SEM-EDX study
Scanning electron micrograph study along with EDX was used to evaluate the morphology of PET carbon microsphere and it is presented in Figure 1. From the Figure 1, it is clearly demonstrated that PET plastic carbon microsphere are uniformly sphere with diameter 2-8 µm. On the other hand, EDX signature revealed the existence of carbon. Almost similar result was reported by Wei et al. (2011) for the thermal dissociation of PET in a supercritical-CO2 system at 500°C for 3h. They also reported that perfect spherical carbon with 1-5 µm sized can be achieved at 600°C and these carbon spheres are smoother surface. Present study structurally and morphologically exactly same as reported by Wei et al. (2011). Very minute observation also revealed that the existence of small particles on the surface of the majority of the spheres. These small particles on the spheres are may be the carbon pieces (Wei et al. 2011).
FTIR study
Fourier transform infrared spectroscopy is an important analytical study by which important functional groups associated with nanoparticles can be identified (Bhaumik et al. 2017). Figure 2a clearly demonstrated the FTIR of PET plastic carbon microsphere. The sharp peaks at 3166 cm−1 and 1440 cm−1 are due to aromatic C-H and aromatic ring, respectively (Wei et al. 2011). Another strong peak was observed at 1705 cm−1 which also indicate the presence of –C=O vibration of carbonyl group (Fig. 2a). Present findings showed good agreement with the findings of Wei et al. (2011) and Ng et al. (2018).
XRD study
The XRD diagram of the PET plastic origin carbon microsphere sample is shown in Fig. 2b. The broad diffraction peaks of this XRD at 22.97 and 42.78 2θ which correspond to the (002) and (100) reflections, respectively for graphene. This diffraction peaks is very similar to amorphous graphite like carbons (Zhang et al. 2016; Sergiienko et al. 2009). Moreover, the broad (002) peak, in particular, encompasses diffuse sets of inter-layer distances which, on average, are larger than those in crystalline graphite (typically 0.344–0.355 nm).
Germination study
Germination of Cicer aritenium was conducted (Eq. 1) under different doses of carbon microsphere (Fig. S 1). The entire germination was studied in three different time intervals (24 h, 32 h and 40 h) and results highlighted in Fig. S1. From the Fig. S1, it is clear that only T3 treatment showed higher germination (53.33 %) with respect to control (T1 50%) during 24h of incubation and no statistical difference (p < 0.069) was observed. Almost similar nonsignificant (p < 0.069) of germination pattern of Cicer aritenium was recorded during 32 h of incubation. However, during 40 h actual germination picture was recorded and results revealed that with increasing CMS dose, germination increased and maximum germination was recorded at T4 treatment (96.67 %), but no statistical significant difference (p < 0.502) was observed. These results are not strictly agreed with previous study (Magrabosco et al. 2020) where, carbon nanotube (bionic carbon microsphere) does not greatly influence the germination. However, Nair et al. (2012) successfully addressed that carbon nanostructure significantly increased the water content in seeds of rice during germination compared to control.
Root and shoot length
The variation of root and shoot length was recorded under difficult treatment conditions at 10 and 15 days interval (Fig. S 2 and S 3). After 10 days, root length of all treatments showed significantly (p < 0.001) different from control (Fig. 3). Similar, significant different (p < 0.039) in root length was also recorded after 15 days (Fig. 3). On the other hand, the variation of shoot length in both time intervals (10 and 15 days) showed non-significant variation (Fig. 3). Present results showed good agreement with the earlier study as reported by Haghighi et al. (2014) who demonstrated the significant increment of root length of tomato under higher concentration (40 mg/L) of carbon nanotubes. Similarly, Jiang et al. (2014) showed the enhancement of both root and shoot of rice with the treatment of carbon nanotubes at 100 µg/mL. Therefore, those results clearly demonstrated that PET plastic origin carbon microsphere has remarkable impact on root length over shoot length of cicer arietinum L.
Root morphology
The morphology of root was assessed under treatment condition in comparison to control and photograph was presented in Figure S 4. From the figure S4 it is revealed that both root length and seminal root number decrease with increasing PET plastic origin carbon microsphere. Moreover, the overall health of the root is also deteriorated with increasing the dose of carbon microsphere dose. Almost similar observation of root morphology was demonstrated by Hajra and Mondal (2017) under treatment with ZnO on Cicer arietinum L.
Fresh and dry biomass
Fresh and dry biomass of shoot and root are presented in Table 1. The results revealed that highest fresh weight at treatment T3 compared to control. However, highest root weight was recorded at treatment T4 with respect to control. Similar non-significant results were recorded for dry mass of both root (p < 0.625) and shoot (p < 0.417). Therefore, present finding suggest that fresh weight is accelerated than dry weight. Almost similar improvement in fresh weight under treatment of carbon nanotubes on radish (Haghighi et al. (2014) and switchgress (Pandey et al. 2018) was reported.
Table 1
Variation of fresh and dry weight of roots and shoots of Cicer arietinum under different treatments.
Treatment | Fresh weight (g) | Dry weight (g) |
Root | Shoot | Root | Shoot |
T1 | 0.943 | 1.057 | 0.097 | 0.087 |
T2 | 0.770 | 0.933 | 0.073 | 0.063 |
T3 | 0.780 | 1,267 | 0.100 | 0.060 |
T4 | 0.957 | 1.230 | 0.101 | 0.074 |
ANOVA (F) | 1.210 | 1.695 | 0.614 | 1.063 |
P value | < 0.367 | < 0.245 | < 0.625 | < 0.417 |
Photosynthetic pigment
In photosynthesis I and II, chlorophyll played vital role for communication of light energy to chemical energy through absorption of visible light (Komenda and Sobotka, 2019). Pigment levels (chl ‘a’, chl ‘b’, total chl and carotenoid) under different doses of plastic carbon microsphere were measured and results depicted in Fig. 4. From the Fig. 4, it is clear that all the pigments gradually increased with increasing concentration of nanocomposite dose. One way ANOVA analysis revealed that the chlorophyll ‘a’ levels under all treatments are statistically significant (p < 0.001) than control and higher level of cholorophyll ‘a’ was recorded in treatment T4. Similarly, chlorophyll ‘b’ level also increased with increasing the concentration of nanocomposite dose. However, treatment wise variation of chlorophyll ‘b’ not statistically significant. But one way ANOVA study indicate that the total chlorophyll level is statistically significant (p < 0.001) among different treatments. Almost similar statistical significant (p < 0.001) difference among different treatments was recorded for carotenoid also. This pigment level also showed dose dependent, i.e. level of carotenoid increase with increasing the concentration of carbon microsphere dose. Almost similar results of enhancement of carotenoid was reported by Siddique et al. (2019) with application of graphene oxide in carrot seedling. Very recently, Gonzalez-Garcia et al. (2019) demonstrated the application of graphene and carbon nanotube on tomato seedlings leads to increase chlorophyll content (chl ‘a’, chl ‘b’, total chl). However, they also suggested that the level of chlorophyll is higher in graphene application than carbon nanotube.
Carbohydrate, protein and ascorbic acid level
Carbohydrate is an indispensable component in our daily diet (Mehmood and Murtaza, 2017). The level of carbohydrate was recorded in higher concentration (T4) (Fig. 5). One way ANOVA results suggested a statistical significant difference (p < 0.0001) among the different treatments. Almost similar dose dependent results was reported by Mehmood and Murtaza (2017).
Similarly protein level was assessed under different concentration of carbon microsphere (Fig. 5). Results also indicates that with increasing concentration, protein level decrease. However, variation of protein under different treatments are statistically significant (p < 0.037). Mehmood and Murtaza (2017) concluded in their research that biosynthesis of silver nanoparticles can enhance the protein level. Very recently, Gonzalez-Garcia et al. (2019) also endorse the same positive effect of carbon nanoparticles application on protein level in tomato seedlings. However, almost opposite results was reported by Mehrian et al. (2015). They highlighted that, with increasing the concentration of silver nanoparticles on tomato plants, total soluble protein level significantly decreased.
Ascorbic acid analysis
Level of ascorbic acid under different concentrations of carbon microsphere is presented in Fig. 5. Higher level of ascorbic acid was recorded in lower concentration (T2) and it is significantly (p < 0.0001) higher control. However, in higher concentration, ascorbic acid level reduced or reach at the level of control. This result indicate that lower dose of nanocomposite can increase ascorbate-glutathion cycle (Sharma et al. 2016). That means it improves osmoregulation, enhancement of nutrient use efficiency and photosynthetic performance (Hasanuzzaman et al. 2019). Present result is in agreement with the earlier study reported by Gonzalez-Garcia et al. (2019). They reported that application of carbon nanomaterials can trigger the level of ascorbic acid in tomato seedlings.
Amino acid, proline and MDA level
Present study also assessed the amino acid level under different treatment conditions. From the present finding, it has been found that amino acid level gradually increase with increasing treatment concentration. (Table 2). One way ANOVA analysis revealed that there is significant (p < 0.0001) variation among different treatments (Table 2). Present study results are very much consistent with the earlier findings as reported by Mehrian et al. (2015).
Table 2
Variation of RIL, MDA, proline, amino acid and catalase under different treatments.
Treatments | RIL (%) | MDA (%) | Proline (µg/g) | Amino acid (µg/g) | Catalase (µM/min) |
T1 | 25.267d | 0.017d | 0.507b | 0.243c | 1.500d |
T2 | 28.567c | 0.023c | 0.380c | 0.267d | 2.010c |
T3 | 33.767b | 0.050b | 0.477b | 0.483b | 2.327b |
T4 | 53.133a | 0.094a | 1,307a | 0.727a | 2.477a |
ANOVA (F) | 2042.32 | 996.94 | 1302.29 | 225.173 | 27.846 |
P value | <0.0001 | < 0.0001 | < 0.0001 | < 0.0001 | < 0.0001 |
RIL: Root ion leakage; MDA: Malondialdehyde |
Proline is multifunctional amino acid which is also a good plant stress (biotic or abiotic) indicator (Senthil Kumar and Mysore, 2012). Proline level under treatment of various doses of PET plastic carbon microsphere leads to gradual increase of proline levels (Table 2). These data clearly suggested that under treatment of carbon microsphere, some sort of stress is generated inside the plant. One-way ANOVA (< 0.0001) clearly endorse the same. During stress condition, the enhancement of proline level is due to proper maintenance of osmotic balance, reduction of ROs, stabilization of membrane (Ahanger and Agarwal, 2017) and protein also proper maintenance of redox potential (Wahid et al. 2007). Shabnam and Kim (2018) also recorded the similar enhancement of proline level in mung bean under the exposure of nano aluminium.
Generation of ROS leads to the membrane damage which is again assessed through estimation of MDA level (Huang et al. 2018; Benson and Brenner, 2004). In this study, MDA level was assessed under different treatment conditions and results are depicted in Figure 5. One way ANOVA results revealed that, there is strong statistical significant (p < 0.0001) in MDA exists among different treatments (Table 2). Results also suggests that, MDA level increase with increasing concentration i.e. absolutely dose dependent variation was recorded. This enhancement of MDA level in the form of lipid peroxidation leads to the generation of oxidative stress under higher dose of carbon microsphere (Debnath et al. 2020). Shabnam and Kim (2018) highlighted that application of nano alumina on mungbean seedlings leads to enhancement of MDA level. Similarly, Souza et al. (2019) highlighted that the up regulation of MDA in Lemna minor under treatment of iron oxide nanoparticles.
Catalase
Antioxidant defense system of plants can function as enzymatic or non-enzymatic activity towards control on reactive oxygen species. Present outcome highlighted in Table 2 where it is clearly revealed that with increasing CMS dose, catalase level increased statistically (p < 0001). Almost opposite results was reported by Shabnam and Kim (2018). They recorded that, nano aluminium has no impact on antioxidant enzyme including catalase.
Cell death
The assessment of cell death was measured through the estimation of root ion leakage (Mishra et al. 2021) and results are depicted in Table 2. From the experimental results it has been found that the percentage of root ion leakage significantly (p < 0.0001) increase with increasing CMS dose. This may be attributed by the fact that with increasing the CMS dose, generation of reactive oxygen species (ROS) was increased (Begum et al. 2012). Moreover, this ROS accumulation not only enhanced the electrolyte leakage, but also causes cell death (Tan et al. 2009; Kawai-Yanada et al. 2004).