3.1. Characterization of synthesized ZnO-NPs
The surface morphology of ZnO-NPs was determined and shown in Fig. 2. The clear crystals of triangle-like shape ZnO-NPs produced by the extracts of the mint and basil were visible in the SEM images. In SEM images, the particles are depicted in a uniform state. As is typical of nanoparticles made through green synthesis, the produced ZnO-NPs occasionally showed considerable aggregation formation. This is due to the fact that biosynthetic NPs have a higher surface area and that their long-lasting affinities cause them to aggregate or clump together (Vidya et al. 2013). Particle agglomeration is caused by the polarity of the nanoparticles and their electrostatic attraction (Aminuzzaman et al. 2018). As the pH value rises, the amount of H+ and OH− ions varies, which has an impact on the structure, morphology, and formation of ZnO, and smaller nanoparticles form at high pHs. Because there are more OH ions present, they are more strongly attracted to the positively charged Zn2+, which encourages the development of strong Zn-O bonds in the structure (Wahab et al. 2009). According to Wang et al. (2008), altering the basicity of the solution allows for the customization of various ZnO morphologies, including rods and flowers. In SEM images, the average particle size of pure ZNO-NPs was 94.11 nm, while the average particle size of M-ZnO-NPs was 73.76 nm and the average particle size of B-ZnO-NPs was 86.82 nm. This result revealed that pure ZnO-NPs had larger particle size, the grain size of ZnO-NPs produced by green synthesis decreased, and the smallest grain size was in M-ZnO-NPs.
3.1.1. XRD analysis
The hexagonal wurtzite structure of ZnO nanoparticles is characterized by these peaks (JCPDS card number: 36-1451) (Kaliraj et al. 2019). The XRD diffractograms of the prepared green ZnO photocatalysts of B-ZnO-NPs and M-ZnO-NPs and raw ZnO-NPs were shown in in Fig. 3. Figure 3a and b showed distinct peaks in the XRD patterns at 2θ = 31.76o, 34.40o, 36.20o, 47.52o, 56.58o, 62.83o, 66.36o, 67.93o, 69.07o, 72.52o, and 76.94o that match well with planes Miller indices (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) for B-ZnO-NPs and M-ZnO-NPs (Kaliraj et al. 2019). The XRD peaks' distinct line widening validates the material's nanoscale particle size. The strong diffraction peaks and lack of impure reflections respectively validated the high crystallinity and purity of the produced ZnO-NPs. No peaks connected to impurities were discovered, and ZnO made by green synthesis was completely pure and incredibly crystalline in its unaltered native condition. These results support earlier findings (Abdullah et al. 2020; Rana et al. 2016). We conducted the experiment at ambient temperature rather than using high temperatures, in contrast to earlier investigations (Anbuvannan et al. 2015; Karnan et al. 2016). This implies that a quick, easy, and environmentally friendly method of synthesis might be able to create highly crystalline nanomaterial. Therefore, in terms of energy efficiency, this study has proven to be a superior and more viable choice for synthesizing ZnO-NPs.
3.1.2. FTIR analysis
The biomolecules in charge of the synthesis of ZnO-NPs were described and identified using FTIR analyzes in the range of 4000 and 400 cm− 1 (Fig. 4). Because Zn2+ ions from zinc acetate attached with polyphenols present in the plant extract and reduced to Zn+ during the synthesis of ZnO-NPs, it is known as a complex production of Zn+ - polyphenols in the reaction solution. The FT-IR spectra of raw ZnO-NPs and green synthesized ZnO-NPs from plant extracts revealed similar peaks, which suggested this complex phenomenon. The FTIR spectrum of the M-ZnO-NPs (Fig. 4a) showed absorption bands at 3837, 3383, 2979, 2903, 1563, 1405, 1252, 1055, 889, 688, 543 and 484 cm− 1. The FTIR spectrum of the B-ZnO-NPs (Fig. 4b) showed absorption bands at 3385, 1563, 1414, 877, 688, 582, 552 and 481 cm− 1.The broad and very intense bands at 3383 and 3385 cm− 1 could be attributed to the presence of the hydroxyl functional groups of alcohols and phenolic compounds that may exist in plant extracts. The peaks that appeared at around 2979 − 2903 cm− 1 are assigned to C–H stretching in alkanes respectively (Abdullah et al. 2020). The peaks located at 1563 cm− 1 are related to the C = C stretching vibrations of aromatic rings in polyphenolic compounds (Matinise et al. 2017). The peaks at 1405 and 1414 cm− 1 arise from the COO- stretching mode of symmetrical acids. The peak of 1252 cm− 1 may be related to the optical character identification of C-O stretching (Bharathi and Bhuvaneshwari 2019). The marked peak at FTIR spectrum of mint 1055 cm− 1 can also be related to the stretching of the C–O, C-C and C-O-C bonds from saturated esters, alcohols, phenols, cycloalkanes and acid anhydrides in the plant extract (Aramugam et al. 2021; Moghaddam et al. 2017). ZnO's primary absorption band is located in the 400–600 cm− 1 range (Ishwarya et al. 2018; Kumar et al. 2019). The Zn-O vibration is associated to the wide, strong band at 500 cm− 1 (Tantiwatcharothai and Prachayawarakorn 2019). The bending vibrations of alkanes and alkenes (C = C bending) are observed at 889, 877 cm− 1 and 688 cm− 1 (Abdullah et al. 2021; Bharathi and Bhuvaneshwari 2019; Larkin 2017) (Table 1)
Table 1
The FTIR spectrum of the biosynthesized ZnO-NFs
Wavenumber (cm− 1)
|
Type of vibration
|
Functional groups
|
References
|
M-ZnO-NPs
|
B-ZnO-NPs
|
484, 543
|
481, 552, 582
|
stretching
|
Zn-O
|
Tantiwatcharothai and Prachayawarakorn, 2019
|
688
|
688
|
deformation (γ) not in plain CAr-H
deformation (γ) O–H not in plain
deformation (γ) = C-A not in plain
|
benzene-H
liquid alcohols or
phenols
cykloallenes
polienes
|
Larkin, 2017
|
889
|
877
|
C = C
|
bending alkene
vinylidene
|
Abdullah et al., 2021
Bharathi and Bhuvaneshwari, 2019
|
1055
|
-
|
stretching (ν) C–O
skeletal (ν) C–C
stretching (ν) C–O–C
|
saturated esters
alcohols and phenols
cycloalkanes
acid anhydrides
|
Aramugam et al., 2021; Moghaddam et al., 2017
|
1252
|
-
|
stretching (ν) C–O
stretching (γ) C–C
deformation (γ) not in plain = C–H
|
saturated esters–acetates
dimers
alcohols and phenols
alkanes
|
Bharathi and Bhuvaneshwari, 2019
|
1405
|
1414
|
stretching (ν) –COO-
|
salts of symmetrical acids
|
Matinise et al., 2017
|
1563
|
1563
|
stretching (ν) –COO-
stretching (ν) C = O
|
aromatic acids salts
β-diketones (phenolic forms)
|
2903–2979
|
-
|
stretching (ν) C–H
|
-CH2-
|
Bharathi and Bhuvaneshwari, 2019; Abdullah et al., 2020
|
3383
|
3385
|
stretching (ν) O–H
|
free –OH group
|
Abdullah et al., 2020
|
3.2. Seed germination and seedling parameters
The seed germination tests were conducted to determine the toxic effects of green synthesized ZnO-NPs on wheat seeds. According to the seed germination test results, it was determined that the M-ZnO-NPs and B-ZnO-NPs had not any significantly toxic effects on wheat seed germination. Green synthesized ZnO-NPs using mint plant extract was significantly increased seed germination of wheat (p < 0.05). Maximum and minimum seed germination percentages were determined as 100% in 400 mg/L M-ZnO-NP concentration and 83.3% in control groups, respectively. In addition, the synthesized B-ZnO-NPs significantly increased seed germination at 400 mg/L concentration (p < 0.05). Maximum and minimum seed germination were determined as 100% at 400 mg/L B-ZnO-NPs concentration and 80% at 50 mg/L concentration. The raw ZnO-NPs enhanced the seed germination of wheat, compared to control. However, this enhancement did not reach to the M-ZnO and B-ZnO-NPs treatments (Fig. 5a). Maximum seed germination percentage was determined at the concentration of 25, 50, and 200 mg/L ZnO-NPs (as 96.7%). It was determined that the best yield of seed germination percentage was obtained at all concentrations of M-ZnO-NPs, compared to control groups, raw ZnO-NPs, and B-ZnO-NPs. In addition, it was determined that the seed germination percentage of wheat increased with increasing M-ZnO-NPs concentrations. Results also showed that the presence of ZnO-NPs in three types increased the seed germination percentage of wheat at the almost all concentrations (Fig. 5a). These results can be explained by the nanoparticles can penetrate through the seed pores depending on their sizes. Also, since Zn is an essential micronutrient for living organisms, seeds are willing to take up these materials. The seed germination test is an important parameter to determine the positive, negative or no effects on plants of external materials that release to the environment. In addition, germination percentage is a parameter used to evaluate seed viability (Meher et al. 2020). Literature studies showed that the different Zn sources (bulk ZnO, ZnO-NPs, ZnSO4, etc.) effects the seed germination process of different plants (Du et al. 2019; Adrees et al. 2021). However, there are little studies that conducted about green synthesized ZnO-NPs on wheat seed germination process (Meher et al. 2020; Sharma et al. 2022). Itroutwar et al. (2020) showed that seed germination percentage of maize was promoted by the green synthesized ZnO-NPs using seaweed, compared to bulk ZnO and control groups. Singh et al. (2019) reported the similar results, the authors showed that green synthesized ZnO-NPs using aloe vera extract caused better common bread wheat seed germination than control groups. In the current study, seed germination results showed the same trend as the others. Meher et al. (2020) reported that green synthesized ZnO-NPs using periwinkle leaf extract had no significant effect on seed germination of wheat (Triticum aestivum-WH1105 variety) at different concentrations (20–100 mg/L). These differences are estimated to depend on the biological source of the synthesis materials, the size and shape of the ZnO-NPs, as well as the wheat species.
In this study, seedling growth parameters such as root-stem length and seedling viability index were evaluated after seed germination. Measurements showed that applications of both M-ZnO-NPs and B-ZnO-NPs and ZnO-NPs increased root elongation at concentrations of 50 mg/L and 100 mg/L. Maximum root elongation was determined as 34.7% at 50 mg/L M-ZnO-NPs concentration, 30.43% at 100 mg/L B-ZnO-NPs concentration and 27.37% at ZnO-NPs concentration. Root elongation is very important for healthy plants, as roots are the first organs to respond to environmental factors and affect the entire life cycle of plants (Takatsuka and Umeda 2019; Taylor et al. 2021). These results demonstrated that there is a threshold to increase root-shoot elongation and increase metabolic/enzymatic activity. In this study, the root threshold was determined as 50 mg/L for the M-ZnO-NPs, while 25 mg/L was determined for the other ZnO-NPs types (Fig. 5b). In addition, the shoot threshold values were determined as 100 mg/L for M-ZnO-NPs and 50 mg/L for B-ZnO-NPs (Fig. 5c). It was determined that the application of B-ZnO-NPs (p > 0.05) and ZnO-NPs (p < 0.05) inhibited the shoot elongation of wheat at almost all concentrations compared to the control. Maximum shoot inhibition was observed as 14.45% at 400 mg/L B-ZnO-NPs concentration and 16.3% at 200 mg/L ZnO-NPs concentration. Asmat-Campos et al. (2022) reported this threshold of tomato seeds at the concentration of 100 mg/L green synthesized ZnO-NPs using coriander plant extract. Other authors reported that the maximum root and shoot elongation was determined at the concentration of 100 mg/L green synthesized ZnO-NPs as 12.1 cm and 20 cm, respectively (Itroutwar et al. 2020). Depending on root and shoot length and germination percentage, the maximum seedling viability index (SVI) was measured at concentrations of 50 mg/L for M-ZnO-NPs and 100 mg/L for B-ZnO-NPs and ZnO-NPs (Fig. 5d).
3.3. Plant growth parameters
In order to determine the effects of foliar treatment of M-ZnO-NPs, B-ZnO-NPs and ZnO-NPs on wheat seedlings, some plant growth parameters such as chlorophyll content, plant height, plant fresh and dry weight were investigated. The chlorophyll content was measured using a chlorophyll meter and the results were given as the SPAD value (Fig. 6). The plants were grown for four weeks after the germination period. The foliar treatments were employed to seedlings sown in pots after first week, and the chlorophyll content was measured before every foliar treatment. Results showed that the chlorophyll contents of wheat treated with M-ZnO-NPs at different concentration decreased with time (from first week to fourth week) (Fig. 6a), while the chlorophyll content of control groups increased. The maximum chlorophyll content was determined at the concentration of 25 mg/L M-ZnO-NPs as 37.46 SPAD value at 1st week (before foliar treatment) (Fig. 6a) (p < 0.05). After the foliar treatment (2nd week), results showed that the foliar treatment caused to decrease on the chlorophyll value. It was assumed that the ZnO-NPs prevented the sunlight to enter the plant cells by covering the leaf surface (just like sunscreen). Thus, the chlorophyll synthesis decreased in plant cells. It was also determined that the chlorophyll content did not significantly change at the foliar treatment of 2nd and 3rd weeks, compared to control group (p > 0.05). However, it decreased at the 4th week, compared to control significantly with the treatment of M-ZnO-NPs. The minimum chlorophyll content was determined at the concentration of 100 mg/L M-ZnO-NPs (p < 0.05). In addition, while the chlorophyll content values of wheat were similar with M-ZnO-NPs in the 1st week compared to the control, it increased with increasing B-ZnO-NPs in the 2nd week as in the 4th week (Fig. 6b) (p < 0.05). It is clearly seen that, the increase in chlorophyll content were more evident with increasing M-ZnO-NPs concentration at the 4th week. The minimum chlorophyll contents were determined at the ZnO-NPs foliar treatments between three types of nanomaterials (Fig. 6). It was thought that these differences depend on their sizes and the ability to stay on the leaf surface of the materials. In this study, it was determined that the size of green synthesized ZnO-NPs smaller than the raw ZnO-NPs (Fig. 2), as like Sahoo et al. (2021). The authors reported that the foliar treatments of green synthesized ZnO-NPs to mung bean plants showed more chlorophyll content than raw ZnO-NPs, because of their sizes. The increasing time and increasing ZnO-NPs concentration caused decreases of chlorophyll content of wheat, especially after 4 weeks (Fig. 6c).
Zinc is an essential micronutrient for plant that have key role on photosynthetic activities (Itroutwar et al. 2020). However, toxic effects can be observed on different plants, especially at nanoscale and high concentrations. Wang et al. (2016) reported that ZnO-NPs showed inhibitory effects on chlorophyll synthesize of Arabidopsis plants at the concentration of 300 mg/L. In addition, Arumugam et al. (2021) showed that green synthesized ZnO-NPs using Syzygium cumini inhibited the chlorophyll content of Sesamum indicum while increasing plant growth. The decreases in chlorophyll content of plants may related with DNA damage (Arumugam et al. 2021) and the electron transport chain in plant cells (Azarin et al. 2023).
In order to determine the effects of raw ZnO-NPs and green synthesized ZnO-NPs on wheat growth, the plant heights and fresh and dry weights of plants were also evaluated. Figure 7a and Fig. 8 clearly showed the differences of wheat plant height after three foliar treatments of M-ZnO-NPs, B-ZnO-NPs, and ZnO-NPs. It was determined that the foliar treatment of M-ZnO-NPs did not significantly affect the plant height compared to control (p > 0.05), but there were significantly differences between groups depending on M-ZnO-NPs concentrations (p < 0.05). Maximum plant height was measured at a concentration of 200 mg/L M-ZnO-NP (Fig. 7a). Application of B-ZnO-NPs showed an increase in plant height with increasing B-ZnO-NP concentration (Fig. 7a), but this increase did not reach the control plants. Foliar application of ZnO-NPs also did not significantly affect wheat plant height (p > 0.05). There are conflicting results in the literature about the effects of foliar applications of ZnO-NPs on different plants (Adil et al. 2022; Keerthana et al. 2021; Azarin et al. 2023). It is assumed that the amount of Zn required for auxin production, which plays a role in plant elongation, may not be adequately supplied by the tested chemicals (Meher et al. 2020).
Shoo et al. (2021) reported that the economic yield of cereal crops is related to the dry matter of the plant. The authors noted that the dry matter of mung bean plants treated by foliar exposure of green synthesized ZnO-NPs was higher than that of raw ZnO-NPs. In the current study, significant decreases were determined in dry weights of 23.68% (50 mg/L) and 19.74% (100 mg/L) in M-ZnO-NPs application (p < 0.05), while dry weight values increased by 6.6% (25 mg/L), 17.1% (200 mg/L) and 7.9% (400 mg/L) (p < 0.05) compared to control plant (Fig. 7c). In addition, fresh weights decreased 8.1% (25 mg/L), 33.2% (50 mg/L) (p < 0.05), 24.4% (100 mg/L) (p < 0.05), and 11.1% (400 mg/L) compared to control except 200 mg/L concentration of M-ZnO-NPs (fresh weights increased as 14.5%) (Fig. 7b). Foliar treatment of B-ZnO-NPs at almost all concentrations decreased the fresh (p < 0.05) and dry weights of the wheat plant compared to the control, as did the raw ZnO-NPs (Fig. 7b and c). Although there are studies in the literature showing the effects of ZnO-NPs on wheat seedling growth parameters, no studies were found in which green synthesized ZnO-NPs were exposed to leaves on wheat and pot trials were conducted. Studies conducted in pots have shown differential effects of green synthesized or raw ZnO-NPs on wheat. Meher et al. (2020) reported that green synthesized ZnO-NPs increased the fresh and dry weight of wheat seedlings. The most important factor affecting the effect of these parameters is the concentration of ZnO-NPs. Doğaroğlu and Köleli (2017) examined the ZnO-NP concentrations affecting the shoot dry weight of wheat, and they reported that the maximum and minimum dry weights were obtained at 40 mg/L and 10 mg/L ZnO-NP concentrations, respectively. Du et al. (2019) reported that 20 mg/L ZnO-NP and 200 mg/L ZnSO4 concentration increased the growth parameters of wheat. However, the highest concentration of both Zn sources (1000 mg/L) caused an inhibition on the dry weight of wheat. Plant dry weight can be used as an indicator of accumulation of ZnO-NPs by plant tissue. Knowledge of the mechanism of penetration of ZnO-NPs into plant tissue is still unclear (Du et al. 2019; Zhu et al. 2020). For this reason, Zhu et al. (2020) provided information on the mechanism of entry of ZnO-NPs into the wheat leaf. According to the authors, ZnO-NPs can penetrate in the wheat cells via stomal aperture. After penetrating into cells, they accumulate in the apoplast and are released as Zn ions and can be absorbed by mesophyll cells (Zhu et al. 2020). In the present study, the ZnO-NP concentration in the leaves of the wheat plant was also determined. The results showed that all sources of ZnO-NPs entered wheat leaves at almost all concentrations (Fig. 9). It was determined that the M-ZnO-NPs significantly penetrated in wheat leaves low concentrations (25mg/L and 50 mg/L) compared to control, however at the higher concentrations, the Zn uptake was decreased. However, these uptakes were lower than the other types of ZnO-NPs. The order of Zn concentration in plants was found as ZnO-NPs > B-ZnO-NPs > M-ZnO-NPs. In B-ZnO-NPs treatment, Zn uptake decreased with increasing concentration, but was still higher than in control. The maximum Zn uptake by wheat was determined at the concentration of 50 mg/L ZnO-NPs, and it increased with increasing ZnO-NPs concentrations. The results showed that phytotoxic effects were related to Zn uptake by plants, depending on other parameters. It is known that nanoparticle sizes are effective in penetration into the stomatal opening and accumulation in plant cells. However, in this study, it was determined that three types of ZnO-NPs were not bioavailable for plants.
3.4. Antibacterial Activity
Antimicrobial activity tests were performed to determine the effects of ZnO-NPs and green synthesized M-ZnO-NPs and B-ZnO-NPs on two different bacteria. For this, Staphylococcus aureus as gram positive bacteria and Escherichia coli as gram negative bacteria were chosen because they are the most used bacterial species in the literature (Siddiqi et al. 2018; Dabhane et al. 2021; Lal et al. 2022). There are various studies in the literature about the antibacterial potential of different NPs synthesized via green approach or chemically, such as Fe2O3, Co3O4 (Batool et al. 2022), Cu-Ni hybrid nanomaterials (Abdullah et al. 2022), Co-ZnO-NPs (Ameur et al. 2019), Ag NPs (Bankar et al. 2010), etc. Synthesized ZnO-NPs via green approach or chemically route have the largest share among these studies due to the properties mentioned in the introduction (Joshi et al. 2018; Ansari et al. 2020; Dmochowska et al. 2020; Daphane et al. 2021; Lal et al. 2022). In these reports, it was stated that metallic NPs have a good antimicrobial potential compared to the positive control (antibiotics) (Batool et al. 2022). It is known that the magnitude of the effects (zone inhibition) here usually depends on the NPs concentrations. In the current study, the bacterial strains exposed to 0.01, 0.02, 0.04 g/mL synthesized ZnO-NPs. Results showed that the 0.01 g/mL and 0.02 g/mL concentration of M-ZnO-NPs was not sufficient amount to determine the effectiveness on E. coli. It was determined that the M-ZnO-NPs negatively affected on gram-negative bacterial (E. coli) strains at the concentration of 0.04 g/mL, while the zone inhibition of gram positive bacterial (S. aureus) strains slightly increased with increasing M-ZnO-NPs (Fig. 10). The inhibition zone diameters were determined at the concentration of 0.01, 0.02, and 0.04 g/mL M-ZnO-NPs on S. aureus as 14.73 mm (45.9%), 15.37 mm (47.9%), and 17.83 mm (55.6%), respectively. In addition, the synthesized B-ZnO-NPs negatively affected the activation of E. coli only at the highest concentration (0.04 g/mL, 71.42%), while M-ZnO-NPs were more effective on S. aureus at all concentrations. The inhibition zone diameters were measured as 14.51 mm, 15.24 mm, and 16.14 mm for S. aureus and the percentage was calculated as 44.08%, 46.26%, and 48.99%, depending on the increasing B-ZnO-NPs concentration, respectively. The maximum inhibition zone diameters were determined in the treatment of raw ZnO-NPs compared to M-ZnO and B-ZnO-NPs. It was measured as 81.67% for E. coli and 72.81% for S. aureus at the concentration of 0.04 g/mL ZnO-NPs. It was determined that the synthesized three types of ZnO-NPs can be toxic on both E. coli and S. aureus bacteria when there were enough materials in the growth media. These results showed that there is a threshold concentration in the resistance of each bacterium to different materials. The results also indicated that gram-positive bacteria (S. aureus) were more susceptible to all three types of ZnO-NPs compared to gram-negatives (E. coli), as reported by Ansari et al. (2020). In this study, although the effect mechanism of ZnO-NPs on bacterial activation was not studied, the other researchers explained the effect mechanisms. The effect mechanisms of external materials on bacterial activities can be explained as cell membrane and cell wall destruction due to NPs deposited on the bacterial surface, electrostatic interaction between Zn + and cell wall, cellular destruction caused by the production of reactive oxygen species in the bacterial cell and/or cell wall structures of the bacteria (Ansari et al. 2020; Aldeen et al. 2022). Table 2 shows the comparative study of the antibacterial activity of ZnO-NPs produced by green synthesis in previous studies.
Table 2
Comparative study of antibacterial activity of green synthesized of ZnO-NPs with previous works
Plant
|
Morphology
|
Crystal Size
|
Antibacterial assay (zone diameter) (mm)
|
Effective concentration of ZnO-NPs (mg/mL)
|
Ref.
|
Gram-positive
|
Gram-negative
|
Thymus syriacus
|
Spherical-like
|
17.19
|
35.00
|
19.33
|
500 mg/mL
|
Şahin et al., 2022
|
Phoenix roebelenii leaves
|
Spherical
|
15 nm
|
15.8
|
15.00
|
4 mg/mL
|
Aldeen et al., 2022
|
Garcinia Cambogia fruit pulp
|
Semi-spherical
|
32.8 nm
|
-
|
19.00
|
0.1 mg/mL
|
Sasi et al., 2022
|
Cinnamomum verum
|
Hexagonal
Wurtzite
|
̴ 45 nm
|
16.75
|
13.25
|
2 mg disc− 1
|
Ansari et al., 2020
|
Mentha spicata extract
|
Hexagonal
Wurtzite
|
|
17.83
|
13.51
|
40 mg/mL (0.04 g/mL)
|
Current study
|
Ocimum basilicum extract
|
Hexagonal
Wurtzite
|
|
16.14
|
14.21
|
40 mg/mL (0.04 g/mL)
|
Current study
|