The visual observations did not show any differences in plants (Fig. 1). Microscopic observation of roots confirmed that MPs were absorbed into the roots and the number of MP particles observed inside the roots increased with increasing MP concentration (Fig. 2); however, the stem cross-sections observed did not confirm the existence of MPs in tissues. Shoot lengths showed an increasing trend with the exposure MP concentration; however, the ANOVA test was not grouped (ANOVA P > 0.05, F = 2.880; Fig. 3). However, there were significant differences between the roots. Lengths of the roots were the highest with 1.25 mg L− 1 MP exposure, whereas 6 mg L− 1 exposure also showed longer roots than with 0, 0.05, and 0.25 mg L− 1 MP exposure conditions. The ANOVA testing grouped 0, 0.05, and 0.25 mg L− 1; 0, 0.05, and 6 mg L− 1; and 1.25 mg L− 1MP exposure conditions (ANOVA P < 0.01, F = 11.723).
The Fv/Fm was reduced significantly with 1.25 mg L− 1 MP exposure compared to the rest of the exposure conditions in which the Fv/Fm values were unchanged. The ANOVA test grouped 1.25 mg L− 1; and 0, 0.05, 0.25, and 6 mg L− 1 MP concentrations (ANOVA P < 0.01, F = 60.583; Fig. 4).
The Chl-a values in the above- and below-water parts showed different trends with increasing MP concentrations. Chl-a, Chl-b, and Car increased with increasing MP concentration, whereas the Chl a/b ratio decreased. Below-water parts showed inconsistent Chl-a, Chl-b, Car, and Chl a/b ratio trends with increasing MP concentration (Fig. 5). The Chl-a, Chl-b, Car, and Chl a/b ratios of the above-water parts were statistically insignificant between treatment groups (ANOVA P > 0.05; F = 0.790, 1.548, 2.328, and 1.657 for Chl-a, Chl-b, Car, and Chl a/b ratios, respectively). The below-water parts showed significant differences, except for Chl-a (Chl-a ANOVA, P > 0.05, F = 0.780). The ANOVA test for Chl-b grouped 0, 0.25, and 6 mg L− 1; 0.25, 1.25, and 6 mg L− 1; and 1.25 and 6 mg L− 1 MP exposure conditions (ANOVA P < 0.01, F = 9.444). The Car was grouped as 0 and 0.25 mg L-1; 0.5 and 1.25 mg L− 1; and 0.5 and 6 mg L− 1 MP exposure conditions (ANOVA P < 0.01, F = 11.599). The Chl a/b ratios were grouped 0.5 and 1.25 mg L− 1; 0.25 and 6 mg L− 1; and 0 and 0.25 mg L− 1 MP exposure conditions (ANOVA P < 0.01, F = 21.959).
The anthocyanin content was significantly higher in below-water parts than in the above-water parts under every treatment condition including 0 mg L− 1 MP exposure (t-test P < 0.01 for 0, 0.05, 0.25 and 1.25 mg L− 1 and P < 0.05 for 6 mg L− 1). The 1.25 mg L− 1 MP-exposed above-water parts had a higher anthocyanin content than the rest of the exposure conditions. Below-water parts showed decreasing anthocyanin content till 0.25 mg L− 1 MP concentration and increased till 6 mg L− 1 MP concentration (Fig. 6). The ANOVA test grouped 1.25 mg L− 1 and 0, 0.05, 0.25, and 6 mg L− 1 of above-water parts (ANOVA P < 0.01; F = 8.846). The anthocyanin contents of below-water parts were not different (ANOVA, P > 0.05, F = 0.828).
The cellular H2O2 content varied under different MP exposure conditions, although it did not follow a trend related to the MP concentration. Under the 1.25 mg L− 1 MP exposure condition, both above- and below-water parts showed the highest H2O2 content (Fig. 7). The below-water parts of 1.25 mg L− 1 MP-exposed plants had higher H2O2 content than the respective above-water portions (t-test P < 0.01 for 0, 0.05, 0.25 and 6 mg L− 1 MP; and P < 0.05 for 1.25 mg L− 1 MP). The ANOVA test grouped the below-water parts as 0, 0.05, and 6 mg L− 1; and 0, 0.25, 1.25, and 6 mg L− 1 MP treatments (ANOVA P < 0.05, F = 3.482) and above-water parts as 0, 0.05, and 0.25 mg L− 1; 0.05, 0.25, and 6 mg L− 1; and 1.25 and 6 mg L− 1 MP treatments (ANOVA P < 0.05, F = 5.616).
The GPX activity of both the above- and below-water parts showed the same trend, increasing until the maximum GPX was reached at 0.25 mg L− 1 MP concentration and recording lower activity at 1.25 and 6 mg L− 1 MP concentrations (Fig. 8a). The GPX activities of 0.05 and 0.25 mg L− 1 MP concentrations of the above-water parts were significantly higher than the respective below-water parts (t-test P < 0.01). The ANOVA test of above-water parts grouped 0 and 1.25 mg L− 1; 1.25 and 6 mg L− 1; and 0.05, 6, and 0.25 mg L− 1 (ANOVA P < 0.01, F = 18.748), whereas below-water parts did not show differences (ANOVA P > 0.05, F = 3.250).
The CAT activity showed an increasing trend with increasing MP concentration, peaking at 1.25 mg L− 1 in both above- and below-water parts. The CAT activity was then reduced close to the level of that with the 0.05 mg L− 1 concentration (Fig. 8b). There were no significant differences between the above- and below-water parts for any of the MP concentrations. The ANOVA test for above-water parts grouped 0, 0.05, and 6 mg L− 1; 0.25 and 6 mg L− 1; and 0.25 and 1.25 mg L− 1 MP exposure conditions (ANOVA P < 0.01, F = 9.020) and below-water parts were grouped as 0, 0.05 and 6 mg L− 1; 0.25 mg L− 1; and 1.25 mg L− 1 (ANOVA P < 0.01, F = 51.218).
The APX activities of the above and below water parts showed different trends with increasing MP concentrations. The APX activity of 0.05 and 0.25 mg L− 1 MP exposure conditions showed significantly higher activity in below-water parts than above-water parts (t-test P < 0.01). The above-water parts did not show a change in APX activity under 0.05 and 0.25 mg L− 1 MP exposure from that at 0 mg L− 1 exposure and then increased under 1.25 and 6 mg L− 1 MP exposure conditions (Fig. 8c). The below-water parts showed a higher APX activity than at the 0 mg L− 1 MP exposure at every MP concentration. The ANOVA testing grouped 0, 0.05, and 0.25 mg L− 1; 1.25 mg L− 1; and 6 mg L− 1 MP exposure conditions of above-water parts (ANOVA P < 0.01, F = 27.644) and 0 mg L− 1; and 0.05, 0.25, 1.25, and 6 mg L− 1 MP exposure conditions of below-water parts (ANOVA P < 0.01, F = 18.579).
Relationships and Correlations
The regression relationships of CAT, APX, anthocyanin, Chl-a, Chl-b, Chl a/b, Car, and Fv/Fm of the above-water parts with the MP concentrations could be explained by second-order polynomial distributions, whereas the elongation relationship was linear. The R2 values of the relationships were 0.8213, 0.9591, 0.9644, 0.9161, 0.9868, 0.9768, 0.8905, 0.9692, and 0.8908 for CAT, APX, anthocyanin, Chl-a, Chl-b, Chl a/b, Car, Fv/Fm, and elongation, respectively. The below-water part parameters, H2O2, CAT, and root length relationships with MP concentrations could be explained using second-order polynomial distributions. The R2 values for H2O2, CAT, and root lengths were 0.8909, 0.9556, and 0.9291, respectively (Table 1). When the 6 mg L− 1 MP exposure condition was omitted, the relationships could be explained by linear distributions. The CAT, APX, anthocyanin, Chl-a, Chl-b, Chl a/b, Car, and Fv/Fm linear regression relationship R2 values were 0.7902, 0.9665, 0.9742, 0.8349, 0.9641, 0.9352, 0.7608, and 0.9808, respectively. The R2 values of the linear relationships of H2O2, CAT, and root lengths were 0.8806, 0.9321, and 0.9442, respectively (Supplementary data 1).
The linear correlations between H2O2 and the remaining parameters were tested. The water CAT, anthocyanin, and Fv/Fm showed a strong correlation with the H2O2 content, with R values of 0.7894, 0.7304, and 0.7419, respectively. The APX, Chl-a, Car, and root lengths showed moderate relationships with R values of 0.5682, 0.5159, 0.5464, and 0.6404, respectively. The H2O2 content in below-water parts was strongly correlated with CAT, APX, Fv/Fm, and root length, and the R values were 0.8435, 0.7272, 0.8590, and 0.8893, respectively.