Effects of inorganic arsenic species on the antioxidant enzyme system of the Amazon Sword Plant (Echinodorus amazonicus Rataj)

Arsenic is one of the elements that can be found in water and has a high toxic effect, especially in inorganic forms. USEPA has classified arsenic and arsenic compounds as carcinogenic to humans (USEPA 1998a; WHO 2011; Jang et al. 2016; Sarkar & Paul 2016). Many terrestrial and aquatic ecosystems around the world are contaminated with arsenic from either anthropogenic or natural sources (Mandal & Suzuki 2002). Arsenic enters the ecosystem from natural sources such as rocks, hot springs, volcanic activities or anthropogenic activities such as agricultural activities, coal burning, oil refining, paint industry, copper smelting and mining activities (USEPA 1998b; USEPA 2002; Rose et al. 2007; Yabanlı 2010; Juncos et al. 2016).

Arsenic is found at higher levels in aquatic ecosystems than in terrestrial areas because it has a very high solubility in water and can derive from rocks rich in arsenic by the waves (Rose et al. 2007). Especially As⁺³ and As⁺⁵ forms of arsenic, which are generally found in the inorganic form in water resources, are more dominant (Viraraghavan et al. 1999; Ötleş & Çağındı 2010). For all organisms, including photosynthetic microorganisms, inorganic arsenic species are generally more toxic than organic arsenic species, and As⁺³ is 5–10 times more toxic than As⁺⁵ (Rossman 2003; Cordos et al. 2006; Jang et al. 2016).

The toxic effect of arsenic on plants depends on its oxidation state (Duman et al. 2010). As⁺³ and As⁺⁵ forms are bioavailable forms for plants and are easily taken up by plant roots (Rahman & Hasegawa 2011; Finnegan & Chen 2012). Given that As⁺⁵ is chemically similar to ⁠, it is easily incorporated into the cell by active uptake with phosphate transporters and prevents the passage of phosphate (Rosen et al. 2011). In this way, it can interfere with important cellular events such as ATP synthesis, oxidative phosphorylation and consumes cell energy (Duker et al. 2005; Tripathi et al. 2007). As⁺³ enters the cell via passive uptake pathways with aquaporins (Rahman & Hasegawa 2011). As⁺³, taken into the cell, reacts with the sulfhydryl groups (–SH) of enzymes and tissue proteins, causing inhibition of cellular function and death, thereby preventing tissue growth (Meharg & Hartley-Whitaker 2002). The interaction of As⁺³, with the cell membranes of plants by inactivating microbial enzymes results in necrosis in the leaves. On the other hand, As⁺⁵ does not react with sulfhydryl groups, so it has no direct effect on membranes, but it affects phosphorylation in mitochondria (Sizova et al. 2002). Inhibition of proteins, higher solubility, faster cellular uptake and slower excretion rate compared to arsenate increase the toxicity of arsenite (Rossman 2003; Gupta 2018; Coelho et al. 2020).

The accumulation of non-essential arsenic by plants negatively affects the processes of cellular metabolism, leading to the production of reactive oxygen species (ROS) and thus oxidative stress (Meharg & Hartley-Whitaker 2002; Yu et al. 2012; Andrade et al. 2016; Abbas et al. 2018). The excessive increase in the amount of ROS under stress conditions leads to lipid and protein oxidation in cell membranes, degradation of chlorophyll, inhibition of enzymes, damage to DNA and RNA, and as a result, cell death (Cakmak 2000; Mittler 2002; Leão et al. 2014; Andrade et al. 2016). ROS causes lipid peroxidation and the release of malondialdehyde (MDA), which is a toxic product (Hartley-Whitaker et al. 2001; Zhang et al. 2007). Evaluation of lipid peroxidation can be done by determining the amount of MDA (Hu et al. 2007).

Plants have a variety of enzymatic and non-enzymatic antioxidant systems to eliminate the increased ROS under oxidative stress conditions (Blokhina et al. 2003; Ren et al. 2021). Antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) increase under the oxidative stress conditions and have an important role in the scavenging of ROS (Das & Roychoudhury 2014; Sarker & Oba 2018).

Echinodorus amazonicus, also known as The Amazon Sword, is a submerged aquatic plant that can reach 40 cm in length and usually grows in wetlands (Leekroh 2017; AquaticCommunity 2020). In previous studies, heavy metal tolerance and accumulation of E. amazonicus were investigated (Sapci & Ustun 2012; Yang & Ye 2015). This plant was used as a test material in the present study such as heavy metal tolerance and their accumulation because it easily adapts to different environmental conditions and for this it is widely used by aquarists. This study was aimed to reveal the effects of As⁺³ and As⁺⁵ ions at different concentrations on the antioxidant enzyme activity, lipid integrity, chlorophyll and carotenoid content of E. amazonicus.

The samples of E. amazonicus were obtained from a company selling aquarium products (Aquainturkey company, Turkey). In order to remove the organisms (e.g., bacteria) on the plants, they were rinsed with distilled water and the deformed leaves were removed by sorting. For the adaptation of the plants to the laboratory conditions, a 45-day acclimatization period was applied in 40-l plastic aquariums in the Water Quality Laboratory (Faculty of Fisheries, Mugla Sıtkı Kocman University, Turkey). During the acclimatization period, water with a temperature of 24–28 °C and pH in the range of 7–8 was used. Photoperiod application was carried out with 14:10 light:dark (Yang & Ye 2015). 20% Hoagland solution was used as nutrient solution (Hoagland & Arnon 1950; Li et al. 2011).

After the acclimatization period, the plants were rinsed with distilled water before starting the experiments. Then, they were placed in plastic aquariums filled with 40 l of water, with their roots buried in the sand. Plants were exposed to 0, 6, 18 and 54 μM concentrations of As⁺³ and As⁺⁵ ions separately for 17 days in three repetitions. As⁺³ and As⁺⁵ ions were applied by taking certain amounts from stock solutions prepared from NaAsO₂, HAsNa₂O₄·7H₂O, respectively. Necrosis and chlorosis were observed in the leaves of the plants in the groups where high concentrations (54 μM) were applied, and therefore, the plants were harvested on the 17th day. In order to perform protein, chlorophyll, MDA and enzyme analysis, undamaged leaves were taken and stored in a deep freezer at −20 °C for further analysis. All analyses were performed considering fresh weight (F.W.).

MDA contents in the samples were measured according to the thiobarbituric acid (TBA) method determined by Heath & Packer (1968) and Du & Bramlage (1992). According to this method, 0.5 g of fresh leaf samples were weighed and homogenized in 10 ml of 0.1% TCA (trichloroacetic acid) and then centrifuged at 10,000 ×g (relative centrifugal force) for 10 min. After centrifugation, 1 ml of the supernatant was taken and 4 ml of 0.5% TBA prepared in 20% TCA was added. The mixture was kept in a water bath at 95 °C for 30 min, and the reaction was terminated by quickly placing it into ice. This mixture was centrifuged again at 10,000 ×g (relative centrifugal force) for 15 min. Absorbance values were recorded at 532 and 600 nm wavelengths in the spectrophotometer. The concentration of MDA was calculated from the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155 mM⁻¹ cm⁻¹. MDA content expressed as μmol g⁻¹ F.W.

0.5 g of leaf samples were homogenized in 5 ml of 50 mM potassium phosphate buffer (pH: 7) containing 1 mM of disodium EDTA and 2% (w/v) polyvinylpyrrolidone. These homogenates were centrifuged at +4 °C 10,000 × g (relative centrifugal force) for 20 min (Peixoto et al. 1999; Zhang et al. 2007). Obtained supernatants were used for enzyme analysis. The protein content of the samples was determined according to Bradford (1976) using bovine serum albumin as a standard. The results were expressed as mg g⁻¹. SOD activity was analyzed by measuring the ability of nitroblue tetrazolium to inhibit photochemical reduction at 560 nm, as determined by Beauchamp & Fridovich (1971). POX activity was determined by the change in absorbance of guaiacol oxidation at 470 nm according to the method determined by Chance & Maehly (1955). Buffer solutions were prepared by using the appropriate pH for each enzyme (50 mM phosphate buffer with a pH of 6.5 for POX, pH of 7 for CAT and pH of 7.8 for SOD, respectively). According to the method determined by the method of Aebi (1984), the decomposition of H₂O₂ was observed by decline at 240 nm for the CAT activity. POX and CAT activities are expressed as unit mg⁻¹ protein.

0.5 g of the plant leaves was extracted in 90% chilled acetone and chlorophyll and carotenoid contents were calculated according to the formulas determined by Arnon (1949) and Lichtenthaler (1987). Pigment contents are expressed as mg g⁻¹ F.W.

STATISTICA Stat 7.0 program was used for the statistical analysis of data obtained from the experimental groups, and analysis of variance (ANOVA) test followed by Post Hoc Tukey test were used to reveal the differences between the groups. A value of p < 0.05 was considered statistically significant.

Similar to this study, Meneguelli-Souza et al. (2016) reported that chlorosis and necrosis were observed at the end of 20 days in Eichhornia crassipes plant treated with 20 mg l⁻¹ of As⁺⁵, and there were significant decreases in photosynthetic pigment content compared to the control (a decrease around 51.17% for chlorophyll a, 42.72% for chlorophyll b, 49.04% for total chlorophyll and 7.77% for carotenoids). However, unlike the current study, researchers observed an increase in photosynthetic pigment contents in the group treated with 0.025 mg l⁻¹ of As⁺⁵. This increase can be due to hormesis, known as the stimulating effect of low doses of toxic substances on photosynthetic pigment contents (Callbrese & Baldwin 2003; Duman et al. 2010). In the current study, decreases were observed in the photosynthetic pigments content in the groups treated with the lowest concentration (6 μM). Divergence in the results may be due to the higher concentration used in the present study (6 μM) if compared with that reported by Meneguelli-Souza et al. (2016) (0.025 mg l⁻¹). Similarly, Malar et al. (2016), reported that the photosynthetic pigment contents of E. crassipes plant decreased with increasing Pb concentrations and that chlorosis and drying occurred in the plant after 10 days in the group treated with the highest concentration (1,000 mg l⁻¹). ROS that increase with oxidative stress cause DNA, protein and lipid damage, as well as necrosis and chlorosis in plant leaves by affecting metabolic processes and photosynthesis (Nath et al. 2014; da-Silva et al. 2017; Roychowdhury et al. 2018). The elements that form the basis of the pigment structure of the plants and heavy metals are replaced, and because the light uptake is prevented due to the deterioration of the pigment structure, photosynthesis cannot be performed and this causes the death of the plant (Prasad 1998).

As a result of the antioxidant enzyme analysis, SOD enzyme activity decreased in all As⁺³ applied groups compared to the control. The most significant decrease was 64.98% (0.43 ± 0.05 units mg⁻¹ protein F.W.) in the 54 μM As⁺³ groups. This decrease was found to be statistically significant when compared to the control (p < 0.05). The continuous decrease in SOD activity may be due to the fact that it is the first line of defense against the toxicity of ROS and the longer treatment period (Khan et al. 2009; Leão et al. 2017). An increase of 32.13% (1.61 ± 0.03 units mg⁻¹ protein F.W.) of SOD activity was observed in the group treated with 6 μM As⁺⁵ compared to the control. Furthermore, SOD activity was almost at the same level as the control in the group treated with 18 μM. A decrease of 26.96% (0.89 ± 0.09 units mg⁻¹ protein F.W.) was detected in the activity of SOD for the group treated with 54 μM As⁺⁵ compared to the control group (p < 0.05). The SOD enzyme is the first line of defense against environmental stress factors and converts the superoxide radical to hydrogen peroxide (Singh et al. 2018). The decreases observed in all the groups treated with As⁺³ in this study may be due to inhibition of the proteins (Srivastava et al. 2007). As⁺⁵ does not bind to protein groups. Therefore, the increase in the group treated with 6 μM As⁺⁵ may be due to the defense mechanism occurred under the oxidative stress conditions, and the decrease in the group treated with 54 μM As⁺⁵ may be due to the oxidation of proteins, damage to nucleic acids and enzyme inhibition caused by ROS, which increased due to the long treatment period (Sizova et al. 2002; Sharma et al. 2012).

For the CAT enzyme acitivity, the highest increase was around 185% (22.83 ± 0.30 units mg⁻¹ protein F.W.) in the group treated with 6 μM As⁺⁵ and followed by the group treated with 6 μM As⁺³ with an increase of 91.3% (15.31 ± 1.22 units mg⁻¹ protein F.W.). The enzyme activity decreased in other application groups, and the CAT activity in the group treated 54 μM As⁺³ was at a lower level than the control group, with a rate of 21.64% (6.27 ± 0.14 units mg⁻¹ protein F.W.).

The POX enzyme activity was increased around 201.5% (104.04 ± 2.62 units mg⁻¹ protein F.W.) in the group treated with 6 μM As⁺⁵ compared to the control group, and there was an increase of around 91.45% in the group treated with 6 μM As⁺³ compared to the control (66.28 ± 1.95 units mg⁻¹ protein F.W.) (p < 0.05). In the groups treated with 18 and 54 μM, the enzyme activity was higher than the control, but the enzyme activity decreased compared to the 6 μM applied group. In the samples treated with 54 μM As⁺³, a decrease of 21.29% (27.25 ± 1.78 units mg⁻¹ protein F.W.) was detected in the activity POX compared to the control group.

Similarly to this study, Tripathi et al. (2014) reported that SOD enzyme activity increased in the first 2 days and decreased in the following days in N. indica aquatic plant treated with different concentrations of As⁺³ and As⁺⁵ for 7 days. Unlike the current study, the decrease in SOD enzyme activity in all groups treated with As⁺³ may be due to the fact that it is the first line of defense against the toxicity of ROS and the longer treatment period (Khan et al. 2009; Leão et al. 2017). Also Farnese et al. (2013) stated that the SOD activity of the water lettuce (Pistia stratiotes L.) treated with different concentrations of As⁺⁵ (0, 5, 10, 15 and 20 μM) increased at the group of 10 μM and decreased at higher concentrations (15 and 20 μM). The increase in CAT and POX activities observed at low concentrations can be evaluated as the plant's ability to cope with arsenic stress by keeping the increased H₂O₂ radicals under control (Praveen et al. 2019). While SOD catalyzes superoxide radicals to H₂O₂ and O₂, CAT and POX enzymes break down H₂O₂ radicals into harmless molecules O₂ and H₂O (Zhang et al. 2007; Ighodaro & Akinloye 2018). Like the current study, da-Silva et al. (2017) found that CAT activity increased at low concentration (0.5 mg l⁻¹) and decreased at high concentrations (1, 1.5 and 2 mg l⁻¹) in Spirodela intermedia aquatic macrophyte, which they treated with As⁺⁵ for 24 h. Duman et al. (2010) found that there was a 65% increase in CAT enzyme activity at the end of the 6th day in the 1 μM As⁺³ group, and 84% increase in the 16 μM As⁺⁵ applied group at the end of the 4th day in duckweed (L. minor L.). In the present study, CAT activity increased around 185% in the group treated with 6 μM As⁺⁵ and around 91.3% in the group treated with 6 μM As⁺³. The increase or decrease level of antioxidant enzyme activities in plants under oxidative stress may vary according to metal and plant species, metal exposure time and concentrations (Radić et al. 2010). The decreases in the antioxidant enzyme activity at high concentrations are due to the fact that ROS, which increase with oxidative stress, bind to the active sites of proteins and inactivate enzyme structures (Khan et al. 2009; Tripathi et al. 2014; Leão et al. 2017).

As a result of the study, it was observed that there were morphological and physiological changes at high concentrations in E. amazonicus aquatic macrophyte treated to different concentrations of As⁺³ and As⁺⁵ ions. A significant decrease in protein, chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents was observed with increasing of arsenic concentrations. The antioxidant enzymes SOD, CAT and POX act as a defense mechanism of the plant against stress. The antioxidant defense system of E. amazonicus dealed with the oxidative stress conditions at low concentration (6 μM) of inorganic arsenic, but at high concentrations (18 and 54 μM), the defense system was not effective against increased ROS. The amount of MDA, which is an indicator of lipid peroxidation, increased in all the groups of treated with As⁺³ and As⁺⁵ ions. The increase in the As⁺³ group was higher than in the As⁺⁵ group. As⁺³ and As⁺⁵ ions were compared with each other and the toxicity of As⁺³ was higher. Compared to other studies, the decrease observed in SOD activities in all the groups treated with As⁺³ even at the lowest concentration (6 μM) shows that As⁺³ is more toxic, and the antioxidant defense system loses its effectiveness as the treatment time gets longer. Considering the natural environment conditions, it is suggested that plants will not survive if exposed to arsenic for a longer period of time. In conclusion, the findings of current studies contribute to the understanding of the effects of pollutants on living things in nature and the mechanisms of possible reactions to these effects, thus helping the survival of living organism in aquatic ecosystems. Since the consequences of adverse events occurring in aquatic ecosystems will cause the disruption of the natural balance, there may be danger to all the living organisms.

The authors would like to thank Dr Ümit ACAR (Çanakkale Onsekiz Mart University, Çanakkale, Turkey) and Dr Murat Can SUNAR (Mugla Sitki Kocman University, Mugla, Turkey) for their useful support in the trial design. The authors also appreciate Dr Daniela Giannetto (Mugla Sitki Kocman University, Mugla, Turkey) for her helpful comments on the manuscript. This study was supported by Mugla Sıtkı Kocman University Scientific Research Projects Coordination Department (BAP 15/242). This study reports part of the results obtained within the MSc thesis of Mr Idris Şener obtained at Muğla Sıtkı Koçman University, Graduate School of Natural and Applied Sciences. The current study was also presented as an oral presentation at the third International Symposium on EuroAsian Biodiversity (SEAB 2017).

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The authors declare that they have no conflict of interest.