Zinc Uptake, Photosynthetic Efficiency and Oxidative Stress in the Seagrass Cymodocea nodosa Exposed to ZnO Nanoparticles

We characterized zinc oxide nanoparticles (ZnO NPs) by dynamic light scattering (DLS) measurements, and transmission electron microscopy (TEM), while we evaluated photosystem II (PSII) responses, Zn uptake kinetics, and hydrogen peroxide (H2O2) accumulation, in C. nodosa exposed to 5 mg L−1 and 10 mg L−1 ZnO NPs for 4 h, 12 h, 24 h, 48 h and 72 h. Four h after exposure to 10 mg L−1 ZnO NPs, we noticed a disturbance of PSII functioning that became more severe after 12 h. However, after a 24 h exposure to 10 mg L−1 ZnO NPs, we observed a hormetic response, with both time and dose as the basal stress levels needed for induction of the adaptive response. This was achieved through the reduced plastoquinone (PQ) pool, at a 12 h exposure, which mediated the generation of chloroplastic H2O2; acting as a fast acclimation signaling molecule. Nevertheless, longer treatment (48 h and 72 h) resulted in decreasing the photoprotective mechanism to dissipate excess energy as heat (NPQ) and increasing the quantum yield of non-regulated energy loss (ΦNO). This increased the formation of singlet oxygen (1O2), and decreased the fraction of open reaction centers, mostly after a 72-h exposure at 10 mg L−1 ZnO NPs due to increased Zn uptake compared to 5 mg L−1.


Introduction
The small size of nanoparticles (NPs) provides them with special physical and chemical properties that are not found in bulk materials allowing their utilization, among others, in agricultural products, catalysis, cosmetics, electronics, energy production, engineering, food industry, pharmaceutics and textiles [1][2][3][4][5].

Zinc Oxide Nanoparticles Characterization
Primary particle size, and the morphological and structural characteristics of ZnO NPs were investigated by transmission electron microscopy (TEM). Samples were prepared by drop-casting dispersions of the NPs onto carbon-coated Cu grids after a 3 min sonication with an ultrasonic (VibraCell 400 W, Sonics & Materials Inc., Newtown, CT, USA), applying a microtip probe under intensity settings 4 [36]. Finally, TEM images were obtained with a Jeol JEM 1010 microscope (Jeol, Tokyo, Japan) [37].
Dynamic light scattering (DLS) analysis was used to define the size-distribution profile of ZnO NPs (5 mg L −1 , 10 mg L −1 and 50 mg L −1 ). Zeta (ζ) potential measurements were conducted to assess the surface charge of the particles as described previously [5]. All measurements were performed in Milli-Q water after brief sonication at 25 • C [5]. Results are presented as means (±SD) of three measurements.

Zinc Determination
Intermediate blades after wet digestion were processed following the methodology described previously [38,39]. Zinc concentrations were determined by flame atomic absorption spectrophotometry (AAnalyst 400 FAAS, Perkin-Elmer, Waltham, MA, USA) with the procedure described in detail before [38,39].

Zinc Leaf Uptake Kinetics
Zinc leaf uptake kinetics was fitted to the Michaelis-Menten equation: (C max × t)/(K m + t), as described in detail previously [5]. Briefly, C represents Zn leaf concentration reached in time t, K m the time taken to reach half of the value of C max , and C max the maximum or saturation Zn concentration. The rate of the initial uptake (C max /2 × K m ), the time needed to get equilibrium (T eq ), the equilibrium concentration (C eq ) and the mean rate of uptake (V c ) were also estimated. Equilibrium concentration (C eq ) is a concentration where the hourly increase is less than 1% compared to the previous hour [38][39][40][41]. The time required to reach equilibrium (T eq ) was assessed as the time needed to get the C eq , and the mean rate of uptake (V c ) was assessed as C eq /T eq [38,39]. Bioconcentration factor (BCF) was estimated as (C eq − C i )/C w , where C i is the initial Zn tissue concentration and C w is the Zn concentration in water [38,39].

Chlorophyll Fluorescence Imaging Analysis
An Imaging-PAM Chlorophyll Fluorometer (Walz, Effeltrich, Germany) was used for photosynthetic efficiency measurements as previously described [5]. In C. nodosa dark-adapted (15 min) leaf samples, we selected six areas of interest, and the allocation of absorbed light energy to photochemistry (Φ PSII ), non-photochemical energy loss as heat (Φ NPQ ), and non-regulated energy loss (Φ NO ), were calculated as described previously [29]. Relative PSII electron transport rate (ETR), non-photochemical quenching (NPQ), and photochemical quenching (q p ), were also measured [42].

Imaging of Hydrogen Peroxide Generation
For the estimation of H 2 O 2 production, C. nodosa leaves were treated with 25 µM 2 ,7 -dichlorofluorescein diacetate (Sigma) in the dark for 30 min, as described previously [43,44].

Statistical Analyses
Zinc leaf uptake kinetics data were analyzed using IBM Statistics SPSS ® 24 (New York, NY, USA). The significant differences on the fluorescence variables, between control and different incubation time in each concentration and between different concentrations at the same exposure time were tested at the 5% level of probability using t-test analysis (IBM Statistics SPSS ® 24). Modal analysis by using NORMSEP computer program was employed to estimate particle size (nm) distribution of ZnO NPs [45].

Characterization of ZnO NPs
The size and morphology of ZnO NPs was measured in stock solution by TEM (Figure 1). Modal analysis was used in data emerged from TEM micrographs, in order to determine the ZnO particle diameter distribution (Figure 2). A percentage of 92.8% of the particles was generally in agreement with manufacturer's characteristics (size < 50 nm). The computed mean (±SD) NPs size group with the highest frequency was 20.44 nm ± 7.95 nm ( Figure 2). Zinc leaf uptake kinetics data were analyzed using IBM Statistics SPSS ® 24 (New York, NY, USA). The significant differences on the fluorescence variables, between control and different incubation time in each concentration and between different concentrations at the same exposure time were tested at the 5% level of probability using t-test analysis (IBM Statistics SPSS ® 24). Modal analysis by using NORMSEP computer program was employed to estimate particle size (nm) distribution of ZnO NPs [45].

Characterization of ZnO NPs
The size and morphology of ZnO NPs was measured in stock solution by TEM (Figure 1). Modal analysis was used in data emerged from TEM micrographs, in order to determine the ZnO particle diameter distribution (Figure 2). A percentage of 92.8% of the particles was generally in agreement with manufacturer's characteristics (size < 50 nm). The computed mean (±SD) NPs size group with the highest frequency was 20.44 nm ± 7.95 nm ( Figure 2). The hydrodynamic size of 5 mg L −1 , 10 mg L −1 and 50 mg L −1 (stock) ZnO NPs solutions, ranged from 220.6 nm to 225.0 nm ( Table 1). The negative surface charge (ζ potential) ranged from −17.5 mV to −18.13 mV (Table 1). This similarity among the different NPs concentrations was indicative of the colloidal stability of the different populations. The size distribution by intensity of ZnO NPs is shown in Figure 3a  The hydrodynamic size of 5 mg L −1 , 10 mg L −1 and 50 mg L −1 (stock) ZnO NPs solutions, ranged from 220.6 nm to 225.0 nm ( Table 1). The negative surface charge (ζ potential) ranged from −17.5 mV to −18.13 mV (Table 1). This similarity among the different NPs concentrations was indicative of the colloidal stability of the different populations. The size distribution by intensity of ZnO NPs is shown in Figure 3a,b. Zinc leaf uptake kinetics data were analyzed using IBM Statistics SPSS ® 24 (New York, NY, USA). The significant differences on the fluorescence variables, between control and different incubation time in each concentration and between different concentrations at the same exposure time were tested at the 5% level of probability using t-test analysis (IBM Statistics SPSS ® 24). Modal analysis by using NORMSEP computer program was employed to estimate particle size (nm) distribution of ZnO NPs [45].

Characterization of ZnO NPs
The size and morphology of ZnO NPs was measured in stock solution by TEM ( Figure 1). Modal analysis was used in data emerged from TEM micrographs, in order to determine the ZnO particle diameter distribution ( Figure 2). A percentage of 92.8% of the particles was generally in agreement with manufacturer's characteristics (size < 50 nm). The computed mean (±SD) NPs size group with the highest frequency was 20.44 nm ± 7.95 nm ( Figure 2). The hydrodynamic size of 5 mg L −1 , 10 mg L −1 and 50 mg L −1 (stock) ZnO NPs solutions, ranged from 220.6 nm to 225.0 nm ( Table 1). The negative surface charge (ζ potential) ranged from −17.5 mV to −18.13 mV (Table 1). This similarity among the different NPs concentrations was indicative of the colloidal stability of the different populations. The size distribution by intensity of ZnO NPs is shown in Figure 3a

Zinc Leaf Uptake Kinetics
Leaf Zn uptake at both ZnO NPs concentration displayed a time dependent variation ( Figure 4). The uptake kinetics at both exposure concentrations was fitted to the Michaelis-Menten equation (r 2 :0.744 for 5 mg L −1 , and 0.681 for 10 mg L −1 , p < 0.01; Figure 4, Table 2). Zinc uptake of C. nodosa leaves increased more rapidly at the beginning of the experiment in the lower ZnO NPs solution (5 mg L −1 ), while it showed a higher and more than doubled initial rate [Cmax/(2 × Km)], and a higher mean rate (Vc) in comparison to the 10 mg L −1 solution ( Figure 4, Table 2). At 5 mg L −1 exposure concentration, the uptake reached the equilibrium concentration earlier (Teq = 28 h) than in the higher solution (42 h) ( Table 2). However, both the maximum concentration (Cmax) and the equilibrium concentration (Ceq) displayed their higher values at the higher ZnO NPs concentration (10 mg L −1 ) ( Figure 4, Table 2). During the experiment, the highest uptake was observed after 72 h of exposure, at both ZnO NP treatments (748.7 ± 29.7 μg g −1 dry wt at 5 mg L −1 and 1086.0 ± 33.7 μg g −1 dry wt at 10 mg L −1 ) ( Figure 4). Moreover, BCF value was higher at the lower exposure concentration ( Table 2).

Zinc Leaf Uptake Kinetics
Leaf Zn uptake at both ZnO NPs concentration displayed a time dependent variation ( Figure 4). The uptake kinetics at both exposure concentrations was fitted to the Michaelis-Menten equation (r 2 :0.744 for 5 mg L −1 , and 0.681 for 10 mg L −1 , p < 0.01; Figure 4, Table 2). Zinc uptake of C. nodosa leaves increased more rapidly at the beginning of the experiment in the lower ZnO NPs solution (5 mg L −1 ), while it showed a higher and more than doubled initial rate [C max /(2 × K m )], and a higher mean rate (V c ) in comparison to the 10 mg L −1 solution ( Figure 4, Table 2). At 5 mg L −1 exposure concentration, the uptake reached the equilibrium concentration earlier (T eq = 28 h) than in the higher solution (42 h) ( Table 2). However, both the maximum concentration (C max ) and the equilibrium concentration (C eq ) displayed their higher values at the higher ZnO NPs concentration (10 mg L −1 ) ( Figure 4, Table 2). During the experiment, the highest uptake was observed after 72 h of exposure, at both ZnO NP treatments (748.7 ± 29.7 µg g −1 dry wt at 5 mg L −1 and 1086.0 ± 33.7 µg g −1 dry wt at 10 mg L −1 ) ( Figure 4). Moreover, BCF value was higher at the lower exposure concentration (Table 2).

Zinc Leaf Uptake Kinetics
Leaf Zn uptake at both ZnO NPs concentration displayed a time dependent variation ( Figure 4). The uptake kinetics at both exposure concentrations was fitted to the Michaelis-Menten equation (r 2 :0.744 for 5 mg L −1 , and 0.681 for 10 mg L −1 , p < 0.01; Figure 4, Table 2). Zinc uptake of C. nodosa leaves increased more rapidly at the beginning of the experiment in the lower ZnO NPs solution (5 mg L −1 ), while it showed a higher and more than doubled initial rate [Cmax/(2 × Km)], and a higher mean rate (Vc) in comparison to the 10 mg L −1 solution ( Figure 4, Table 2). At 5 mg L −1 exposure concentration, the uptake reached the equilibrium concentration earlier (Teq = 28 h) than in the higher solution (42 h) ( Table 2). However, both the maximum concentration (Cmax) and the equilibrium concentration (Ceq) displayed their higher values at the higher ZnO NPs concentration (10 mg L −1 ) ( Figure 4, Table 2). During the experiment, the highest uptake was observed after 72 h of exposure, at both ZnO NP treatments (748.7 ± 29.7 μg g −1 dry wt at 5 mg L −1 and 1086.0 ± 33.7 μg g −1 dry wt at 10 mg L −1 ) ( Figure 4). Moreover, BCF value was higher at the lower exposure concentration (Table 2).  Table 2. Kinetics of Zn accumulation in C. nodosa leaf blades exposed to 5 mg L −1 and 10 mg L −1 ZnO NPs. The fits correspond to a Michaelis-Menten equation: C = (C max × t)/(K m + t); C, in µg g −1 dry wt; K m , in hours; t, in hours; C eq , in µg g −1 dry wt; T eq , in hours; V c , concentration/hours. In parentheses, standard errors are given.

Allocation of Absorbed Light Energy in Leaf Blades of Cymodocea nodosa Exposed to ZnO NPs
The allocation of absorbed light energy at PSII in leaf blades of C. nodosa exposed to ZnO NPs for 4 h, 24 h, 48 h and 72 h is shown in Figures 5 and 6. Exposure to 5 and 10 mg L −1 ZnO NPs significantly decreased the quantum efficiency of PSII photochemistry (Φ PSII ) compared to control values, with the exception of the 24 h treatment that Φ PSII increased at 10 mg L −1 ZnO NPs and did not differ compared to control values at 5 mg L −1 ZnO NPs (Figure 5a). Thus, at the 24-h treatment, Φ PSII in 10 mg L −1 ZnO NPs was higher than in 5 mg L −1 (Figure 5a). ZnO NPs ± SD (n = 3); dashed and bold lines are the uptake kinetics calculated using Michaelis-Menten equation. Table 2. Kinetics of Zn accumulation in C. nodosa leaf blades exposed to 5 mg L −1 and 10 mg L −1 ZnO NPs. The fits correspond to a Michaelis-Menten equation: C = (Cmax × t ) / (Km + t); C, in μg g −1 dry wt; Km, in hours; t, in hours; Ceq, in μg g −1 dry wt; Teq, in hours; Vc, concentration/hours. In parentheses, standard errors are given

Allocation of Absorbed Light Energy in Leaf Blades of Cymodocea Nodosa Exposed to ZnO NPs
The allocation of absorbed light energy at PSII in leaf blades of C. nodosa exposed to ZnO NPs for 4 h, 24 h, 48 h and 72 h is shown in Figure 5 and Figure 6. Exposure to 5 and 10 mg L −1 ZnO NPs significantly decreased the quantum efficiency of PSII photochemistry (ΦPSΙΙ) compared to control values, with the exception of the 24 h treatment that ΦPSΙΙ increased at 10 mg L −1 ZnO NPs and did not differ compared to control values at 5 mg L −1 ZnO NPs (Figure 5a). Thus, at the 24-h treatment, ΦPSΙΙ in 10 mg L −1 ZnO NPs was higher than in 5 mg L −1 (Figure 5a).  The quantum yield of regulated non-photochemical energy loss (Φ NPQ ) increased significantly at 5 mg L −1 ZnO NPs compared to control, while at 10 mg, L −1 increased after 24 h of exposure, but decreased at further exposure time (48 h and 72 h) (Figure 5b). Φ NPQ after 4 h, 48 h and 72 h exposure to the low concentration was significantly higher than in the high concentration (Figure 5b). The quantum yield of regulated non-photochemical energy loss (ΦNPQ) increased significantly at 5 mg L −1 ZnO NPs compared to control, while at 10 mg, L −1 increased after 24 h of exposure, but decreased at further exposure time (48 h and 72 h) (Figure 5b). ΦNPQ after 4 h, 48 h and 72 h exposure to the low concentration was significantly higher than in the high concentration (Figure 5b). The non-regulated energy loss (ΦNO) did not differ compared to control values after exposure to 5 mg L −1 ZnO NPs ( Figure 6), but decreased compared to the control values after 24 h of exposure to the high NPs concentration, and increased at further exposure time (48 h and 72 h) (Figure 6). At 48 h and 72 h treatment ΦNO of C. nodosa leaf blades exposed to the high concentration was significantly higher than in the low concentration ( Figure 6).

Electron Transport Rate and Non-Photochemical Quenching in Leaf Blades of Cymodocea Nodosa Exposed to ZnO NPs
The relative electron transport rate (ETR) of PSII decreased significantly at both ZnO NPs concentrations, with the exception of the 24 h exposure, where the ETR increased at the high concentration and did not differ compared to control values at the low concentration (Figure 7a). With the 24 h treatment, the ETR of C. nodosa leaf blades exposed to the high concentration was significantly higher than in the low concentration (Figure 7a).
The non-photochemical quenching (NPQ) increased at the low concentration compared to control, while at the high concentration it increased after 24 h of exposure, but decreased significantly at further exposure time (48 h and 72 h) (Figure 7b). After 4 h, 48 h and 72 h treatment, the NPQ of C. nodosa leaf blades exposed to 5 mg L −1 was significantly higher than 10 mg L −1 (Figure 7b).  Figure 6. The quantum yield of non-regulated energy dissipated in PSII (non-regulated heat dissipation, a loss process due to PSII inactivity) (Φ NO ) in control leaf blades of C. nodosa (Cymodocea nodosa) and in leaf blades exposed to 5 mg L −1 and 10 mg L −1 ZnO NPs for 4 h, 24 h, 48 h and 72 h. Symbol explanations as in Figure 5.

The Redox State of Plastoquinone Pool of Cymodocea nodosa Leaf Blades Exposed to ZnO NPs
The non-regulated energy loss (Φ NO ) did not differ compared to control values after exposure to 5 mg L −1 ZnO NPs ( Figure 6), but decreased compared to the control values after 24 h of exposure to the high NPs concentration, and increased at further exposure time (48 h and 72 h) (Figure 6). At 48 h and 72 h treatment Φ NO of C. nodosa leaf blades exposed to the high concentration was significantly higher than in the low concentration ( Figure 6).

Electron Transport Rate and Non-Photochemical Quenching in Leaf Blades of Cymodocea nodosa Exposed to ZnO NPs
The relative electron transport rate (ETR) of PSII decreased significantly at both ZnO NPs concentrations, with the exception of the 24 h exposure, where the ETR increased at the high concentration and did not differ compared to control values at the low concentration (Figure 7a). With the 24 h treatment, the ETR of C. nodosa leaf blades exposed to the high concentration was significantly higher than in the low concentration (Figure 7a).
The non-photochemical quenching (NPQ) increased at the low concentration compared to control, while at the high concentration it increased after 24 h of exposure, but decreased significantly at further exposure time (48 h and 72 h) (Figure 7b). After 4 h, 48 h and 72 h treatment, the NPQ of C. nodosa leaf blades exposed to 5 mg L −1 was significantly higher than 10 mg L −1 (Figure 7b). The quantum yield of regulated non-photochemical energy loss (ΦNPQ) increased significantly at 5 mg L −1 ZnO NPs compared to control, while at 10 mg, L −1 increased after 24 h of exposure, but decreased at further exposure time (48 h and 72 h) (Figure 5b). ΦNPQ after 4 h, 48 h and 72 h exposure to the low concentration was significantly higher than in the high concentration (Figure 5b). Figure 6. The quantum yield of non-regulated energy dissipated in PSII (non-regulated heat dissipation, a loss process due to PSII inactivity) (ΦNO) in control leaf blades of C. nodosa (Cymodocea nodosa) and in leaf blades exposed to 5 mg L −1 and 10 mg L −1 ZnO NPs for 4 h, 24 h, 48 h and 72 h. Symbol explanations as in Figure 5.
The non-regulated energy loss (ΦNO) did not differ compared to control values after exposure to 5 mg L −1 ZnO NPs ( Figure 6), but decreased compared to the control values after 24 h of exposure to the high NPs concentration, and increased at further exposure time (48 h and 72 h) (Figure 6). At 48 h and 72 h treatment ΦNO of C. nodosa leaf blades exposed to the high concentration was significantly higher than in the low concentration ( Figure 6).

Electron Transport Rate and Non-Photochemical Quenching in Leaf Blades of Cymodocea Nodosa Exposed to ZnO NPs
The relative electron transport rate (ETR) of PSII decreased significantly at both ZnO NPs concentrations, with the exception of the 24 h exposure, where the ETR increased at the high concentration and did not differ compared to control values at the low concentration (Figure 7a). With the 24 h treatment, the ETR of C. nodosa leaf blades exposed to the high concentration was significantly higher than in the low concentration (Figure 7a).
The non-photochemical quenching (NPQ) increased at the low concentration compared to control, while at the high concentration it increased after 24 h of exposure, but decreased significantly at further exposure time (48 h and 72 h) (Figure 7b). After 4 h, 48 h and 72 h treatment, the NPQ of C. nodosa leaf blades exposed to 5 mg L −1 was significantly higher than 10 mg L −1 (Figure 7b).   10 mg L −1 ZnO, but increased after a 24 h exposure (Figure 8). After a 72 h exposure of C. nodosa to ZnO NPs, the PQ pool was more oxidized at 5 mg L −1 than at 10 mg L −1 , but less than controls (Figure 8). The redox state of plastoquinone (PQ) pool (a measure of PSII open reaction centers) (qP), did not differ compared to control values after 4 h, 24 h, and 48 h exposure to 5 mg L −1 ZnO NPs but reduced after 72 h (Figure 8). PQ pool reduced compared to control values after 4 h, 48 h and 72 h exposure to 10 mg L −1 ZnO, but increased after a 24 h exposure (Figure 8). After a 72 h exposure of C. nodosa to ZnO NPs, the PQ pool was more oxidized at 5 mg L −1 than at 10 mg L −1 , but less than controls (Figure 8).

Chlorophyll Fluorescence Images of Cymodocea nodosa Leaf Blades Exposed to ZnO NPs
We did not detect any spatial heterogeneity of ΦPSII, ΦNPQ, ΦNO, and qP images in the control leaf blades of C. nodosa measured at 200 μmol photons m −2 s −1 actinic light (Figure 9). In addition, exposure to both NPs concentrations for 4 h, 24 h, 48 h and 72 h did not significantly alter these patterns ( Figure  9). A temporal heterogeneity was observed at the images of qP and ΦNPQ (Figure 9). The lowest ΦPSΙΙ with simultaneous low qP values was observed after a 12 h exposure at the high concentration ( Figure  10a). At the same time, the highest levels of H2O2 were detected (Figure 10b). Immediately after this, we noticed that the 24 h exposure to 10 mg L −1 ZnO NPs resulted in the highest ΦPSΙΙ values with the highest qP values, both of them being higher than the control values ( Figure 9). A parallel decreased ΦNO was detected (Figure 9).

Imaging of Hydrogen Peroxide Production After Exposure of Cymodocea nodosa Leaf Blades to ZnO NPs
No noteworthy quantities of H2O2 could be noticed in the control leaf blades of C. nodosa ( Figure  11a). Exposure at the low concentration for 4 h did not result in any change to H2O2 production (Figure 11b), while the same exposure time at the high concentration, resulted in increased production of H2O2 (Figure 11c). After a 24-h exposure to 5 mg L −1 ZnO NPs, H2O2 levels were the same as the control ones (Figure 11d), while they also dropped at 10 mg L −1 and could not be detected at all (Figure 11e). Later, after a 48-h exposure to ZnO NPs, there was an increase in the accumulation of H2O2 being higher at 5 mg L −1 (Figure 11f), than at 10 mg L −1 ZnO NPs (Figure 11g). However, after a 72-h exposure to ZnO NPs the accumulation of H2O2 decreased at 5 mg L −1 (Figure 11h), but increased

Chlorophyll Fluorescence Images of Cymodocea nodosa Leaf Blades Exposed to ZnO NPs
We did not detect any spatial heterogeneity of Φ PSII , Φ NPQ , Φ NO , and q P images in the control leaf blades of C. nodosa measured at 200 µmol photons m −2 s −1 actinic light (Figure 9). In addition, exposure to both NPs concentrations for 4 h, 24 h, 48 h and 72 h did not significantly alter these patterns (Figure 9). A temporal heterogeneity was observed at the images of q P and Φ NPQ (Figure 9). The lowest Φ PSII with simultaneous low q P values was observed after a 12 h exposure at the high concentration (Figure 10a). At the same time, the highest levels of H 2 O 2 were detected (Figure 10b). Immediately after this, we noticed that the 24 h exposure to 10 mg L −1 ZnO NPs resulted in the highest Φ PSII values with the highest q P values, both of them being higher than the control values (Figure 9). A parallel decreased Φ NO was detected (Figure 9).

Imaging of Hydrogen Peroxide Production After Exposure of Cymodocea nodosa Leaf Blades to ZnO NPs
No noteworthy quantities of H2O2 could be noticed in the control leaf blades of C. nodosa ( Figure  11a). Exposure at the low concentration for 4 h did not result in any change to H2O2 production (Figure 11b), while the same exposure time at the high concentration, resulted in increased production of H2O2 (Figure 11c). After a 24-h exposure to 5 mg L −1 ZnO NPs, H2O2 levels were the same as the control ones (Figure 11d), while they also dropped at 10 mg L −1 and could not be detected at all (Figure 11e). Later, after a 48-h exposure to ZnO NPs, there was an increase in the accumulation of H2O2 being higher at 5 mg L −1 (Figure 11f), than at 10 mg L −1 ZnO NPs (Figure 11g). However, after a 72-h exposure to ZnO NPs the accumulation of H2O2 decreased at 5 mg L −1 (Figure 11h), but increased at 10 mg L −1 ZnO NPs (Figure 11i). The highest H2O2 accumulation was detected after a 12-h exposure to 10 mg L −1 ZnO NPs (Figure 10b) and the lowest after a 24-h exposure to 10 mg L −1 (Figure 11e).

Discussion
Previously, extensive literature survey has demonstrated both the positive and detrimental impacts of NPs on terrestrial and aquatic plants, which are due to size and type of NPs (especially their specific surface area) and the plant species [5,7,8,12,22,25]. Toxicity of ZnO NPs is determined to be due to the dissolution, release and uptake of free Zn ions, but specific nanoparticulate effects may be hard to unravel from effects due to free zinc ions [46,47]. Thus, ZnO NPs effects in C. nodosa were correlated to both applied ZnO NPs concentration and to Zn uptake.
In C. nodosa cellular, physiological and biochemical measurable responses (biomarkers) to metallic elements (e.g., Cd, Cr, Cu, Ni) have been proposed as early warning signals of alterations in seawater quality [32,33,38]. However, there has been little consideration of the seagrasses, especially C. nodosa, as the test material in evaluating the effects of metal oxide nanoparticles [5], despite the fact that coastal ecosystems are expected to be the destination of the majority of the nanoparticles, mainly ZnO and TiO2 NPs, discharged by industry [46]. NPs are released into aquatic environment either by direct uses or wastewater plant effluents [47][48][49].

Imaging of Hydrogen Peroxide Production After Exposure of Cymodocea nodosa Leaf Blades to ZnO NPs
No noteworthy quantities of H 2 O 2 could be noticed in the control leaf blades of C. nodosa (Figure 11a). Exposure at the low concentration for 4 h did not result in any change to H 2 O 2 production (Figure 11b), while the same exposure time at the high concentration, resulted in increased production of H 2 O 2 (Figure 11c). After a 24-h exposure to 5 mg L −1 ZnO NPs, H 2 O 2 levels were the same as the control ones (Figure 11d), while they also dropped at 10 mg L −1 and could not be detected at all (Figure 11e). Later, after a 48-h exposure to ZnO NPs, there was an increase in the accumulation of H 2 O 2 being higher at 5 mg L −1 (Figure 11f), than at 10 mg L −1 ZnO NPs (Figure 11g). However, after a 72-h exposure to ZnO NPs the accumulation of H 2 O 2 decreased at 5 mg L −1 (Figure 11h), but increased at 10 mg L −1 ZnO NPs (Figure 11i). The highest H 2 O 2 accumulation was detected after a 12-h exposure to 10 mg L −1 ZnO NPs (Figure 10b) and the lowest after a 24-h exposure to 10 mg L −1 (Figure 11e).

Discussion
Previously, extensive literature survey has demonstrated both the positive and detrimental impacts of NPs on terrestrial and aquatic plants, which are due to size and type of NPs (especially their specific surface area) and the plant species [5,7,8,12,22,25]. Toxicity of ZnO NPs is determined to be due to the dissolution, release and uptake of free Zn ions, but specific nanoparticulate effects may be hard to unravel from effects due to free zinc ions [46,47]. Thus, ZnO NPs effects in C. nodosa were correlated to both applied ZnO NPs concentration and to Zn uptake.
In C. nodosa cellular, physiological and biochemical measurable responses (biomarkers) to metallic elements (e.g., Cd, Cr, Cu, Ni) have been proposed as early warning signals of alterations in seawater quality [32,33,38]. However, there has been little consideration of the seagrasses, especially C. nodosa, as the test material in evaluating the effects of metal oxide nanoparticles [5], despite the fact that coastal ecosystems are expected to be the destination of the majority of the nanoparticles, mainly ZnO and TiO 2 NPs, discharged by industry [46]. NPs are released into aquatic environment either by direct uses or wastewater plant effluents [47][48][49].
be due to the dissolution, release and uptake of free Zn ions, but specific nanoparticulate effects may be hard to unravel from effects due to free zinc ions [46,47]. Thus, ZnO NPs effects in C. nodosa were correlated to both applied ZnO NPs concentration and to Zn uptake.
In C. nodosa cellular, physiological and biochemical measurable responses (biomarkers) to metallic elements (e.g., Cd, Cr, Cu, Ni) have been proposed as early warning signals of alterations in seawater quality [32,33,38]. However, there has been little consideration of the seagrasses, especially C. nodosa, as the test material in evaluating the effects of metal oxide nanoparticles [5], despite the fact that coastal ecosystems are expected to be the destination of the majority of the nanoparticles, mainly ZnO and TiO2 NPs, discharged by industry [46]. NPs are released into aquatic environment either by direct uses or wastewater plant effluents [47][48][49].  Photosynthetic organisms via the process of photosynthesis, transform the light energy into chemical energy with the collaboration of PSII and PSI, while the most susceptible constituent of the photosynthetic apparatus to environmental stresses is believed to be PSII [15][16][17][18]50]. PSII functionality estimated by chlorophyll fluorescence imaging has been considered as the most suitable method to identify NPs toxicity effects on plants [5,21,22]. Exposure of plants to NPs can have positive or negative effects on the light reactions of photosynthesis [51].
At the beginning of the experiment, Zn uptake kinetics at 5 mg L −1 ZnO NPs with a more than twice initial rate and higher mean rate (V c ) than 10 mg L −1 (Figure 4, Table 2), resulted in a significant lower quantum efficiency of PSII photochemistry (Φ PSII ) compared to 10 mg L −1 , after a 24 h exposure (Figure 5a). However, at 5 mg L −1 exposure concentration, Zn uptake reached the equilibrium concentration earlier (T eq = 28 h) than at 10 mg L −1 (T eq = 42 h), resulting in a significantly higher regulated non-photochemical energy loss as heat (Φ NPQ ) after 48 h and 72 h exposure, compared to 10 mg L −1 (Figure 5b). This lower Φ NPQ at 10 mg L −1 ZnO NPs, resulted in significantly higher non-regulated energy loss (Φ NO ) of C. nodosa leaf blades exposed to 10 mg L −1 for 48 h and 72 h ( Figure 6), since there was no difference in Φ PSII (Figure 5a). The significantly increased Φ NO (Figure 6) implies higher singlet oxygen ( 1 O 2 ) production. Φ NO consists of chlorophyll fluorescence internal conversions and intersystem crossing, indicative of 1 O 2 formation via the triplet state of chlorophyll ( 3 chl*) [42,[52][53][54]. The increased 1 O 2 formation, mostly after 72 h exposure to 10mg L −1 ZnO NPs, was due to an increased Zn uptake compared to 5 mg L −1 (Figure 4).
A reduced photosynthetic efficiency, measured as the maximum quantum efficiency of PSII (F v /F m ), and the redox state of plastoquinone (PQ) pool (q p ), was also observed previously due to increased Zn accumulation [7]. Furthermore, ZnO NPs treatments enhanced generation of H 2 O 2 [7]. A relationship between closed reaction centers (q p ) and increased H 2 O 2 generation was noticed (Figures 9-11). The PQ pool is considered as the component integrated in plant antioxidant defense [55], which at a 12 h of exposure mediated the generation of chloroplastic H 2 O 2 [22], acting as a fast acclimation-signaling molecule [55,56].
After 12 h exposure of C. nodosa to 10 mg L −1 ZnO NPs, leaf Zn uptake was higher than in 5 mg L −1 (Figure 4), resulting in the lower Φ PSII and the lowest q p values (Figure 10a). At the same time, an increased accumulation of H 2 O 2 was detected in the leaves of C. nodosa (Figure 10b). Closed reaction centers (q p ) indicate excess photon supply and associated ROS production [57][58][59]. This photosynthesis derived H 2 O 2 is moving throughout leaf veins, serving as a signaling molecule and triggering a stress-defense response [22,35,44,55,56,60,61]. Thus, this stress-defense response triggered a significant increase of the fraction of open PSII reaction centers (q p ) ( Figure 8) and a significant increase of Φ PSII (Figure 5a) and ETR (Figure 7a) observed after 24 h exposure of C. nodosa to 10 mg L −1 ZnO NPs; while at the same time, H 2 O 2 generation decreased to control levels ( Figure 11e). However, we have to emphasize that antibacterial activity of ZnO and ZnO NPs is due to ROS generation [62,63], which also orchestrates a regulatory action at various plant developmental stages [64].
Hormesis has been extensively documented in plants, revealing that biphasic dose-responses occur commonly [65,66]. Thus, at low-level stress, plants are activating responses at the cellular and molecular level that enhance adaptation and plant tolerance [65,66]. The photoprotective mechanism of non-photochemical quenching (NPQ) which is closely related to ROS, follows a biphasic dose-response pattern typical of hormesis [67]. NPQ in C. nodosa leaf blades exposed to 10 mg L −1 ZnO NPs depicts a hormetic response. A hormetic response suggests that a basal stress level is needed for adaptive responses [68,69]. This basal stress level was 10 mg L −1 ZnO NPs and the time required for the induction of this mechanism was 24 h exposure.