Genotoxic and Anatomical Deteriorations Associated with Potentially Toxic Elements Accumulation in Water Hyacinth Grown in Drainage Water Resources

: Potentially toxic elements (PTEs)-induced genotoxicity on aquatic plants is still an open question. Herein, a single clone from a population of water hyacinth covering a large distribution area of Nile River (freshwater) was transplanted in two drainage water resources to explore the hazardous e ﬀ ect of PTEs on molecular, biochemical and anatomical characters of plants compared to those grown in freshwater. Inductivity Coupled Plasma (ICP) analysis indicated that PTEs concentrations in water resources were relatively low in most cases. However, the high tendency of water hyacinth to bio-accumulate and bio-magnify PTEs maximized their concentrations in plant samples (roots in particular). A Random Ampliﬁed Polymorphic DNA (RAPD) assay showed the genotoxic e ﬀ ects of PTEs on plants grown in drainage water. PTEs accumulation caused substantial alterations in DNA proﬁles including the presence or absence of certain bands and even the appearance of new bands. Plants grown in drainage water exhibited several mutations on the electrophoretic proﬁles and banding pattern of total protein, especially proteins isolated from roots. Several anatomical deteriorations were observed on PTEs-stressed plants including reductions in the thickness of epidermis, cortex and endodermis as well as vascular cylinder diameter. The research ﬁndings of this investigation may provide some new insights regarding molecular, biochemical and anatomical responses of water hyacinth grown in drainage water resources.


Introduction
The fast global discharge of potentially toxic elements (PTEs) into the hydrosphere has attracted serious focus due to their high bioavailability, bioaccumulation, and biomagnification potentials [1][2][3]. variability has advantageous due to their sensitivity and rapid responses. PTEs-induced phytotoxicity caused significant alterations in leaf protein profiles including reduction of photosynthetic protein and cellular damage at the DNA level and organelles such as mitochondria or lysosomes [25]. In this regard, the behavior of PTEs on biochemical parameters of water hyacinth is contradictory. For instance, the interference of Cd and Mn ions with protein synthesis caused inhibition in RNA and DNA content. However, Zn ions caused the opposite effect through increasing RNA and DNA content and protein synthesis [26]. To the best of the authors' knowledge, molecular and anatomical investigations on water hyacinth plants grown under toxic elements stress are still insufficient. Therefore, the main objectives of this study are to investigate the effect of PTEs contamination on DNA pattern, protein profile characters, and anatomical structure of water hyacinth populations grown in two drainage water resources compared to another population grown in freshwater source (Nile River).

Potentially Toxic Elements Concentration in Water and Water Hyacinth Plants
Elemental analysis of water and plant samples is illustrated in Table 1. In general, elements concentration in drainage water samples was greatly higher than freshwater samples. Ca, Na, Mg, and K showed the highest concentrations in water samples. Concentrations of PTEs in fresh and drainage water samples were relatively low in most cases, and some of these PTEs were below detection limit. Elamom drain recorded the highest Ca, Mg, K, Na, and P levels (3.11, 4.08, 4.74, 8.34, and 8.0-fold of the freshwater source). Arsenic (As) and nickel (Ni) were detected only in Elamom drain. This drain recorded the highest concentrations of Ba, Sr, and V. Concentrations of Ca, Mg, K, Na, and P showed higher values in root and leaf samples, particularly in drainage water sources. These elements showed higher accumulation in leaves than roots suggesting their nutritional functions in plant [27]. PTEs showed a high biomagnification potential based on their high concentration in plant organs. Immobile PTEs (e.g., Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Sb, Ti, V, and Zn) showed higher accumulation in roots; however, mobile PTEs (e.g., Ba and Sr) exhibited higher accumulation in leaves. The bioaccumulation factor (BAF) of PTEs (Al, Ba, Cu, Mn, Sr, Ri, V, and Zn) confirmed the hyperaccumulating potentials of water hyacinth (Table 2). According to Ma et al. [28], plants are classified as hyperaccumulators when BAF value is higher than 10.

Molecular Analysis
A single clone from a population covering the entire distribution area in Nile River was selected as a source of the studied populations. Nine random primers were used to differentiate among populations grown in fresh and drainage resources (Table 3 and Figure 1). The OPA-01 primer gave 8 different bands sized from 1560 to 250 bp. Two of which were polymorphic bands with 25% polymorphism percentage and the other one was monomorphic. It was clear that the sizes of 100 bp were existed only in the freshwater population and disappeared in other drainage water populations. On the other hand, the band size of 375 bp was existed only in both drainage water populations. The application of OPA-02 primer indicated the amplification of seven bands with a size ranged from 1400 to 375 bp, which one of them was polymorphic with 14.3% polymorphism percentage. The Band with the size of 375 bp was unique and found once in the population of Elamom drain. The OPB-05 primer separated six amplified bands with a size ranging from 1300 to 370 bp. Two of these bands were polymorphic (33.3% polymorphism percentage) and four were monomorphic. The band with a size of 1000 bp was absent in the population of Nashart drain. However, the band with molecular size of 370 bp was unique in the population of Elamom drain. The OPA-04 primer exhibited five different bands ranging from 1450 to 600 bp, where one of them was polymorphic (22% polymorphism percentage). Moreover, it is cleared that the band with a size of 1200 bp was existed only in both populations grown in drainage water resources. was polymorphic (22% polymorphism percentage). Moreover, it is cleared that the band with a size of 1200 bp was existed only in both populations grown in drainage water resources. The OPC-03 primer separated five different bands with a size ranging from 1500 to 600 bp. One of them was polymorphic (20% polymorphism percentage) and another one with the size of 1500 bp was absent only in the population of Elamom drain. Data presented in Table 2 showed that the nine used RAPD primers produced a total of 57 amplified bands. Among them, five were polymorphic with 12.3% polymorphism percentage. The OPB-05 primer gave the highest total polymorphic bands, whereas OPA-02 primer recorded the lowest ones. Furthermore, the primers OPA-06, OPA-07, OPC-14 and OPV-07 produced only monomorphic bands, and these primers were not able to differentiate among the populations of fresh and drainage water resources.

Total Protein Analysis by SDS-PAGE
The electrophoretic profiles and banding pattern of total protein isolated from leaves and roots of water hyacinth grown in fresh and drainage water resources are presented in Figure 2 and Table  A3. SDS-PAGE banding patterns of leaves revealed 23 bands with different molecular weights. Among them, five bands showed high variability; however, the other 18 bands were commonly detected in the studied populations. Both sources of drainage water caused changes in the protein electrophoretic profiles. These changes include alteration in band intensities (66, 65.  The OPC-03 primer separated five different bands with a size ranging from 1500 to 600 bp. One of them was polymorphic (20% polymorphism percentage) and another one with the size of 1500 bp was absent only in the population of Elamom drain. Data presented in Table 2 showed that the nine used RAPD primers produced a total of 57 amplified bands. Among them, five were polymorphic with 12.3% polymorphism percentage. The OPB-05 primer gave the highest total polymorphic bands, whereas OPA-02 primer recorded the lowest ones. Furthermore, the primers OPA-06, OPA-07, OPC-14 and OPV-07 produced only monomorphic bands, and these primers were not able to differentiate among the populations of fresh and drainage water resources.

Total Protein Analysis by SDS-PAGE
The electrophoretic profiles and banding pattern of total protein isolated from leaves and roots of water hyacinth grown in fresh and drainage water resources are presented in Figure 2 and Table A3. SDS-PAGE banding patterns of leaves revealed 23 bands with different molecular weights. Among them, five bands showed high variability; however, the other 18 bands were commonly detected in the studied populations. Both sources of drainage water caused changes in the protein electrophoretic profiles.

Anatomical Structure
The anatomical investigation of water hyacinth plants (roots, leaves, and petioles) in transverse sections is illustrated in Figure 3 and Table 4. It is realized that the epidermis of root, leaf, and petiole consists of one layer of quadrangular cells. Unlike leaf and petiole, root epidermis was not covered by cuticle. Root diameter showed a noticeable reduction in plants grown in drainage water. This reduction was associated with a notable decrease in epidermis, cortex and endodermis thickness as well as vascular cylinder diameter. Additionally, number of metaxylem and diameter of xylem vessels were higher in plants grown in freshwater compared to those grown in drainage water resources (Figure 3a-c and Table 4). Furthermore, epidermis of leaf lamina in transverse section has one layer with very thin cuticle layer, and the mesophyll tissue was distinguished into a palisade and spongy tissue. Substantial reductions in lamina thickness, upper epidermis, lower epidermis and mesophyll tissue thickness as well as length and width of vascular bundles were observed on plants grown in drainage water relative to those grown in freshwater (Figure 3d,f and Table 4).  Table 4).

Anatomical Structure
The anatomical investigation of water hyacinth plants (roots, leaves, and petioles) in transverse sections is illustrated in Figure 3 and Table 4. It is realized that the epidermis of root, leaf, and petiole consists of one layer of quadrangular cells. Unlike leaf and petiole, root epidermis was not covered by cuticle. Root diameter showed a noticeable reduction in plants grown in drainage water. This reduction was associated with a notable decrease in epidermis, cortex and endodermis thickness as well as vascular cylinder diameter. Additionally, number of metaxylem and diameter of xylem vessels were higher in plants grown in freshwater compared to those grown in drainage water resources ( Figure 3A-C and Table 4). Furthermore, epidermis of leaf lamina in transverse section has one layer with very thin cuticle layer, and the mesophyll tissue was distinguished into a palisade and spongy tissue. Substantial reductions in lamina thickness, upper epidermis, lower epidermis and mesophyll tissue thickness as well as length and width of vascular bundles were observed on plants grown in drainage water relative to those grown in freshwater ( Figure 3D,F and Table 4). Sustainability 2020, 12, x FOR PEER REVIEW 6 of 16   The microphotographs in Figure 3G-I show that the petiole consists of single epidermis layer and parenchyma cells contain vascular bundles. Each bundle was surrounded by bundle sheath of sclerenchyma cells. Epidermis thickness of petiole showed a reduction in plants grown in drainage water compared with plants grown in freshwater. Likewise, length and width of lacuna and vascular bundle of petiole (µ) showed reductions in plants grown in drainage water compared to those grown in freshwater ( Figure 3G-I and Table 4).

Discussion
Large-scale utilization of water hyacinth for contaminated effluent purification is of great importance due to its high tolerance against biotic and abiotic stress conditions [29]. Although PTEs concentration in drainage water resources was relatively low, their mutual effects caused several deteriorations on molecular and anatomical characters of water hyacinth plants. These results are in agreement with [30] as water hyacinth plants can survive under a mixture of PTEs (Cd, Co, Cr, Cu, Mn, Ni, Pb, and Zn) up to 3 mg L −1 and under Pb 2+ stress up to 100 mg L −1 . Concentrations of Ba and Sr were the highest among other PTEs (0.053-0.1016 mg L −1 ) and (0.3437-1.107 mg L −1 ), respectively. The translocation factor (TF) of the PTEs was > 1 suggesting the higher rhizofiltration efficiency of these metals by water hyacinth plants. According to Ma et al. [28], hyperaccumulating plants with TF > 1 are classified as high-efficient plants for PTEs translocation from roots into shoots. PTEs showed higher accumulation in roots compared to leaves. PTEs are mainly localized in vascular tissues and epidermal cells to mediate their translocation to other plant tissues [15]. Additionally, it may be localized as precipitates into metal binding compounds existed in cell walls (carbohydrates, cellulose, hemicellulose and lignin) Results of PCA were performed by applying Varimax rotation with Kaiser normalization to assist the interpretation of PTEs concentration (Tables A1 and A2, and Figure A1, supporting information). PCA is commonly used in such studies to investigate the relationship between elements and their potential origins. Data as the different groups of elements that correlate together might have a similar common origin and similar behavior. The initial principal components (PC1 and PC2) explained about 87.5% of the variation (72.17 and 15.32%, respectively). In addition, PC1 and PC2 had the highest e eigenvalues (11.55 and 2.45, respectively) as indicated in Table A1 in Appendix A, supporting information. Principle components 1 (PC1) showed high positive correlation and was loaded with Ba, Sr, Cu, Mn, Zn, Sb, and Ni, and it is suggested that these elements are derived from natural and anthropogenic origins. However, the rotated component matrix revealed that Fe, Cd, Cr, As, Al, Co, Ti, and Mo showed negative correlation and strongly correlated with principal component 2 (PC2), and these elements might be derived from industrial origins.
The obtained results illustrated the high tolerance of water hyacinth against PTEs stress. Genotoxicity of toxic elements can be assessed by molecular techniques such as Randomly Amplified Polymorphic DNA (RAPD). RAPD analysis showed generation of new DNA bands in plants grown in drainage water resources. These bands have not existed in plants grown in freshwater. Some of these bands have only existed in plants grown in the higher contaminated drainage water (Figure 1). [31] illustrated that DNA alterations detected by RAPD analysis offered a useful biomarker assay for the evaluation of genotoxic effects of PTEs in Capsicum annum. It was also reported that Cd has the capacity not only to cause morbidity to the exposed organisms but also has the potential to induce genotoxic adverse effects [32,33]. RAPD assay indicated damages and mutations in DNA induced directly and/or indirectly by the phytotoxic effect of copper [34]. RAPD assay also showed variations in band intensity, loss of typical bands and appearance of new bands suggesting several damages in DNA of barley seedling treated with Cd (30-120 mg/L −1 ) [35].
The present study demonstrated the presence of 21 protein bands separated from leaves of water hyacinth plants grown in freshwater. Meanwhile, other plants grown in Elamom and Nashrat drains showed the presence of 22 and 23 protein bands, respectively. The protein profile of roots revealed detection of three protein bands of control plants. However, 16 protein bands were detected in roots of water hyacinth plants grown in drainage water resources. These findings are consistent with several reports confirming that PTEs exposure might cause complete elimination of some protein bands and creation of new ones [36,37]. Proteins are directly responsible for most biological processes in living cells. Therefore, it is necessary to conduct proteomic studies, which elucidate protein presence and role under certain environmental conditions [38]. It is well known that PTEs stress can activate a range of potential cellular mechanisms in plants, some of which being the mobilization of specific molecules such as stress proteins that play a very significant role in Cd detoxification and tolerance in plants [39,40]. The changes in protein banding patterns have been attributed to the occurrence of either gene mutation or induction of cytological aberrations. The absence of some bands might be due to the deletion of their corresponding genes.
Anatomically, water hyacinth has an epidermis layer, which is not covered by a cuticle or only covered with thin cuticle layer to support gas and nutrients absorption from surrounding water. The adverse effects of PTEs on cell growth and sizing caused a noticeable reduction in root diameter as well as thickness in cortex and vascular cylinder diameter in plants grown in drainage water. So far, no sufficient details are available regarding the anatomical responses of water hyacinth under PTEs stress. According to [41] no injurious effects on root anatomy of water hyacinth grown under the presence of As. Contrariwise, [42] reported that arsenic (As) stress caused several deteriorations in anatomical characters of water hyacinth leaves due to the reduction of cell size. The harmful effect of PTEs on the anatomical structure of water hyacinth may be attributed to the adverse effects on cell organelles and the nutrients imbalance in plant tissues [43,44]. The same harmful effects on anatomical structure were recorded under differs biotic and abiotic stresses in numerous plants [45][46][47][48][49]

Elemental Analysis
Uniform-sized bottles were rinsed with the representative resources before water sampling. Water samples were taken at a depth of 10 cm from the surface of fresh and drainage water. Representative water samples were inserted into bottles leaving an appropriate head-space, and the bottles were tightly closed by caps to avoid potential contamination. Bottles were placed directly in the fridge until elemental analysis.
Water hyacinth plants were divided into roots and leaves in order to distinguish between the accumulation potentials of different plant organs. Plant samples (roots and leaves) were air-dried for a week, oven-dried at 70 • C until weight constant and mechanically ground to a fine powder using a stainless steel grinder. Plant samples were acid-digested for elemental determination of Ag, Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Sb, Sr, Ti, V and Zn. Subsamples (1.0 g) of roots and leaves were digested using the mixture of concentrated nitric acid (HNO 3 ), hydrochloric acid (HCl) and 30% hydrogen peroxide (H 2 O 2 ) [50,51].
Total elements concentration in water and plant samples was determined using Inductively where C water , C root , and C leaves are PTEs concentrations in water (mg L −1 ), leaves (mg kg −1 ), and roots (mg kg −1 ), respectively [28]. Quality control of data was performed with the use of repeat measurements for all obtained data. The mean relative standard deviation (RSD) was less than 5%. Principal component analysis (PCA) was performed using MVSP software ver 3.13 (Kovach Computing Services, Pentraeth, UK) [52] to assist the interpretation of PTEs concentration in water and plant samples.

Total DNA Extraction
Total DNA was extracted from young leaves using the modified cetyl trimethylammonium bromide (CTAB) method [53] with some modifications. Leaves were wrapped in filter papers under hand pressure for five min to remove moisture, and 150 mg of samples were ground using pistil and mortar. Thereafter, 600 µL of preheated (65 • C) extraction buffer (2% CTAB, 20 mM EDTA, 100 mM Tris-HCl, 1.4 M NaCl, 2% polyvinylpyrrolidone, and 0.2% mercaptoethanol) were added. The mixture was transferred to a centrifuge tube (2 mL), incubated for 30 min in a 65 • C water bath, and samples were inverted every 5 min. 600 µL of chloroform-isoamyl alcohol (24:1) was added and mixed by inverting the tubes carefully for 8 times, and the mixture was centrifuged at 12,000 rpm for 10 min at room temperature. The supernatant was collected, carefully mixed with a two-third volume of ice-cold isopropanol, and the DNA samples were collected by centrifuging for 10 min. RNase (10 µg/mL) was added to the 50 µL of TE buffer (10 mM Tris and 0.1 mM EDTA) prior to dissolving the DNA to remove any RNA, and the mixture was incubated at 37 • C for 30 min. After incubation, 100 µL and 750 µL volumes of 3.0 M sodium acetate and ice-cold absolute ethanol were added, respectively. The DNA was collected by high-speed centrifugation for 10 min, carefully washed with ice-cold absolute and 70% ethanol and centrifuged at 120000 rpm for 10 min. Finally, samples were dried at room temperature and dissolved in 50-100 µL of TE buffer. The quality and concentration of DNA were determined by a P330 photometer (EMPLEN, Munich, Germany).

RAPD-PCR
For DNA amplification, nine decamer RAPD primers (Operon technologies, CA, USA) were used (Table 1). PCR was performed as follows: initial denaturation at 94 • C for 5 min; followed by 35

Total Protein Extraction
Total proteins were extracted from water hyacinth leaves and roots. Briefly, approximately 0.5 g powder of fresh leaves and roots were homogenized by mechanical grinding and mixed well with 500 µl of the protein extraction buffer (62.5 mM Tris-Hcl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 5 M Urea and 0.01% bromophenol blue) by vortexing. Protein extracts were centrifuged at 14,000 rpm for 10 min at 4 • C and separated by 12% (SDS-PAGE) according to [54]. Molecular weights of different bands were calibrated with a mixture of standard protein markers (Molecular Weight Marker, M. W. 14.000-66.000; Catalog No. SDS7, Sigma-Aldrich, Munich, Germany). The banding profile was stained by Coomasie blue dye then photographed and scored.

Anatomical Studies
Roots, leaves, and petioles of water hyacinth plants were sampled for anatomical characterization. Samples (0.5 cm length) were placed in FAA solution (killing and fixing), washed in 50% ethyl alcohol, and dehydrated in butyl alcohol series. Samples were impeded in paraffin wax (56-58 • C). Transverse sections (15 microns thick) were done with rotary microtome model 820, fixed with albumin, stained with a combination of safranin and light green, and finally fixed in Canada balsam [55]. The sections were investigated microscopically and photomicrographed (Leica, Wetzlar, Germany) [48,56,57].

Conclusions
Water hyacinth (Eichhornia crassipes (Mart.) Solms) is one of the most critical aquatic plants in Egypt and worldwide. Due to its hyper accumulating potentials of PTEs and its high resistance against biotic and abiotic stress conditions, water hyacinth has received significant attention to clean-up contaminated water effluents. However, research trials undertaken to improve adaptability of water hyacinth against PTEs stress are still very few. The novelty aspects of this research are to study molecular, biochemical and anatomical characters of water hyacinth subjected to PTEs stress. Although PTE's concentration in drainage water resources was relatively low, the hyperaccumulating potentials of water hyacinth maximized their concentration in plant tissues. Immobile PTEs (e.g., Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Sb, Ti, V, and Zn) showed higher accumulation in roots; however, mobile PTEs (e.g., Ba and Sr) exhibited higher accumulation in leaves. DNA alterations detected by RAPD analysis confirmed the genotoxic effects of PTEs. Protein profile alterations in electrophoretic profiles and banding patterns were observed on plants grown in drainage water resources, especially protein isolated from roots.
The ultrastructural analysis also showed several deteriorations on the anatomical structure of plants grown under PTEs stress. Further investigations should be undertaken to explore molecular and biochemical characters of plants grown under higher PTEs concentrations.     Table A3. SDS-PAGE banding patterns of total protein isolated from leaves and roots of water hyacinth grown in fresh and drainage water resources.