, and 665 nm is a parameter that measured the cell concentration [47]. This parameter depends on the type of culture (autotrophic, heterotrophic, or mixotrophic) and CO 2 concentration, light penetration, and the presence or absence of inorganic or organic compounds can affect it [48]. If the algae concentration is too low, they will not be established in an anaerobic condition of cultures because the dissolved oxygen quantity is too low for the overall cell respiration [49]. For this mechanism, when concentration hits 1.9 × 10 7 cell mL −1 , it is important to keep an active growth phase with density around 1-2 × 10 4 cell mL −1 . (2) Neubauer haemocytometry using Neubauer improved chamber is currently used as a standard in laboratories for manual cell counting under a light microscope. The literature mentions that the Neubauer chamber showed the best overall performance [50].

Biomass Quantification
Spectrophotometric analysis of the microalgae containing pigment such as Chlorophyll A has been developed to quantitative the biomass culture of Chlorella vulgaris by measuring the optical density (OD) as absorbance [42]. Chlorophyll A's wavelengths in a range of 400-460 nm and 650-680 nm have been reported frequently. [37]. The OD of microalgae culture at 680 nm (OD680) is measured to determine the dry biomass concentration of microalgae, according to Gao et al. [41].

Pigments
Microalgae have pigment contents, for example, chlorophylls and carotenoids. Of the total amount of wet biomass, the pigments can be a percentage of 0.1-9.7% [37]. The viability of cells is an essential parameter for determining the growth rate of algae. This index is known as chlorophylls content (sum of Chlorophyll A and Chlorophyll B) of the algal cells [51]. Several methods are available to determine Chlorophyll A, for example, fluorometry, high-performance liquid chromatography (HPLC) using a fluorescence detector, and spectrophotometry.
The chlorophyll content is measured after centrifugation (mechanical breaking of the collected cells), followed by extracting the chlorophyll from the broken cells into alcohol or acetone. The extract is analyzed spectrophotometrically measuring absorbance at 665 nm and 750 nm [52]. Ethanol extraction is less harmful than the acetone method. The Chlorophyll A content (using ethanol extraction method) was calculated using Equation (1) [53].
where Chla is Chlorophyll A concentration (mg·m -3 ), V ethanol is the constant volume of the extract (mL), V water is the volume of filtered water (L), A 665 is the absorbance of the samples in 665 nm wave, similar to A 750 . E 665 and E 750 indicate the absorbance of the samples acidified with 1 mol/L HCl at 665 nm and 750 nm, respectively [53]. Another method of determining algae pigments described by Wellburn [54] uses dimethylsulphoxide (DMSO). The supernatant extract was diluted with DMSO, and spectrophotometric tests were done at 649, 665, and 480 nm. The pigment content was calculated using the formulae [37]: Total pigment (mg/L) = Chla + Chlb + Total carotenoids (5) where Chla and Chlb are Chlorophyll A and B, respectively, concentration (mg·m −3 ); and OD 649, OD 665 , and OD 480 are the optical densities measured spectrophotometrically at 649, 665, and 480 nm, respectively.

Chemistry and Composition of Chlorella vulgaris
Chlorellavulgaris biomass contained 25-30% protein, 6-10% carbohydrate, and 30-40% lipid. While growing, Chlorella vulgaris can reach 5-40% [55] lipids per dry weight of biomass mainly composed of glycolipids, hydrocarbons, phospholipids, and free fatty acids [13]. To determine the lipids, Blight and Dyer described the extraction process of total lipids from Chlorella vulgaris using a mixture of chloroform and methanol. Quantification is conducted gravimetrically after evaporating the extracting solvent [56]. Bradford [44] and Dubois [45] methods are used for proteins and sugars, respectively.

Toxicity of Photocatalyst Nanoparticles to Chlorella vulgaris
The main source of nanoparticles in the environment is engineering nanoparticles for water and air pollution, contaminated soil remediation with hazardous substances, sensors for environmental application, biomedical imaging, and drug production [57].
Heterogeneous photodegradation based on nanoparticles (NPs) as photocatalytic materials is also a well-known practice, and they are utilized for this purpose. Rogozea et al. used NiO/ZnO NPs modified silica for photodegradation purposes. A high surface area of nanoparticles facilitates the efficient photodegradation reaction because of their small size (<10 nm) [58]. In addition to photocatalytic activity of nanoparticles, these have been described by the same group as having optic, fluorescence proprieties, and applications for compound degradation [59,60].
Heterogeneous photocatalysis is an advanced oxidation process for pollutant degradation by generating hydroxyl radicals HO · , electron-hole pairs (e − /h + ), and superoxide radicals (O ·− 2 ) [61]. The hydroxyl radicals react with organic pollutants and transform them into mineral constituents or in less toxic compounds [1]. The mechanism of photocatalysis are explained in Section 4. A fair number of metal oxides have been used as photocatalytic materials for water treatment since the year 1972, e.g., titanium dioxide (TiO 2 ), zinc oxide (ZnO), iron (III) oxide (Fe 2 O 3 ), vanadium oxide (V 2 O 5 ), tungsten trioxide (WO 3 ), etc. [62,63]. The photocatalytic materials have unique properties such as chemical stability, non-toxicity, photosensitivity, and low cost. For example, ZnO nanoparticles have a ∼3.22eV bandgap, and they have a small size, increasing the specific surface area. Higher numbers of active surface sites can facilitate absorption and degradation of pollutants, making photogenerated charge carriers able to react with pollutants [64].
Between photocatalysts, TiO 2 and ZnO semiconductors present a higher efficiency for pollutant degradation [65], they are UV-light active materials and absorb a small portion of the solar spectrum (4-5%) [66]. In the past twenty years, numerous bodies of research were also developed to efficiently use solar energy and design new photocatalyst activity under visible light irradiation [67].
The continuous exposure of TiO 2 nanoparticles increases the probability of contaminating the environment. For example, because of Ag nanoparticles' use in consumer products, some of them are released into the aquatic environment, thus becoming a source of dissolved Ag that exerts toxic effects on organisms like algae, bacteria, daphnia, and fish [68]. The interactions between the particles and algal cells will lead to aggregation and sedimentation reducing their availability in the system. All these previously mentioned factors contribute to diminishing the toxicity of the nanoparticles during the algae exposure. The oxidative stress indicators with decreased levels across the cycles manifest the decrease in toxic effects. Thiagarajan et al. demonstrate that the nanoparticles' continuous exposure to algal cells (e.g., Chlorella vulgaris cells) would significantly reduce their toxic impact [69].
Chlorella vulgaris has a high growth rate, a simple cell cycle, and can respond quickly to different nanoparticles [53,70]. When algae are incubated with a different type of nanoparticles, they have specific interactions like nanoparticles-nanoparticles homo-aggregation [71], algal cell-cell interaction [72], and nanoparticles-algae hetero-aggregation [73]. For TiO 2 nanoparticles, homo-aggregation interaction is caused by the electrical double layer that is compressed around the nanoparticles [74]. The medium pH influences the interaction between the nanoparticles and algae [75]. pH and ionic strength are parameters that play an essential role in aggregation states of nanoparticles in water. Nanoparticles have toxic effects on the microalgae which is important to determine their impact on water. Moreover, decreasing the nanoparticles concentration will result in lower toxicity across the microalgae cycles from aggregation and sedimentation that plays a vital role in the growth rate of Chlorella vulgaris [76].
Although research papers have addressed the toxic effects of nanoparticles on plant cells, the toxicological studies are still limited.
The ecotoxicity of nanoparticles to algae has attracted the attention of scientists. For different types of nanoparticles, they have different toxicity behaviors. They are identified as causing growth inhibition and further algal deaths at the nanoparticle-dependent concentration and their properties (Table 3) [77]. Depending on the type of nanoparticles and the algae's experimental conditions, the toxic effects may vary. Some studies demonstrate the toxicity of nanoparticles on algae growth and development (EC 50 ). Table 3. Toxic effect of nanoparticles (NPs) to Chlorella vulgaris algae.  The most probable toxic effects of nanoparticles can be determined using a mathematic model (pattern) of the probability theory or calculating the EC 50 value. EC 50 is the concentration of test substance, resulting in a 50% reduction in either growth (EbC 50 ) or growth rate (ErC 50 ) relative to the control within 72-96 h exposure. Ecotoxicity tests are quantified by determining the EC 50 values in several replicates, defined as the concentration that produces a toxic effect in 50% of the tested population [87]. Toxicity tests on the algae must be consistent with the OECD TG 2011 guide [18]. ThI is a test that aims to determine the effects of a substance on the growth of freshwater microalgae.

Synthesis Method/NP Characteristics Growth Conditions Toxic Effect References
Most toxicity tests are done using a single type of nanoparticle. Ko et al. tested the toxicity of a mixture of ZnO, NiO, CuO, TiO 2 nanoparticles, and Fe 2 O 3 on microalgae Chlorella vulgaris growth. The parameter that was taken into consideration was the chloro-phyll content. The tests highlighted the high toxicity of the probes that contained ZnO nanoparticles [79]. Table 3 reviews some types of nanoparticles used in photocatalytic processes; the conclusions resulting from their toxicity studies are the following.
Titanium oxide significantly decreased the growth and biomass (dry weight, Chlorophyll A, and total chlorophyll) of Chlorella vulgaris. TiO 2 nanoparticles can adsorb Zn and P from the algal growth medium owing to surface adsorption of the NPs on the algal cell promoting growth inhibition. The acidity significantly increased the oxidative damage of TiO 2 NPs on the algal cells. Metzler et al. [88] disclose that the "young" (3-5 days) and the "old" (15-26 days) algal cultures were less resistant to the stress imposed by TiO 2 nanoparticles than the "mid-age" (8-14 days) culture.
The interaction between the ZnO NPs and algae altered the morphological characteristics after 72 h treatment. The toxicity of ZnO nanoparticles was attributed to dissolved Zn 2+ . The oxidative stress induced by ZnO was ten-fold higher than that by TiO 2 NPs.
Nickel oxide nanoparticles induced inevitably influenced the growth of aquatic photoautotroph, cellular toxicity, and morphological alteration, cell deterioration, and oxidative stress, which were related to NPs concentration and reactive oxygen species (ROS) production, resulting in biotoxicity on Chlorella vulgaris.
Ag nanoparticles changed Chlorella vulgaris growth kinetics and cell metabolism shown in photosynthetic pigments and chemical composition because it can affect carbon acquisition, photosynthesis, and respiration processes. AgNPs could affect their toxic effects to Chlorella vulgaris by increasing cells' aggregation, especially at higher concentrations.

Mechanism of Photocatalyst Nanoparticles during Oxidative Stress of Algae
The application of photocatalysis in wastewater remediation aimed to evaluate the risk of nanoparticles used during the photodegradation process. Despite the merits of environmental remediation based on photocatalytic materials like TiO 2 and ZnO, there are a few drawbacks. Bioaccumulation and toxicity are significant shortcomings that affect the aquatic environment when wastewater is treated by photocatalysis, especially if contaminants' complete mineralization is not ensured.
Heterogeneous photocatalysis relies on the generation of electron-hole pairs when a photon of energy higher or equal to the bandgap energy of the semiconducting photocatalyst (E g ) is absorbed (Equation (6)). The electron-hole recombination on the surface (or in bulk) of the nanoparticles represents the main mechanism responsible for the photocatalyst's deactivation. The electron-hole pair trapping in surface states leads to reactions with chemisorbed O 2 and HO -/H 2 O to generate reactive species such as superoxide O ·− 2 and hydroxyl radicals HO · (Equations (7)-(10), Figure 1).
ROS interaction with cellular components may lead to toxic effects during oxidative stress and act as secondary reactions in numerous cellular processes. ROS disrupt cell function by lipid peroxidation, oxidizing proteins and damaging nucleic acid. Oxidative stress in algal cells is monitored using common markers including lipid peroxidation monitoring by quantifying malondialdehyde content and antioxidant enzyme (glutathione S transferase and peroxidase) activities [78]. Chlorella vulgaris has been reported by Dauda et al. [78] to produce polyunsaturated fatty acids sensitive to reactive oxygen species.

Conclusion and Perspectives
This review reflects an image of scientific progress in Chlorella vulgaris toxicity studies and its impact in the aquatic ecosystem. This microalga is a simple plant because of its unicellular structure, but it has a complex role in oxygen production and as a food chains producer. The most common parameters for Chlorella vulgaris algae growth are Bold's basal medium (BBM); the temperature of 20-30 °C; and the light intensity of around 150 μE·m 2 ·s −1 under a 16/8 h light/dark cycle. Considering that the nanoparticles are used in a wide variety of fields (engineering, construction, medical, electric etc.), toxicity tests became very important when we discussed their involvement in algae or other microorganisms growth. Ecotoxicity tests were conducted to measure the impact of each powder on aquatic and terrestrial ecosystems. The most tested nanoparticles for algae toxicity are the Numerous studies confirm that dissolved Ti 4+ , Zn 2+ , W 3+ , Sn 4+ , and ROS contribute to the nanoparticles' toxicity on green algae.
ROS interaction with cellular components may lead to toxic effects during oxidative stress and act as secondary reactions in numerous cellular processes. ROS disrupt cell function by lipid peroxidation, oxidizing proteins and damaging nucleic acid. Oxidative stress in algal cells is monitored using common markers including lipid peroxidation monitoring by quantifying malondialdehyde content and antioxidant enzyme (glutathione S transferase and peroxidase) activities [78]. Chlorella vulgaris has been reported by Dauda et al. [78] to produce polyunsaturated fatty acids sensitive to reactive oxygen species.

Conclusion and Perspectives
This review reflects an image of scientific progress in Chlorella vulgaris toxicity studies and its impact in the aquatic ecosystem. This microalga is a simple plant because of its unicellular structure, but it has a complex role in oxygen production and as a food chains producer. The most common parameters for Chlorella vulgaris algae growth are Bold's basal medium (BBM); the temperature of 20-30 • C; and the light intensity of around 150 µE·m 2 ·s −1 under a 16/8 h light/dark cycle. Considering that the nanoparticles are used in a wide variety of fields (engineering, construction, medical, electric etc.), toxicity tests became very important when we discussed their involvement in algae or other microorganisms growth. Ecotoxicity tests were conducted to measure the impact of each powder on aquatic and terrestrial ecosystems. The most tested nanoparticles for algae toxicity are the oxides, for example, ZnO, TiO 2 , Ag, NiO, etc. Analysis of the algae showed that the application of more than 20 ppm of nanoparticles could negatively impact aquatic biology's growth and physiology.
The nanoparticle exposure concentrations to Chlorella vulgaris is challenging to define, especially in low concentration. The toxicity is estimated based on the effects on the growth rate of algae. Chlorella vulgaris is a valuable bioindicator of nanoparticle suspensions, permitting a better understanding of nanoparticles' toxicity risks to the aquatic environment.
Author Contributions: L.A. designed and carried out this research, supervised, provided advice, helped in this paper's revision and editing, and funding acquisition. C.A. prepared and analyzed the data, and conducted theoriginal draft preparation. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.