Characterization of CuO NPs and BPs
SEM images showed more or less circular crystalline structure of NPs and BPs. The sizes of the CuO NPs and BPs obtained by SEM analysis were 49 ± 3.4 nm and 1 ± 0.2 µm respectively (Supplementary Fig. 1). The zeta potential value and PdI of CuO NPs in deionized water suspension analyzed by DLS were -30.6 mV and 0.134 respectively and these values for CuO BPs were -33.4 mV and 0.121 respectively. The MHDs of NPs were 191 ± 2.3, 193 ± 3.1, 190 ± 3.4, 192 ± 4.1, and 195 ± 4.1 nm at 0 hours for 20, 40, 60, 80, and 100 mg L⁻1 concentrations respectively. In terms of hydrodynamic stability of dispersions, MHDs of NPs after six hours of treatment were found to be 192 ± 3.8, 192 ± 4.3, 194 ± 3.7, 196 ± 2.9, and 199 ± 4.2 nm for chemical suspensions of 20, 40, 60, 80, and 100 mg L⁻1 respectively. The MHDs of particles at 0 and 6 hour did not show any significant statistical difference. This means that particles did not initiate agglomeration during the treatment period. Thus, dispersions were considered stable during the treatment period.
A detailed microscopic observation of smears prepared from treated secondary root tips of V. faba var. Pusa Sumit provided an overview of cytogenotoxic potential of CuO NPs and BPs. Different genetic endpoints like MI, CAs, and micronuclei were analyzed in root meristems exposed to graded concentrations of NPs and BPs suspensions viz. 20, 40, 60, 80, and 100 mg L⁻1 including control. Each index showed a dose-dependent correlation to chemical exposure. The correlation coefficients (r-value) of MI were found to be -0.99 and -0.95 against the treatment of CuO NPs and BPs respectively. Maximum MI was recorded in control, i.e., 34.0 ± 0.62 and it reduced gradually in treated root cells at 20, 40, 60, 80, and 100 mg L⁻1 concentrations. At 100 mg L⁻1 exposure, MI was minimum and found to be 9.9 ± 0.57 (NPs) and 21.3 ± 0.78 (BPs) (Table 1 and Fig. 1A). Reduction in MI with increasing concentration of CuO NPs and BPs was found statistically significant (p≤0.05).
The microscopic studies on mitotic phases in cells of secondary root meristems of V. faba revealed several CAs including stickiness, fragmentation, diagonal anaphases, precocious chromosomes, laggards, bridges, C-metaphase, spindle deformities, clumping, and micronuclei formation (Fig. 5). The correlation coefficient (r-value) of CAs was found to be 0.99 in both CuO NPs and BPs treated roots. Average percentage of CAs were minimum at 20 mg L⁻1 concentration i.e., 1.93 ± 0.33 (NPs) and 0.89 ± 0.23 (BPs). The percentage of CAs increased with increasing concentrations of chemicals and reached maximum at 100 mg L⁻1 i.e., 7.85 ± 0.54 (NPs) and 4.3 ± 0.33 (BPs) (Table 2 and Fig. 1B). The increase in the occurrence of these abnormalities was found significant in statistical reference (p≤0.05). At 20 mg L⁻1 exposure of CuO NPs, several aberrations viz. chromosome break, disturbance in the organization of metaphase chromosomes and movement of chromosomes at anaphase/spindle disturbance, diagonal anaphases, and micronuclei were observed. However, frequency of these aberrations was low in comparison to that observed at higher doses of chemicals. Additionally, several other aberrations such as fragmentation, stickiness, clumping of chromosomes, precocious chromosomes, bridges, laggards, and C- metaphase were observed at 40 and 60 mg L⁻1 exposure. At the highest dose exposure of 80 and 100 mg L⁻1, the average frequency of aberrations was much high and interestingly, multiple chromosomal aberrations like fragments, bridges, and laggards were seen in a single cell. However, the frequency of aberrations was higher in CuO NPs treatment as compared to BPs. The frequency of each aberration at all concentrations of CuO NPs (Fig. 1C) and BPs (Supplementary Fig. 3) has also been recorded during experiment.
Presence of micronuclei in cells was also dose-dependent; being (r-value = 0.99) with CuO NPs and (r-value = 0.98) in case of CuO BPs both being statistically significant. The average formation of micronuclei was 1.13 ± 0.24, 2.20 ± 0.36, 3.66 ± 0.34, 4.98 ± 0.38, 6.15 ± 0.59 in NPs treatment and 0.48 ± 0.21, 0.82 ± 0.29, 1.34 ± 0.31, 2.13 ± 0.28, 3.07 ± 0.35 in BPs treatment at 20, 40, 60, 80, 100 mg L⁻1 concentration of each respectively (Fig. 1D).
Plants exposed to CuO NPs showed a marked reduction in root and shoot lengths and leading to increase in phytotoxicity percentage. However, BPs stress did not show any significant reduction in seedling growth at an initial concentration (20 mg L⁻1), and its effects were less severe than that of NPs. Upon application of 100 mg L⁻1 of CuO NPs and BPs maximum reduction of 3.72 and 1.79 fold in root and 3.29 and 1.64 fold in shoot lengths was recorded over control (Fig. 1E, 1F & supplementary Fig. 2). Phytotoxicity percentage was observed 73.15 and 44.12, respectively at concentration of 100 mg L⁻1 (supplementary Fig. 4).
The content of photosynthetic pigments including chlorophyll and carotenoids significantly decreased under NPs and BPs stress. Plants exposed to NPs had a lower amount of pigments than plants under bulk particle stress. The total decline of 2.99 fold in chlorophyll and 3.93 fold in carotenoids content was recorded over control under NPs stress. While this decline was 1.56 fold and 2.03 fold under BPs stress (Fig. 2A and 2B). The amount of photopigments exhibited a similar trend and decreased with increasing concentration of CuO NPs treatment.
Malondialdehyde and Proline contents
The degree of lipid peroxidation in leaves was recorded in terms of MDA content. It significantly increased throughout the whole range of CuO NPs and BPs concentrations. The highest MDA content was recorded at 100 mg L⁻1 of CuO NPs and BPs, i.e., 2.84 and 2.18 fold over control respectively (Fig. 2C). Moreover, significant alteration in proline content was also recorded in treated leaves. In case of CuO NPs treated plants, maximum accumulation of proline content was recorded at 80 mg L⁻1 concentration, i.e., 2.43 fold over control. While in case of CuO BPs treated plants proline content increased to 1.9 fold over control (Fig. 2D).
Activities of antioxidant enzymes
Among the various antioxidant enzymes assessed in our study, CAT, POD and SOD showed more or less similar trend of activity while GST and GR varied. The activities of these antioxidant enzymes altered significantly and POD activity was found to be more affected in plants leaves under CuO NPs stress. An increase of 3.23 fold in CAT, 3.95 fold in POD and 2.59 fold in SOD activity was recorded over control in NPs exposed leaves (Fig. 3A, 3B and 3C). The enzymatic activity exhibited dose-dependent increase up to 80 mg L⁻1 of CuO NPs while it decreased at 100 mg L⁻1 concentration. Leaves exposed to CuO BPs showed increased activity of these enzymes in each concentration of chemical and the highest recorded increment was 2.43 fold in CAT, 2.98 fold in POD and 2.03 fold in SOD activity as compared control (Fig. 3A, 3B and 3C).
Activities of GST and GR in plant leaves altered significantly at lower concentrations of CuO NPs and the highest activity was recorded at 60 mg L⁻1 NPs. Further raising their concentrations reduced GST and GR activities. At 100 mg L⁻1, GST activity was lower than control, however, the difference was not significant in statistical reference. Under BPs stress, GST and GR activities were less affected at initial concentrations. At 100 mg L⁻1, maximum activity of GST (1.69 fold) and GR (1.51 fold) was recorded over control (Fig. 3D and 3E).
Bio-uptake of CuO NPs and BPs
The ICP-OES analysis showed dose-dependent increase in the amount of CuO NPs and BPs internalized into the plant tissues. For CuO NPs, internalized Cu was 2.79 ± 0.36, 7.44 ± 0.58, 13.5 ± 0.78, 18.45 ± 0.82, and 22.72 ± 0.58 mg L⁻1 in roots and 1.03 ± 0.08, 2.06 ± 0.2, 3.57 ± 0.24, 5.53 ± 0.32, and 7.34 ± 0.33 mg L⁻1 in leaves at 20, 40, 60, 80, and 100 mg L⁻1 respectively (Fig. 4A). However, in the case of CuO BPs, internalized Cu was 1.31 ± 0.1, 2.65 ± 0.31, 4.43 ± 0.7, 6.28 ± 0.45, and 7.62 ± 0.45 mg L⁻1 in roots and 0.56 ± 0.07, 0.88 ± 0.15, 1.79 ± 0.27, 2.71 ± 0.22, and 3.64 ± 0.18 mg L⁻1 in leaves at 20, 40, 60, 80, and 100 mg L⁻1 respectively (Fig. 4B).