Nitrogen Effect on Growth-Related Parameters and Evaluation of Portulaca oleracea as a Phytoremediation Species in a Cr(VI)-Spiked Soil

: In a pot experiment, we assessed the potential of purslane ( Portulaca oleracea ) as a phytoremediation species in Cr(VI)-contaminated soils. We focused on the evaluation of phytotoxic Cr(VI) effects at concentrations reaching 150 mg Cr(VI) kg − 1 and the possible stress amelioration effect of nitrogen on Cr(VI)-stressed plants. Treatments were T-0 (control), T-1 (25 mg Cr(VI) kg − 1 ), T-2 = 50 mg kg − 1 , T-3 = 100 mg kg − 1 , and T-4 = 150 mg kg − 1 . We measured Cr(VI) concentration in aerial and root tissues, a series of parameters related to photosynthesis and plant growth, phosphorus aerial plant tissue content, and we also calculated indices (ratios) related to leaf growth and above ground tissue water content. Cr(VI) almost exclusively was found in root tissues; all physiological and growth parameters studied were severely affected and plants selectively accumulated phosphorus in aerial plant tissues with increasing Cr(VI) soil concentrations. On the other hand, N amendment resulted in improved plant features in some of the measured parameters: chlorophyll index was improved with added N at T-2, plant height was signiﬁcantly higher at T-0, T-1, and T-2, and aerial dry weight and leaf area was higher at T-0; these effects indicate that added N did increase P. oleracea potential to ameliorate Cr(VI) toxic effects. We conclude that purslane showed a potential as a possible species to be successfully introduced to Cr(VI)-laden soils, but more research is certainly necessary.


Introduction
Metal ions can be introduced to surface soils by natural or anthropogenic processes and their environmental impact is greatly affected by their mineralogical and geochemical form [1]. Cr is mainly found in two valence states, namely +3 (chromite (Cr(III)) and +6 (chromate Cr(VI)). However, in natural soil conditions trivalent chromium Cr(III) is the predominant state [2]. Hexavalent Cr compounds are found in wastes of numerous industrial activities (i.e., chromic acid and Cr-pigment production, leather tanning, cement production, metal plating, and stainless-steel production), and its anionic form results in increased possibility of Cr(VI) pollution dispersal [2][3][4].
Cr(III) is an essential element for some redox reactions that serve fundamental cellular functions relevant to sugar, protein and lipid metabolism in humans (recommended adult intake of 50 to 200 µg/d); however it is not an essential element for plants [5][6][7]. Hexavalent chromium (Cr(VI)) is of much higher toxicity (10 to 100 times) compared to Cr(III) for both acute and chronic exposure, posing serious health hazards for humans. Hexavalent Cr has been identified as one of the seventeen chemicals threatening human health and is classified as a human carcinogen causing a variety of cancer diseases in humans that result in increased overall mortality rates. Cr(VI) and Cr(III) in soil are in dynamic equilibrium before in real soil conditions. Thus, it could be the case that well-fertilized plants address Cr(VI) exposure in a way that their developmental (i.e., root and shoot weight, and aerial part height), as well as physiological features (i.e., photosynthetic rate and chlorophyll content) are less severely affected compared to non-fertilized plants; however, due to a lack of evidence from the literature, this potentially beneficial effect of fertilization has to be elucidated [36].
Purslane (Portulaca oleracea) is a halophytic annual plant species, tolerant to several abiotic stresses [22]. This species adaptability is largely attributed to great morpho-cytophysiologic variability that greatly contributes to the rapid growth and propagation under harsh environmental conditions. Key factors that contribute to purslane adaptability involve the production of secondary metabolites and the species ability to switch from C4 to CAM photosynthesis (carbon fixation-photosynthetic mechanism) under drought stress [21,37]. Salinity, drought and metal stress induce common physiological responses from plants [38]. Tolerant plants to other abiotic stresses can be possible candidates to be tested for phytoremediation purposes. To the best of our knowledge, despite the literature reports regarding the accumulation and toxic effects of chromium on purslane plants [36,[39][40][41], there is a void in the literature concerning the ability of the species to grow under elevated Cr(VI) soil conditions, especially when N-added soils and compared to soil with non N addition.
Furthermore, it may be the case that nitrogen applied to Cr(VI)-stressed purslane could result in higher plant aerial biomass and thus in higher overall removal of Cr(VI) from soil. If this is the case, purslane could act faster as a phytoremediation species for the restoration of a Cr(VI)-laden area, when considering that metal uptake is affected both by plant tissue Cr(VI) content and the aerial biomass. However, this beneficial effect is also not elucidated by current literature evidence. Hence, the aims of this work were to study the developmental and physiological features of purslane, as well as its Cr(VI) content and possible toxicity symptoms resulting from Cr(VI) exposure in a soil well-fertilized with N and compare these effects with those in purslane plants grown in a non-fertilized soil. This study targeted specifically the evaluation of purslane as a potential phytoremediation species towards Cr(VI)-contaminated soils. To the best of our knowledge, although there are some works that have investigated purslane as a phytoremediation species towards Cr(VI) (e.g., [36]), there is no investigation in relation to the effects of added N on the phytoremediation capacity of the species.

Materials and Methods
A 10-treatment (2 levels of nitrogen × 5 levels of Cr(VI)) × 10 replicates) experiment was established. Overall, we had 100 replicates (each in 2-L pots) and for each replicate, a mixture of 1000 g of soil and 800 mL perlite was prepared. Soil was obtained from a field in the agricultural region between Volos and Larisa (39.394925 N, 22.756285 E), an area not affected from any known source of pollution. Soil spiking was performed using Cr(VI) solution of 10,000 ppm Cr(VI), by dissolving 19.22 g of CrO 3 in 1000 mL distilled water. Spiking solution was applied to the soil resulting in 5 Cr(VI) treatments (T-0: control; T-1: 25 mg Cr(VI) kg −1 , with 2.5 mL spiking solution per pot; T-2: 50 mg kg −1 , with 5 mL solution; T-3: 100 mg kg −1 , 10 mL solution; and T-4: 150 mg kg −1 , 15 mL solution.
For each Cr(VI) treatment, half of the replicates (10 out of 20) were amended in rates equivalent to 200 kg of nitrogen per hectare or 100 mg N per kg soil as NH 4 NO 3 salt (thereafter named N-1 treatments and the non-added-N treatments are named N-0). The spiked soil treatments along with the un-amended control were placed in 2-L plastic pots, watered to their holding capacity and the spiked soil was left to equilibrate for 20 days. During the equilibration period, soil was thoroughly mixed three times per week and water was added as needed to keep soil to its water holding capacity.
At the end of the equilibration period, four samples per Cr(VI) treatment were obtained from the pots, air dried, passed through a 2-mm sieve in order to determine the initial (Day 0) hexavalent chromium (Cr(VI)) soil concentration.
Plants were grown in an unheated greenhouse. On Day 0, P. oleracea plants, already sown 25 days before Day 0 in peat-filled seedling trays, were transplanted in pots (one plant per pot). Transplantation took place when plants reached a height of 12 cm.
During the growth period, to compensate for any light and temperature differences in the greenhouse, plant positions were exchanged regularly, and water was applied to the plants according at regular intervals in amounts that depended on weather conditions (50-250 mL per pot). One month prior to harvest date, we measured plant height in cm, photosynthetic rate (µmol CO 2 m −2 s −1 ) at a constant light intensity (250 µmol cm −2 s −1 ) using the LI-Cor LI-6400XT Portable Photosynthesis System (LI-Cor, Lincoln, NE, USA)) and chlorophyll content (SPAD index) was measured using the OPTI-SCIENCES CCM-200 plus chlorophyll content meter (Opti-sciences, Hudson, NH, USA).
Plants were grown in the Cr(VI) spiked soil for 50 days-from 14 October 2019 (establishment of seedlings in the pots) to 4 December 2019, when plants were harvested. On harvest day we measured the weight of stems, the weight of leaves and the leaf area per plant. Then, aerial plant tissues were washed with deionized water and root tissues were meticulously washed so that no soil particles remained attached and further rinsed with deionized water.
Aerial and root plant tissues were dried in an air-forced drying oven at 70 • C for 96 h. Both aerial and root tissues were weighted and pulverized. Then, 1.00 g samples of plant tissue were dry-ashed at 500 • C for 4 h and ash was extracted using 10 mL of 20% HCl. Plant tissue K, P and Cr(VI) concentrations were estimated according to established laboratory protocols-dry ashing at 500 • C for 5 h, and then ash extraction with 20 mL of 20% HCl [44]. Due to the lack of sufficient plant tissue mass, especially in the high Cr(VI) treatments, out of the 10 replicates initially sown, 5 replicates for extraction and measurement were formed by combining tissues from every two pots. Furthermore, out of the primary data, we calculated a secondary index, i.e., tolerance index (TI), equal to dry aerial biomass in contaminated soil over that in control. Because of the fact that we had effectively two controls, typical to a two-factor experiment like ours, i.e., (a) no Cr(VI) with no N, and (b) no Cr(VI) with added N, we calculated TI as two independent factors, one for soils without N and one for soils with N.

Quality Assurance and Statistical Analyses
For data quality control purposes in-house plant and soil reference materials were used and recovery rates were within the range of 95% to 105% of the certified value. To rule out any possibility of cross-contamination, for every extraction batch blank samples were also measured. For Cr(VI) calibration curves, Merck standard solutions were used (Merck, Burlington, MA, USA).
Statistical analysis of the data was performed using IBM SPSS Statistics 25 and Excel 2019 software. One-way ANOVA and Duncan's multiple range tests were used to identify statistically significant differences between treatments and two-way ANOVA and Duncan's multiple range tests at p = 0.05 were used to monitor the effect that Cr and nitrogen had on the different parameters studied.

Cr(VI) Concentration in Plant Tissues
Increasing soil Cr(VI), increased aerial tissue Cr(VI) content (p < 0.001), reaching 4.13 mg Cr(VI) kg −1 dry matter (T-4 (Cr(VI)) treatment with no added N), while nitrogen addition had no significant effect in the aerial tissue Cr(VI) content (p = 0.915) (Figure 1a). Furthermore, increasing Cr(VI) soil concentration resulted in significant increase (p < 0.001) in root tissue Cr(VI) concentration and nitrogen addition had no significant effect on root tissue Cr(VI) content (p = 0.109). In root tissues, Cr(VI) levels were orders of magnitude higher compared to aerial tissues, reaching 339 mg kg −1 dry matter at the highest Cr(VI) soil concentration with no added N, while at T-4 Cr(VI) with added N root Cr(VI) concentrations reached 596 mg kg −1 (p < 0.001). This finding indicates that in the highest tested soil Cr(VI) concentrations, N amendment resulted in increased root Cr(VI) concentrations (Figure 1b), significantly higher than the non-added-N treatment, whereas aerial contents of Cr(VI) remained low without being affected by N addition.
Horticulturae 2021, 7, x FOR PEER REVIEW 5 of 13 statistically significant differences between treatments and two-way ANOVA and Duncan's multiple range tests at p = 0.05 were used to monitor the effect that Cr and nitrogen had on the different parameters studied.

Cr(VI) Concentration in Plant Tissues
Increasing soil Cr(VI), increased aerial tissue Cr(VI) content (p < 0.001), reaching 4.13 mg Cr(VI) kg −1 dry matter (T-4 (Cr(VI)) treatment with no added N), while nitrogen addition had no significant effect in the aerial tissue Cr(VI) content (p = 0.915) (Figure 1a). Furthermore, increasing Cr(VI) soil concentration resulted in significant increase (p < 0.001) in root tissue Cr(VI) concentration and nitrogen addition had no significant effect on root tissue Cr(VI) content (p = 0.109). In root tissues, Cr(VI) levels were orders of magnitude higher compared to aerial tissues, reaching 339 mg kg −1 dry matter at the highest Cr(VI) soil concentration with no added N, while at T-4 Cr(VI) with added N root Cr(VI) concentrations reached 596 mg kg −1 (p < 0.001). This finding indicates that in the highest tested soil Cr(VI) concentrations, N amendment resulted in increased root Cr(VI) concentrations (Figure 1b), significantly higher than the non-added-N treatment, whereas aerial contents of Cr(VI) remained low without being affected by N addition.

Chlorophyll Content Index and Photosynthetic Rate
As a result of Cr(VI) exposure, the purslane developmental and physiological parameters studied were significantly affected. Chlorophyll content (SPAD index) was found to gradually decrease (p < 0.001) from 12.

Chlorophyll Content Index and Photosynthetic Rate
As a result of Cr(VI) exposure, the purslane developmental and physiological parameters studied were significantly affected. Chlorophyll content (SPAD index) was found to gradually decrease (p < 0.001) from 12.

Plant Height and Aerial Fresh Weight
Plant height and aerial fresh weight, as expected, followed the same trend observed for photosynthetic rate and chlorophyll content. Increasing Cr(VI) concentrations resulted in lower height values (p < 0.001) and nitrogen amendment had a positive effect in plant height in every Cr(VI) level tested (p = 0.038). For aerial fresh weight the trend was similar, with increasing Cr(VI) concentrations exerting negative effects on the measured values (p < 0.001) and in this case nitrogen addition had also a positive effect (p < 0.001) ( Table 1).

Aerial Dry Weight and Root Dry Weight and Leaf Area Per Plant
Cr(VI) increasing concentrations negatively affected aerial dry matter production (p < 0.001), and nitrogen addition resulted in higher values for every Cr(VI) level studied (p < 0.001). More specifically, in treatments where no nitrogen was added, values decreased gradually from 0.99 g (T-0) to 0.23 g (T-4) and in treatments where nitrogen was added, aerial dry weight values were significantly higher, reaching 2.73 g in the control treatment

Plant Height and Aerial Fresh Weight
Plant height and aerial fresh weight, as expected, followed the same trend observed for photosynthetic rate and chlorophyll content. Increasing Cr(VI) concentrations resulted in lower height values (p < 0.001) and nitrogen amendment had a positive effect in plant height in every Cr(VI) level tested (p = 0.038). For aerial fresh weight the trend was similar, with increasing Cr(VI) concentrations exerting negative effects on the measured values (p < 0.001) and in this case nitrogen addition had also a positive effect (p < 0.001) ( Table 1).

Aerial Dry Weight and Root Dry Weight and Leaf Area per Plant
Cr(VI) increasing concentrations negatively affected aerial dry matter production (p < 0.001), and nitrogen addition resulted in higher values for every Cr(VI) level studied (p < 0.001). More specifically, in treatments where no nitrogen was added, values decreased gradually from 0.99 g (T-0) to 0.23 g (T-4) and in treatments where nitrogen was added, aerial dry weight values were significantly higher, reaching 2.73 g in the control treatment (T-0) and gradually decreased to 0.36 g for the highest soil Cr(VI) concentration used in the experiment (T-4). On the other hand, root dry weight showed an increasing trend with increasing Cr(VI) concentrations even from the lower Cr(VI) level (T-1) (0.39 g per pot), despite the fact that differences failed to escalate in the higher Cr(VI) levels, resulting in marginally higher than 0.05 significance (p = 0.053); nitrogen addition resulted in higher root dry weight values (p = 0.021) ( Table 2). Considering the results presented in Figure 1 and Table 2, the addition of N in plants treated with the highest Cr(VI) concentration may result in the removal of significantly higher amounts of Cr(VI) from contaminated soils. In particular, despite the similar contents of Cr(VI) in the aerial parts the increase in dry weight per pot (from 0.23 g pot −1 to 0.36 g pot −1 in no N and added N plants, respectively) indicates an increase in the removal of Cr(VI) from soils by 56.5%. On the other hand, in the case of roots we noticed an increase in both root dry weight per pot (from 0.5 g pot −1 to 0.96 g pot −1 in no N and added N plants, respectively) and Cr(VI) content in dried tissues (339 mg kg −1 and 596 mg kg −1 in no N and added N plants, respectively), meaning a cumulative increase of 237.6% in Cr(VI) removal from soils. Dry to fresh aerial tissue weight ratio increased significantly (p = 0.002) even from the lowest soil Cr(VI) (T-1) concentration (Table 3). Leaf area was significantly reduced (p < 0.001) even from the lowest level of Cr(VI) soil concentration (T-1), while nitrogen addition had a positive effect, increasing leaf area (p < 0.001) ( Table 3). Apart from leaf area, leaf weight-to-total aerial weight ratio followed the same pattern as the leaf area, with increasing Cr(VI) concentrations resulting in lower (p < 0.001) ratio values and N addition resulting in higher ratio values (p = 0.007) ( Table 3). Leaf weight/total aerial tissue weight ratio decreased gradually with increasing Cr(VI) concentrations (p < 0.001) and nitrogen addition resulted in significantly higher ratio values (Table 3).

Tolerance Index
Tolerance index values (aerial biomass in contaminated soil/aerial biomass in control), complemented the results of physiological and growth parameters. Cr(VI) increasing soil concentrations resulted in lower tolerance index values (p < 0.001) and nitrogen addition had a similar effect (p < 0.001). It seems that nitrogen addition resulted in higher plant growth potential in the control treatment, that was abruptly limited from the toxic effect of Cr(VI), even from the lower level of Cr(VI) applied to the soil (Figure 3).

Tolerance Index
Tolerance index values (aerial biomass in contaminated soil/aerial biomass in control), complemented the results of physiological and growth parameters. Cr(VI) increasing soil concentrations resulted in lower tolerance index values (p < 0.001) and nitrogen addition had a similar effect (p < 0.001). It seems that nitrogen addition resulted in higher plant growth potential in the control treatment, that was abruptly limited from the toxic effect of Cr(VI), even from the lower level of Cr(VI) applied to the soil (Figure 3).

Phosphorus in Plant Tissues
In aerial plant tissues phosphorus content was measured and Duncan post hoc test results indicated that aerial tissue P concentrations increased (p < 0.001) with rising Cr(VI) soil concentrations, while nitrogen addition resulted in lower aerial tissue P concentrations when compared to the no N treated plants (p = 0.034) (Table 4). However, it must be noted that when the N effect was compared between same Cr(VI) additions, no differences

Phosphorus in Plant Tissues
In aerial plant tissues phosphorus content was measured and Duncan post hoc test results indicated that aerial tissue P concentrations increased (p < 0.001) with rising Cr(VI) soil concentrations, while nitrogen addition resulted in lower aerial tissue P concentrations when compared to the no N treated plants (p = 0.034) (Table 4). However, it must be noted that when the N effect was compared between same Cr(VI) additions, no differences were evident. For potassium content, despite the fact that the effect of Cr(VI) and N addition were significant (p < 0.001 and p = 0.002 respectively), the trend was not clear and further data are required to reach conclusive results (Table 4).

Discussion
Cr(VI) levels in root tissues were orders of magnitude higher compared to Cr(VI) concentrations found in aerial tissues, especially when plants were treated with the highest Cr(VI) concentration and fertilized with nitrogen. Increased Cr(VI) concentrations in root tissues have been noticed for a series of plant species, where plants limit the translocation of potentially toxic elements to the aerial plant tissues [2,10,11,15,45]. The physiological parameters studied (chlorophyll content and photosynthetic rate) were significantly affected with increasing Cr(VI) concentrations, while nitrogen amendment had a positive effect. Reduced values of total chlorophyll content, photosynthetic rate, and impediment of plant growth due to Cr(VI) stress have been documented for several plant species [2,8,20,26,45,46].
Values of all the parameters relevant to plant growth (aerial fresh weight, aerial dry weight, root dry weight, plant height, and leaf area) were significantly affected with rising Cr(VI) concentrations; nitrogen addition partly alleviated Cr(VI) toxic effects. Cr(VI) is known to impede several processes essential for plant growth such as photosynthesis, mineral uptake, enzyme and gene function, that inevitably result in reduced plant growth [6,11,45]. According to Kale et al. [47], plant growth of hydroponically grown P. oleracea was severely affected by increasing Cr contents, while plant accumulated significant amounts of Cr compared to other species (up to 190 mg kg −1 dry biomass). Various research articles have reported that nutrient addition to the growth medium alleviated to some degree Cr(VI) stress effects, as was the case with Arabidopsis thaliana [48,49]. In these investigations, the effect of N was non-significant, but the experimental settings were very different from ours (i.e., seedlings were watered with nutrient or Cr(VI) solutions in soilless culture). Contrary to such reports, our findings indicate that nitrogen amendment can support the growth of plants under Cr(VI) stress and partially compensate for the negative effects of Cr(VI) on plant physiological and metabolic processes. Leaf growth characteristics were proposed as bio-indicators of heavy metal stress. Cr stress is known to result in reduced leaf area, leaf size, and total leaf number per plant [2,45,50,51]. In the present experiment, a series of parameters such as leaf area and the ratio of leaf weight/total aerial weight were significantly affected with increasing Cr(VI) concentrations. Nitrogen amendment partly alleviated the effects of Cr(VI) stress. In parallel to the leaf growth restriction, significantly lower water content was noticed in above ground tissues. These results are in accordance with other works supporting that toxic effects of Cr(VI) in root tissues, alterations on the membrane structure of stomatal guard cells and the reduced diameter of tracheary vessels under Cr(VI) stress are the main factors that limit the water supply to aboveground tissues and therefore inhibit plant growth [2,51]. It seems that root tissues are the most affected plant parts since heavy metals are usually accumulated in higher amounts in roots compared to other plants parts. This was also the case in the study of Kale et al. [47], who recorded higher amounts of Cr in root tissues, followed by a reduction in root length with increasing Cr content in nutrient solution. Similar results were reported by Dwivedi et al. [52] who evaluated two Portulaca species (e.g., P. tuberosa and P. oleracea) for their phytoremediation capacity of multiple heavy metals (e.g., Cu, Ni, Hg, and Pb) and suggested that roots accumulated the highest amounts of metals, followed by stems, leaves and flowers, regardless of the studied metal. Based on the findings of Anandi et al. [53], this selective accumulation of heavy metals in plant tissues could be due to differences in tolerance to toxic effects, as aerial tissues are more susceptible to stress than roots.
According to the literature, Portulaca species have been reported for the phytoremediation of heavy metal-polluted soils, since the species seems to be tolerant to toxic effects of increased contents of various metals. For example, Deepa et al. [54] suggested the efficiency of P. oleracea stem cuttings in removing Cu from two different types of soils (e.g., Alfisol and Vertisol), while plant uptake was higher for the Alfisol due to the lower availability of Cu in this particular soil type. Moreover, it is worth to highlight the potency of Portulaca species to hyperaccumulate different heavy metals, e.g., Cd, As and Cr; this indicates the presence of efficient defense mechanisms that alleviate heavy metal toxic effects [55]. The suggested mechanisms for stress alleviation include the biosynthesis of osmoregulators such as proline or the induction of antioxidant enzymes, e.g., guaiacol peroxidase (GPX) [47]. Finally Yang et al. [56] reported that purslane above and below ground plant parts showed a very high concentration in various trace elements, including chromium, and further suggested the use of the species as a potential biomonitor or phytoremediator.
Cr(VI) also limits the uptake of N, P, K, Mn, Fe, Cu, Zn and S. It is referred that Cr(VI) root uptake is mainly performed by phosphate and sulphate transporters due to the structural similarity of Cr(VI) to phosphate and sulfate ions [45,57]. Results of de Oliveira et al. [58] indicated that increasing Cr(VI) concentrations resulted in higher sulfate root uptake and elevated sulfur in aerial plant tissues. Elevated phosphorus plant tissue content exerts positive effects on enzymes involved in Cr(VI) reduction [59] and increased P uptake under Cr(VI) stress was noticed in Citrullus vulgaris [60]. In Arabidopsis thaliana seedlings, high phosphorus concentrations in plant tissues resulted in significantly reduced Cr(VI) tissue concentrations [48]. On the other hand, Brassica napus plants subjected to oxidative stress recorded higher phosphorus cell content and results indicated that elevated P content resulted in lower ROS stress [61]. These results are in accordance with the present experimental results, where purslane seemed to selectively absorb soil phosphorus when under Cr(VI) stress and nitrogen amendment had a positive effect on P accumulation in purslane aerial tissues with increasing Cr(VI) concentrations.

Conclusions
• Cr(VI) contents in root tissues were orders of magnitude higher than the concentrations found in aerial plant tissues. • All physiological and growth parameters measured were severely affected and nitrogen in all cases resulted even partially in Cr(VI) stress alleviation. • Under Cr(VI) stress purslane plants selectively accumulated phosphorus in aerial plant tissues. • Cr(VI) stress resulted in lower water content in aerial plant tissues. • Added N did not result in increased Cr(VI) content in aerial biomass compared to same Cr(VI)-amended treatments without N; however, the fact that added N improved plant's growth and physiological functions even when exposed to high Cr(VI) soil concentrations, means that sufficient N fertilization may be a satisfactory treatment to increased purslane tolerance against Cr(VI) toxicity.
• On the same lines, added N makes purslane a species to be further considered for phytoremediation of Cr(VI)-laden soils; however, we acknowledge that more research is necessary before conclusive decisions may be drawn. This funding is highly appreciated.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The study did not report any data.