1 Introduction

Pollution of irrigation water may affect both the biotic and abiotic components of the ecosystem (Qadir et al. 2010). Farmers in developing countries who require water for irrigation often have no other choice than using wastewater (Keraita et al. 2008; Liu et al. 2005). The wastewater irrigation provides a valuable source of plant nutrients and organic matter needed for maintaining fertility and productivity levels of the soil (Qadir et al. 2010). High concentrations of heavy metals inhibit plant growth and development and cause various morphological, physiological, and biochemical responses (Song et al. 2014; Ahmed and Slima 2018). The degree of plant damage depends on the plant species, the concentration of toxic ion, its stage of growth, climate, and soil conditions (Qadir et al. 2010). It was reported that high concentrations of heavy metals in wastewater affect many physiological and biochemical processes and inhibit plant growth and development (John et al. 2009).

Most of the peri-urban lands are contaminated with pollutants, including copper (Cu), zinc (Zn), lead (Pb), cadmium (Cd), nickel (Ni), and mercury (Hg) (Singh and Kumar 2006) especially in the arid and semi-arid regions (Mojid et al. 2019). The discharges of wastewater is the most hazardous when the heavy metals exceed allowable concentration limits (Barakat 2011). The toxic effects, environmental stability (Esmaeilzadeh et al. 2016), and high potential for being absorbed and bio-accumulated in many aquatic species are important aspects of heavy metals (Esmaeilzadeh et al. 2017). Long-term irrigation with sewage effluent was shown to increase the amount and availability of heavy metals in soils (Liu et al. 2005). Soil fertility and plant growth can be enhanced with properly managed wastewater irrigation through increasing levels of soil organic matter and plant nutrients which may eliminate the need for commercial fertilizers in cropland (Mojid et al. 2019; Eid 2019). To alleviate the problems of land salinization and the contamination of soils with heavy metals, a group of plants has been identified such as halophytes, salt-tolerant plants, and heavy metal hyperaccumulators which can extract heavy metals from the soil (Moray et al. 2015). In coastal saline areas, heavy metal pollution could only be remediated by using halophytes that are probably the only candidate for phytoremediation of heavy metal–polluted saline soils (Sharma et al. 2010; Kumari et al. 2019). Halophytes and hyperaccumulators are found in a diverse range of angiosperm families and use similar mechanisms to combat the salt and heavy metal–borne stresses (Moray et al. 2015).

Salicornia europaea L. is an annual halophyte from the Chenopodiaceae family (Piernik et al. 2017) and one of the plants that has the highest salt tolerance (greater than 1 M NaCl) in the world (Rozema and Schat 2013; Zare-Maivan et al. 2015) and therefore well adapted in an environment where the level of soil salinity is as high as half-strength of seawater (Ebadi et al. 2018). It is widespread in Urmia Lake, Iran (Zare-Maivan et al. 2015), Europe, South Africa, South Asia, and North America and prevalent in salt-affected areas near coastlines, tidal floodways, salt lakes, and brine springs (Piernik et al. 2017). Since S. europaea dominates arid and saline soils of retreated Urmia Lake, its cell membrane integrity and antioxidant potential to acclimated to such conditions (Zare-Maivan et al. 2015). These species can survive in saline environments due to physiological and biochemical adaptations (Smillie 2015). Enzymatic and non-enzymatic antioxidants are important in plant defense under heavy metal stress (Kandziora-Ciupa et al. 2013). Previous reports have shown that the content of free proline depends on the type of metal and its concentration (Sharma and Dietz 2006) and plant species and varies between organs (Kandziora-Ciupa et al. 2013).

Long-term heavy metal exposure can enhance ethylene production and induce oxidative stress in plants (Thao et al. 2015). Higher ethylene production is due to the peaked expression of ethylene-responsive genes (Sharma et al. 2020). Higher production of reactive oxygen species (ROS), induced by some heavy metals, causes oxidative stress (Mittler 2002; Sytar et al. 2018). Heavy metals lead to damaging effects such as photochemical reactions, chlorophyll degradation, enzyme inhibition, disruption of membranes integrity, and reduction in metabolic efficiency and carbon fixation (Liu et al. 2005; Seregin and Ivanov 2001). Antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) along with the non-enzymatic antioxidant are the major components of ROS-scavenging system (Babaei et al. 2017; Seyed Sharifi et al. 2017). In non-hyperaccumulator plants, heavy metals at high concentration like Zn in wheat (Li et al. 2013) and Cu in Solanum nigrum (Fidalgo et al. 2013) led to an inhibition of the antioxidant enzyme. Halophytes are expected to be more capable to cope with heavy metal stress than common plants (Wu et al. 2017) due to a more efficient antioxidant system (Perez-Romero et al. 2016). For example, halophyte Salicornia brachiata can upregulate the activity of CAT and SOD and have lower damage to lipid membrane from ROS in response to heavy metals such as Cd, Ni, and arsenic (As) (Sharma et al. 2010; Wang et al. 2014).

There has been a continuing interest in searching for tolerant native plants to heavy metals. It remains to be further investigated how these metal ions affect plant growth at different developmental stages. Recently, the responses of plants to individual metal were extensively studied (Pedro et al. 2013; Perez-Romero et al. 2016). However, in natural soil-plant systems, they often have to face multiple metal stresses, and the interactive effects of more elements should be studied (An et al. 2004). Consequently, the present study aimed to evaluate the effects of time and duration of heavy metal–polluted wastewater application on the growth parameters (nodes on the main stem, number of pairs of side branches, diameter of the branched area, the plant biomass), enzyme activities, proline, soluble sugars, chlorophyll, and protein contents of Salicornia europaea in coastal areas of Urmia Lake. We hypothesized that the native S. europaea exhibits optimum growth and physiological parameters in urban and industrialized areas with respect to effectively reduce pollution of irrigation water.

2 Materials and Methods

2.1 Study Area

The study site is in the Urmia region (37°42′ N, 45° 19′ E), located in the north west of Iran (Fig. 1). The area is characterized by average annual rainfall of 300–700 mm; average annual temperature is −5 °C to 10 °C and has cold winters and hot summers. This study was conducted at coastal areas of Urmia Lake irrigated with the water contaminated by domestic and industrial effluent.

Fig. 1
figure 1

Location of the sampling sites

Full size image

2.2 Treatments and Experimental Design

The experiment was conducted in a split-plot design with four replications for each treatment. The main-plot factor of the experiment includes control (freshwater irrigation) and wastewater irrigation, and sub-plot factors include the irrigation with sewage at three stages, i.e., short term (20 days after transplanting, up to the vegetative growth stage in 14 August 2019), average term (30 days after transplanting, up to the flowering stage in 24 August 2019), and long term (40 days after sowing up to the reproductive stage), and two sampling times, i.e., 2 days and 4 days after irrigation with sewage. Indeed, wastewater quality parameters change over time due to floating/settling according to the type of media (Sirianuntapiboon and Kongchum 2006; Cortes-esquivel et al. 2012). Each plot consisted of 3 rows with 100-m long. Seeds were sown in 10 July and transplanted in 24 July 2019. Irrigation was done to maintain soil moisture above 80% field capacity. The amount of irrigation water was obtained based on Benami and Ofen (1984).

2.3 Soil and Water Analysis

The soil of the site was clay loam soil (21% sand, 48% silt, and 31% clay) with EC 33.8 dS m−1, pH 8.81, phosphorus 3.7 mg kg−1, and nitrogen 0.01%. Minimum and maximum values of heavy metals in sewage water during the 2019 are shown in Table 1. Farmers in these areas who require water for irrigation often use sewage, and the process is going on for about 30 years.

Table 1 Mean, minimum, and maximum values and standard deviation of heavy metal concentrations in sewage (n = 4)
Full size table

2.4 Plant Analysis

The number of pairs of side branches, nodes on the main stem, stem elongation, diameter of the branched area, and root length was recorded during the growth period. The concentrations of chlorophyll and carotenoids were calculated using the Arnon (1949) method. The total soluble protein content was determined by the method of Bradford (1976). To extract protein, 0.2 g of the plant fresh tissue was crushed by using liquid nitrogen, followed by adding 1 mL of buffer Tris-HCl (0.05 M, pH = 7.5). The obtained mixture was centrifuged for 20 min at 16000×g (13,000 rpm) at 4 °C, and then, the supernatant was utilized for enzyme activity measurements. Further, CAT, POD, and PPO activities were assayed according to Kar and Mishra (1976). Soluble sugars were measured according to Dubois et al. (1956). Leaf proline content was determined based on Bates et al. (1973). Relative water content (RWC) was calculated based on Tambussi et al. (2005). Electrical conductivity was estimated by the method of Jodeh et al. (2015).

2.5 Statistical Analysis

Analysis of variance (ANOVA) and means comparison on data were performed using the SAS 9.1 statistical package program. The least significant difference (LSD) method was used to test the significant differences between means comparison of main effects and interactions.

3 Results

3.1 Morphological Traits

Increasing the time exposure to wastewater improves the diameter of the branched area, but did not present a significant change in response to heavy metals at flowering and reproductive stages (Table 2). The nodes on the main stem and the number of pairs were lower in the reproductive stage than the flowering stage. Time had a significant effect on a number of pairs and the diameter of the branched area.

Table 2 Growth parameters of Salicornia europaea as affected by time and duration of wastewater application
Full size table

3.2 Biomass Components

As presented in Table 3, with wastewater irrigation, up to 16% increase in shoot length is observed, and the longest shoot is recorded at the reproductive stage. Root length was not significantly affected by wastewater irrigation, whereas it significantly decreased at reproductive stage. The highest plant biomass is found in plants irrigated with sewage at the reproductive stage (Table 3). Under the long-term duration of wastewater, aboveground dry biomass of S. europaea reached to 780 mg per plant. At the reproductive growth stage, root biomass of Salicornia reached to the highest weight per plant.

Table 3 Root and shoot length and plant biomass (dry weight) of Salicornia europaea as affected by time and duration of wastewater application
Full size table

3.3 Photosynthetic Pigments and Total Soluble Protein

This study shows that the application of wastewater caused a significant increase in chlorophyll-a, chlorophyll-b, and total chlorophyll compared with the control but 40 days after sowing (reproductive stage) produced a severe reduction in chlorophyll-a, total chlorophyll, and carotenoid by 49%, 44%, and 153%, respectively, than the flowering stage (Table 4). Chlorophyll-a and total chlorophyll increased significantly at 30 days after sowing in plants exposed to wastewater but, at the reproductive stage, 19 to 37% and 14 to 43% reduction was noted in chlorophyll-a and total chlorophyll, respectively, in comparison with the control. The reduction of total chlorophyll was less pronounced in chlorophyll-b than in chlorophyll-a. Chlorophyll-b was 37% lower than the control for longer time (40 days after sowing) of wastewater exposure. A significant increase in leaf total soluble protein was obtained from wastewater-irrigated Salicornia at the reproductive stage. The content of soluble protein was the lowest in untreated control plants after 20 days of sowing (vegetative growth stage).

Table 4 Chlorophyll content, carotenoid, total soluble protein and electrical conductivity of Salicornia europaea as affected by time and duration of wastewater application
Full size table

3.4 Soil Electrical Conductivity

In the present study, although there was no significant effect of time on electrical conductivity (EC), a significant interaction between time and irrigation is observed when wastewater treatment is applied (Table 4). During a growth period of plants, the level of lipid peroxides improves with an increase in the time of exposure to wastewater (Table 4). Long-term expose to wastewater led to 11% increase in EC level compared with the control.

3.5 Proline and Soluble Sugars

The results presented in Fig. 2 show that proline and soluble sugars were significantly increased in leaves of Salicornia plants exposed to wastewater, and they were higher in the reproductive stage within 4 days after irrigation with sewage. Under wastewater irrigation, proline content at the flowering and reproductive stage increases by 33% and 59%, respectively, compared with the control, but at vegetative stage, proline content increases to 5% compared with the control (Fig. 2A).

Fig. 2
figure 2

Proline (A) and soluble sugars (B) of Salicornia europaea as affected by time and duration of wastewater application. Fast, 20 days after sowing (at vegetative stage); average, 30 days after sowing (at flowering stage); long term, 40 days after sowing (at reproductive stage). Each bar indicates mean (n = 4) ± standard error. The different alphabets on bars indicate significant difference (P ≤ 0.05) using LSD

Full size image

In control plants, the level of soluble sugars is markedly decreased, in 4 days after irrigation (Fig. 2B). The accumulation of soluble sugars in the leaves was observed as a response to the long-term effect of wastewater, and maximum level was reached at 4 days after irrigation which was 63% higher than at control.

3.6 The Activity of Catalase, Peroxidase, and Polyphenol Oxidase Enzymes

Activities of antioxidant enzymes are determined over an experimental period in control and wastewater-treated leaves (Figs. 3 and 4). In control plants, the tendency of PPO activity ascended at flowering stage and then declined in 4 days after irrigation of reproductive stage, while POD activity fluctuated in different time and duration, and CAT activity was dropped slightly. In plants growing in the wastewater with excessive heavy metals, the lowest level of CAT, POD, and PPO was observed for the first time in the vegetative stage, but they were still higher than in controls. The data showed that CAT activity increased between 5.5 and 155% in plants treated with wastewaters. The highest CAT activity was observed at the reproductive stage. At this time, CAT activity is approximately 154% higher than in the control plants (Fig. 3A).

Fig. 3
figure 3

Catalase (CAT, A) and peroxidase (POD, B) of Salicornia europaea as affected by time and duration of wastewater application. Fast, 20 days after sowing (at vegetative stage); average, 30 days after sowing (at flowering stage); long term, 40 days after sowing (at reproductive stage). Each bar indicates mean (n = 4) ± standard error. The different alphabets on bars indicate significant difference (P ≤ 0.05) using LSD

Full size image
Fig. 4
figure 4

Polyphenol oxidase enzymes (PPO, A) and RWC (B) of Salicornia europaea as affected by time and duration of wastewater application. Fast, 20 days after sowing (at vegetative stage); average, 30 days after sowing (at flowering stage); long term, 40 days after sowing (at reproductive stage). Each bar indicates mean (n = 4) ± standard error. The different alphabets on bars indicate significant difference (P ≤ 0.05) using LSD

Full size image

Plants exposed to short- and long-term duration did not show significant changes in POD, indicating that the high metal concentrations did not affect the activity of POD at any time (Fig. 3B). At 4 days after irrigation of the flowering and reproductive stages, a 15–56% higher than control of POD was obtained. The maximum increase in POD activity was 32.4% during 4 days after irrigation at the reproductive stage, 16.7% during 4 days after irrigation, and 16.2% during 2 days after irrigation at the flowering stage. The activity of PPO was increased with the wastewater irrigation, but significant changes were not observed at the vegetative stages (Fig. 4A). The enzymatic activity observed at 4 days of irrigation was higher than 2 days of irrigation.

3.7 Relative Water Content

Irrigation with wastewater results in considerable negative water potential within 4 days after irrigation of flowering stage when compared with the untreated control (Fig. 4B). In control plants, RWC was high and then remained unaltered when the plants were irrigated with wastewater for the first time. Long-term exposure to wastewater caused only minor alterations in RWC. RWC in Salicornia was not duration- and time-dependent in wastewater irrigated plants, and there is a gradual decrease in RWC in plant exposed to wastewater at the vegetative and flowering stage. At the 4 days after irrigation of the reproductive stage, RWC considerably decreased by 5.4% in comparison with the control.

4 Discussions

Salicornia plants exposed to time and different duration of heavy metals exhibited morphological, physiological, and biochemical changes. The metal iron (Fe), Cu, Ni, and Zn are essential for plants but are also toxic when found in high concentrations (Kranner and Colville 2011; Yang et al. 2012). The presence of unbalance nutrients can disturb the uptake of necessary elements and consequently plant growth (Mallick 2004). Wastewater-treated plants show a healthy morphology, indicating the non-toxic concentrations of the metals. Based on observed results in response to higher concentrations of Cu-, Cd-, Pb-, and Ni-treated plant, the higher diameter of the branched area, shoot length, and shoot weight may be due to the sufficiency of Fe and Zn in the wastewater. In general, the root-shoot transfer of Zn and Cu was higher than that of Cd, Pb, and Ni (Eissa and Negim 2018). The gradual supply of plants with Zn, Fe, and other heavy metals occurred when plants exposed to the average term duration of wastewater at the flowering stage. Reduction in dry matter yield of Salicornia plants at a higher concentration of heavy metals was observed by Sharma et al. (2011) due to Ni, Ozawa et al. (2009) due to Cd, and by Sharma et al. (2010) due to Cd and Ni. Hatata and Abdel-aal (2008) stated that a high ratio of dry mass to fresh mass is a criterion for stress response, which is indicative of the whole-plant level. Some researchers have shown that biomass estimations have been confined to aboveground biomass and the root system biomass can comprise 30–80% of the total plant biomass (Eltaher et al. 2019). It is interesting to note that higher shoot to root growth ratio occurred in plants that had been treated with Pb (Nicholls and Mal 2003). With the development of plant roots during the growing season, Pb and Cd were uptake much more by Salicornia plants. These above adaptions and improvements make the soil suitable for the growth of healthy plants (Farid et al. 2013). The inhibitory effect of heavy metals on root length seems mainly due to the reduced water uptake (Farid et al. 2013) and reduction in mitotic cells in the meristematic zone of the root (Kabir et al. 2010). Also, it may be correlated with the metal-induced inhibition of the photosynthetic process (Hatata and Abdel-aal 2008) and the respiration in the shoot system and protein synthesis in the root (Drazkiewicz and Baszyński 2005). Due to the presence of large quantities of Ni and Cd in the wastewater (Table 1), only minor amounts of Fe and Zn are translocated to the shoots, and the excess of Fe in roots of plants hinders the development of cells and causes higher plant biomass (Adamski et al. 2012). Nicholls and Mal (2003) also observed that the cells of roots are affected more severely by the exposure to Cu than are other parts of the plant. Thus, if Cu damaged the roots of the treated plants more severely, the roots may not have been able to support the growth of the shoot.

Heavy metals observed in irrigation treatments (Table 1), as micronutrients essential for the plant growth and development, could lead to inhibition of photosynthesis when they were greater in the reproductive stage. It seems that during the flowering stage in Salicornia, the optimum of the metal uptake occurs, causing increased chlorophyll content. During the reproductive stage, where the heavy metals were the strongest inducer, a content of chlorophyll-a, chlorophyll-b, and total chlorophyll was decreased in the leaves of plants. However, the negative effect of heavy metals on chlorophyll-a fluorescence, electron transport, and light-dependent reactions of photosynthesis depend on metal concentrations and exposure time (Hajihashemi et al. 2020). Moreover, the long-term exposure of heavy metals inhibited the activity of PSI (Sharma et al. 2020). In this study, a high concentration of heavy metals at the end of growing stage inhibits uptake and transportation of other metal elements such as Zn and Fe by antagonistic effects, and therefore, the leaves lose the capacity of synthesis of pigments (Zengin and Kirbag 2007; Hatata and Abdel-aal 2008). The higher concentrations of Ni caused serious damage to chloroplasts, carotenoids, and chlorophyll and caused CO2 deficiency due to reduced enzyme activity in the Calvin cycle, as well as changes in the thylakoid membrane that affected the photosynthesis process (Pandey and Sharma 2002). Many studies have shown the influences of a positive effect of Zn and Cd metals on chlorophyll content, but high concentration of Zn in wastewater could be one of the main reasons for the decline in chlorophyll (Hajihashemi et al. 2020). Farid et al. (2013) stated that in Cd-treated wheat plants, increased leaf photosynthesis was observed due to the smaller leaf area. However, the results of this study could be interpreted as the effect of strong oxidation due to the metabolic perturbations and accelerated senescence (Zhou and Qiu, 2018), presence of Cd on the photochemical apparatus (Hatata and Abdel-aal 2008; Singh et al. 2012), and replaced Zn in chlorophylls (Mukhopadhyay et al. 2013) and probably due to the effect of interference of Cu with Fe uptake (Zengin and Kirbag 2007) and interaction of Pb to –SH group of enzymes of chlorophyll biosynthesis (Shu et al. 2012). Zengin and Kirbag (2007) reported that 0.4 mg L−1 of Cu resulted in a significant decrease of chlorophyll, and the minimum value of chlorophyll was observed when plants were exposed to 0.6 mg L−1 copper. The severe effects of Cu on the chloroplast in Salicornia were also reported from other plants (Andosch et al. 2012; Bazihizina et al. 2015). Moreover, numerous studies have reported a direct effect of Cu on the photosynthetic electron transport chain (Li et al. 2015), which may be associated with substantial stress response. Bibi and Hussain (2005) reported that Pb despite of low concentration proved more toxic in effecting photosynthetic pigments compared with Cu- and Cd-treated plants.

Visible symptoms of excessive heavy metals were accompanied by an increased total soluble protein, as an important indicator of the biochemical status of plants (Kandziora-Ciupa et al. 2013). This observation does not exclude the possibility of increased degradation of protein under excess heavy metals. Long-term exposure to wastewater irrigation showed significant increasing either enzyme activity or protein as well as inhibiting the production of H2O2 (El-Amier et al. 2019) to acclimate heavy metal toxicity through metal binding (Kandziora-Ciupa et al. 2013). In this respect, increasing the soluble protein contents was correlated with Zn concentration in Melia azedarach (Doganlar and Atmaca 2011), Pb concentrations in wheat and lentil (Mesmar and Jaber 1991), and Cd concentration in Oryza sativa (Singh et al. 2006).

The treatment with heavy metals can lead to the interruption of activities of several essential enzymes, various aspects of the photosynthetic processes, and water content of cells (Kabir et al. 2010; Verma and Dubey 2003). Heavy metal at high concentrations is a major factor that enhances membrane damage and cell structures (Ekmekçi et al. 2008), which in turn could explain a higher value of EC (Table 1). Deleterious effects of Cu (Nicholls and Mal 2003; Demirevska-kepova et al. 2004), Cd (Hatata and Abdel-aal 2008), and Pb (Kabir et al. 2010) on membrane permeability have been reported. Hatata and Abdel-aal (2008) reported that Cd treatment caused a declined in the efflux of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), and Fe from roots and leaves of plants. In this respect, Nicholls and Mal (2003) stated that Cu could damage the permeability of plasma membranes, which can lead to the leakage of potassium ions and other substances dissolved in the cell. These damaging membrane effects could explain, in part, the reduction of water content in sewage-treated plants affecting cellular turgor; it seems that such damages could be mitigated and repaired by antioxidative enzymes like CAT, POD, and PPO and after that increasing cell enlargement and plant growth. To impose environmental stresses, it is a general strategy to overcome oxidative stress (Verma and Dubey 2003).

The accumulation of proline and total soluble sugars is a well-known biochemical response in plant to HMs-induced stress conditions (Moghaieb et al. 2004; Li et al. 2013). The Cd may bind to the carbohydrates of cell walls and the thiol groups of enzymes that exchanges metal ion concentrations such as Mg, Zn, and Fe (Iannelli et al. 2002). Proline increases the tolerance of plants to heavy metals through metal chelation in the cytoplasm (Zengin and Kirbag 2007), scavenging hydroxyl radicals (Xing et al. 2010), inhibit lipid peroxidation (Li et al. 2013), maintaining the water balance (Mallick 2004), and the protection and stabilization of protein synthesis (El-Amier et al. 2019). According to Tripathi and Gaur (2004), proline reduced the uptake of Cu by plant cells, and probably stabilized the plasma membrane, thus mitigating the toxic effects of Cu to the plant. Induction or activation of proline biosynthesis enzymes along with decreasing proline oxidation under stress conditions may be caused by decreased utilization of proline in protein synthesis and enhanced protein turnover (Li et al. 2013; Xing et al. 2010). Protein degradation contributes to Ni- or Cu-induced proline accumulation (Lin and Kao 2007). Mehta and Gaur (1999) reported that the hierarchy of metal toxicity to Chlorella vulgaris was Cu > Cr > Ni > Zn. They suggested that the intracellular concentration of proline followed by a slow and steady increase with increasing concentration of Zn, Cu andchromium (Cr). The increase in proline in plants may be due to a decrease in the electron transport system activity leading to the accumulation of NADH and H+ (Sharma et al. 2010). Proline accumulation is related to the activation of the gene encoding the OAT in response to stress (ornithine d-aminotransferase) (Thompson et al. 1997), which is catalyzed by the PDH (proline dehydrogenase) in plant cells (Kavi Kishor et al. 2005), and it may be caused by diminished consumption of proline in protein synthesis and higher protein turnover (Delauney and Verma 1993). A previous investigation suggests that the OAT activity, rather than the PDH activity, is associated with the proline accumulation in Ni- (Lin and Kao 2007), Cu- (Ku et al. 2011), and Zn-treated (Li et al. 2013) plants.

Exposure to HMs has been shown to induce the displacement of nutritional elements (Rocha et al. 2014) and oxidative stress in plant cells due to the enhancement in ROS production, such as singlet oxygen (1O2), hydroxyl radical (HO•), hydrogen peroxide (H2O2), and superoxide radical (\( {\mathrm{O}}_2^{-} \)). Increased enzyme activity, as observed in our studies, was either due to the protective measure adopted by Salicornia plants against oxidative damage and/or could be an increased production of ROS (Yan et al. 2008). The change in the activity of antioxidative enzymes is depended on the plant species, age, and duration of the stress (Demirevska-kepova et al. 2004). At the reproductive stage, the activity of the CAT that was approximately four-fold increases than the controls (Fig. 3A). It can be explained by an increase in its substrate to maintain the level of H2O2 as an adaptive mechanism of the plants (Shu et al. 2012) and was related to increased synthesis of glucocorticoid receptor (GR protein) after Cd exposure (Hatata and Abdel-aal 2008). The increase in CAT activity due to Fe (Xing et al. 2010), Pb (Shu et al. 2012), Cd (Singh et al. 2012), Ni, and Cd (Pandey and Sharma 2002) stresses has been observed in other plant species. Higher activities of enzymes in Zn and Fe supplemented wastewater-irrigated plants indicate the specific role of Zn-Fe in promoting ROS detoxification through CAT and PPO enzymes. Cadmium toxicity is moderated trough Zn antioxidant properties (Aravind and Prasad 2005). Induction in peroxidase activity, as stress enzymes (Verma and Dubey 2003), has been documented under toxic levels of Pb, Cu, Cd, Zn, and Fe (John et al. 2009; Jucoski et al. 2013; Shu et al. 2012). Enhancement in the enzyme activity of CAT and PPO suggests that these enzymes serve as an essential defense tool to resist heavy metal–induced oxidative damage in Salicornia plants. Therefore, this also indicates that Salicornia may be more efficient in avoiding damage from heavy metals (Shu et al. 2012). So, under metal toxicity, the level of peroxidase activity has been used as the potential biomarker to evaluate the intensity of stress (Verma and Dubey 2003). The effects on CAT and PPO were similar. CAT eliminates H2O2 by breaking it down directly to form water and oxygen, however, less efficient than POD in H2O2 scavenging because of its low substrate affinity. Therefore, as long as the stress is not too intense for the plant’s defense capacity, the main response to heavy metals is an increase in POD activity (Zhang et al. 2007). On the contrary, the non-significant increase in POD activity in this study might be due to the increasing rate of ROS scavenging by other antioxidative enzymes (CAT, PPO) and the increased H2O2 due to the inactivation of POD. The binding of Cd metal with thiol groups could inactivate these enzymes (Hatata and Abdel-aal 2008). Delay in the elimination of H2O2 and toxic peroxides is mediated by POD and in turn an enhancement in the free radical-mediated lipid peroxidation under Pb toxicity (Verma and Dubey 2003). Enhancement of POD activity under Ni stress was explained by its role in building up a physical barrier against toxic metals entering the cell (Hegedüs et al. 2001) as well as in scavenging H2O2 (Yan et al. 2008). Therefore, PODs serve as a parameter of metabolism activity against metal toxicity.

This study suggested that heavy metals could disturb the plant-water relationships in Salicornia europaea at flowering stage. When proline and soluble sugar contents in the plant leaf reached their highest level, the water content of leaves decreased by 5.12% compared with the control. According to Pedro et al. (2013), Cd decreases the turgor potential and cell wall elasticity, which might result in a smaller of leaf cells with smaller intercellular spaces (Ghnaya et al. 2005). The reduction of xylem vessel area can lead to the reduction of water flow to the leaves (Hajihashemi et al. 2020). The decrease of weight observed at reproductive stage may support the depressive action of Cd on cellular turgor.

5 Conclusions

Based on these results, the tolerance of Salicornia europaea to heavy metal stress relies on physio-chemical mechanisms. Increasing heavy metal exposure at the reproductive stage significantly increases the development of plant biomass of plants, which experience destruction of photosynthetic apparatus. Additionally, the accumulation of total soluble protein, proline, and soluble sugar was also observed at the reproductive stage. The increased activities of antioxidant enzymes and proline may be attributed to the adaptive defense system of S. europaea against the toxic effect imposed by cadmium, copper, nickel, and lead. However, the protection of Salicornia against reactive oxygen species was insufficient, especially at the reproductive stage. The level of electrical conductivity and relative water content explained the greater tolerance of plants to heavy metal stress at the flowering than the reproductive stage. In S. europaea, catalase activity was much higher indicating that it is the most important reactive oxygen species (ROS)-scavenging enzyme. It can be concluded that proper management of wastewater irrigation and quality parameters is required to ensure the successful and long-term use of wastewater for irrigation. These plants grown in sewage-irrigated soils would be an environmentally friendly solution for disposal problems and effectively reduce food chain contamination and risks to human health.

Since roots are the first organ for the hyperaccumulation of different heavy metal ions, therefore, the changes of enzyme activities and metal accumulation in root system need to be determined, and intermittent use of clean water between wastewater irrigation will not only reduce the heavy metal load but could also enhance the soil fertility.