A Study of the Biomarker Responses of Water Spinach Grown in Polluted Water

ABSTRACT

A pond ecosystem is one of the most important freshwater ecosystems. It serves as the home to various flora and fauna. Ipomoea aquatica, or water spinach, is a common pond water plant. It is consumed by people for its taste and health benefits. Pond water also serves as a water resource for everyday domestic activities. These pollute the pond water, endangering the health of the plants and fish in the pond. In our work, we have studied the quality of the water from used and unused ponds. This was correlated with the health quality and stress response of the plants. It was found that decreasing pond water quality leads to a decrease in the quality of aquatic plants.

1. Introduction

Ipomoea aquatica, or the water spinach, is a commonly available plant in the Indian subcontinent and is largely ingested as a green vegetable. It is native to the Indian subcontinent. It is a fast‐growing plant and thrives in a hot and humid climate. I. aquatica is an aquatic species that grows in small rivers and permanent ponds, or in very humid or temporarily flooded lowlands. They are low in calories and fats, but have a significant amount of vitamins, minerals, and antioxidants. I. aquatica is an excellent bioindicator of water pollution, because of its inherent properties of accumulating various water pollutants, and is therefore commonly used as an agent for phytoremediation [1].

Water pollution refers to the introduction of harmful substances, toxic chemicals, heavy metals, or other pollutants into water bodies. It can occur through various sources, of which industries are one of the main contributors. This contamination can have a detrimental effect on the well‐being of plants, human beings, and the ecosystem.

When this polluted water mixes with soil, it makes the soil contaminated. These contaminants have a significant effect on the plant, depending on the classification and level of contamination. Excessive heavy metals present in the water can become toxic for the plants [2]. As these metals cannot be broken down by plants, when these metals are present in high concentrations, they lead to increased oxidative stress, which in turn leads to cellular damage, which is manifested as higher malondialdehyde concentration and greater DNA damage, as reported in a study on the aquatic plants of water bodies of South India. Previous studies have reported such genotoxic and mutagenic effects in plants such as Allium cepa growing in the polluted waters of the Água Boa stream of Brazil [3, 4]. These contaminants can also alter the pH of the soil, which affects plant growth. The altered pH of the soil will affect the availability of certain elements, particularly the micronutrients for uptake by the plants [5]. In addition, water contaminated with high levels of nitrogen and phosphorous, often from fertilizers, can lead to nutrient imbalances. While this may initially promote growth, over time, it may result in poor root development and reduced overall plant health [6, 7].

Water pollution is a major concern for plant growth due to the essential role that water plays in the survival and development of plants. Water contamination poses significant risks to plant growth by introducing toxic substances, pathogens, and imbalanced nutrients into the environment. These issues can lead to reduced growth, poor health, and lower yields, making it a serious environmental and economic concern. The severity of these effects depends on the plant species, the contaminant class, and the accumulation of pollutants in the water. Thus, water pollution poses a significant global problem that requires holistic efforts to diminish its effects and protect water resources. In our present work, we have studied the consequences of water pollutants on the physiology of the water spinach, a common Indian aquatic plant.

2. Materials and Method

2.1. Area of Study

Plant specimens (leaves of the plant) along with water samples (five samples each) were collected from two ponds—Pond 1 from the Hedua area and Pond 2 from the Tala area, which were regularly used for domestic purposes. Plant specimens and a water sample from a pond not regularly used were taken as the control—Pond 3. The samples were collected during the dry season.

2.2. Total Solid and Total Dissolved Solid Content of Water

The total solid concentration of water was estimated by evaporating both investigational and control water samples in a pre‐weighed beaker, cooling, and weighing again. The total dissolved solids were estimated by evaporating both the test and control water samples' filtrate in a pre‐weighed beaker, cooled, and weighed again [8].

2.3. Dissolved Oxygen Content of Water

Manganous sulfate, potassium iodide solution, and concentrated sulfuric acid were added to both test and control samples. This was titrated against 0.025 N sodium thiosulfate with starch as an indicator. The dissolved oxygen (DO) content was measured [9].

2.4. Chemical Oxygen Demand of Water

Potassium dichromate, sulfuric acid, and silver sulfate were dissolved in test and non‐test water samples and refluxed for approximately 2 h. This was titrated against 0.1 (N) ferrous ammonium sulfate solution with two to four drops of ferroin as an indicator dye. The chemical oxygen demand (COD) was determined [10].

2.5. Phosphate Content of Water

To both test and non‐test water samples, ammonium molybdate and stannic chloride solution were added. The optical density value was evaluated at 690 nm. The phosphate concentration was calculated by graphical representation using a standard curve prepared using different concentrations of standard phosphate solutions [11].

2.6. Comet Assay

The plant leaves from all three ponds were taken, ground, and mixed with cetyltrimethylammonium bromide (cTAB) extraction buffer. It was then incubated at 60°C, followed by centrifugation to remove the pellet. Chloroform:isoamyl alcohol was added to the supernatant, followed by centrifugation, and addition of isopropanol to precipitate DNA after washing with 70% ethanol. The extracted DNA was visualized by ethidium bromide staining after running on an agarose gel [12].

2.7. Study of Malondialdehyde Concentration

The plant extract was prepared by crushing the plant specimen, centrifuging, and collecting the supernatant. To this extract was added 20% TCA, 0.5% TBA, and were homogenized. This liquid was boiled and then ice‐cooled. Then centrifugation was done, and the optical density of the supernatant was measured at 532 nm. The concentrations were found by graphical plotting using a standard curve prepared by using different concentrations of standard malondialdehyde [13].

2.8. Statistical Analysis

Triplicate experiments of all tests were performed. The standard deviation was determined, and Student's t‐test at p < 0.05 was applied to find out the statistical significance by Microsoft Excel through its built‐in Data Analysis Tool Pack after testing the normality of the data.

3. Results

3.1. Solid Content of Water

The amounts of total solids and total dissolved solids in the water of Pond 1 were 1000 and 2000 ppm, respectively. The amounts of total solids and dissolved solids in the water of Pond 2 were 1000 and 1000 ppm, respectively. The amount of the entire solid and total dissolved solids in the water of Pond 3 was 500 ppm in both conditions. Thus, the water of the used ponds showed significantly higher solid content as compared to the water of the control pond (Figure 1).

FIGURE 1.

Solid content of pond water.

3.2. Dissolved Oxygen Content of Water

The dissolved oxygen (DO) contents of water from Ponds 1, 2, and 3 were 3, 3.5, and 6.5 mg/dL, respectively. Thus, it was found that the dissolved oxygen content of the water of Ponds 1 and 2 was significantly reduced in comparison to the water of Pond 3 (Figure 2).

FIGURE 2.

Dissolved oxygen content of pond water.

3.3. Chemical Oxygen Demand of Water

The chemical oxygen demand (COD) of water from Ponds 1, 2, and 3 were 400, 350, and 10 mg/L, respectively. Thus, the COD of control pond water was about three to four times less than the water of Ponds 1 and 2 (Figure 3).

FIGURE 3.

Chemical oxygen demand of pond water.

3.4. Phosphate Content of Water

The phosphate contents of water from Ponds 1, 2, and 3 were 0.6, 0.4, and 0.05 mg/L, respectively. Thus, the phosphate content of water in Ponds 1 and 2 were about 10‐fold higher than the water from Pond 3 (Figure 4).

FIGURE 4.

Phosphate content of pond water.

3.5. Comet Assay

The amount of damage to DNA was studied. It was found that the DNA of the plants taken from Pond 1 had the highest DNA damage, followed by plants taken from Pond 2. The plant specimens from Pond 3 did not show any DNA damage (Figure 5).

FIGURE 5.

Estimation of DNA damage by comet assay (T = Pond 2, H = Pond 1, C = Pond 3).

3.6. Study of Malondialdehyde Content

The amounts of malondialdehyde in the plant specimens from Ponds 1, 2, and 3 were 2.5, 1.5, and 0.8 µmol/g, respectively. Thus, the plants growing in the water of Ponds 1 and 2 had three‐fold and two‐fold higher malondialdehyde concentrations than in plants growing in the water of Pond 3 (Figure 6).

FIGURE 6.

Malondialdehyde content of plant specimens.

4. Discussion

4.1. Solid Content of Water

It has been previously reported that regular use of pond waters for domestic purposes, as well as discharge of effluent, deteriorates the quality of water [14, 15]. Water from polluted ponds leads to decreased quality of both flora and fauna growing in such an ecosystem. Increased solid content of water reduces the sunlight penetration in water, thus reducing the rate of photosynthesis in aquatic plants. They also lead to water turbidity. In our work, there was increased solid content in the water of ponds regularly used for domestic purposes. This contamination of pond water leads to a decrease in the physiological functioning of the aquatic plants, which leads to a decrease in their oxygen production, which further leads to a deteriorating quality of the pond ecosystem

4.2. Dissolved Oxygen Content of Water

Dissolved oxygen refers to the level of free, unbound oxygen present in water. Oxygen remains in water in dissolved form as dissolved oxygen (DO). The concentration of dissolved oxygen is a vital water quality determinant because of its direct effect on the growth and metabolism of aquatic animals, as well as its indirect influence on the whole water quality. In other words, it is the amount of oxygen available for sustenance of aquatic life [16, 17]. Both water samples of the used ponds had low DO content, implying that the phytoplanktons of these ponds have a reduced photosynthetic efficiency, and the fish in these ponds have a low survival rate.

4.3. Chemical Oxygen Demand of Water

Chemical oxygen demand (COD) is often used as a measurement of organic pollutants in water, wastewater, and aqueous toxic wastes. COD is a vital determinant of water quality. It depends on the amount of oxidant consumed due to organic substances in the aquatic ecosystem. A higher COD value indicates higher dissolved organic matter in the pond water. Humic substances and surfactants are common pollutants [18]. In our study, we found higher COD values in the water of the used ponds as compared to the unused ponds, indicating that Ponds 1 and 2 had a high content of organic pollutants.

4.4. Phosphate Concentration of Water

The presence of phosphorus (P) in water bodies is the main reason for eutrophication. Presence of nutrients, which are an essential component of fertilizers are a major cause of eutrophication. Eutrophication leads to hypoxia, which in turn may cause increases in harmful algal blooms [19]. Phosphates are a vital component in synthetic detergents, as they are used as additives for enhancing the efficiency of detergents [12]. In our work, we found that the phosphate content of the water of the ponds used for domestic purposes was about 10 times higher than that of the water of the unused ponds.

4.5. Comet Assay

The comet assay is a technique to study the amount of DNA damage caused by exposure to genotoxic chemicals. Pollutants, particularly at high concentrations, can produce free radicals, which lead to DNA damage. The overabundance of reactive oxygen species results in a variety of damage, which can be detected, including DNA strand breaks, modification of nucleobases, DNA–DNA crosslinks, and formation of pyrimidine dimers in a eukaryotic cell [20]. Detergents are amphipathic molecules. This allows them to interfere with and break down biological membranes. Various water pollutants may lead to varied types of DNA damage, which are understood by studying the comet assay results [21]. The basis of the comet assay is the migration of cleaved DNA in an electric field, whereas the intact DNA remains within the nucleoid. An image of the comet's tail and nucleoid helps in the study of the amount of damaged DNA. A very recent review report studied the role of aquatic pollutants on aquatic life. It was found that these pollutants caused increased genotoxicity, which was studied through the comet assay [22]. In our work, we observed the highest DNA damage in the plants growing in Pond 1, followed by DNA damage in the plants growing in Pond 2. The plants growing in Pond 3 showed the least DNA damage. The pollutants in the pond water used for various domestic purposes included detergents and chemicals, which led to DNA damage in the plants growing in those ponds. The presence of pollutants was highest in Pond 1, which was reflected in the highest DNA damage to the plants growing in Pond 1 as compared to the other two ponds.

4.6. Estimation of Malondialdehyde Content

Exposure to varied pollutants leads to stress in plants, which increases free radical generation, leading to cellular damage. Malondialdehyde is a harmful byproduct of this cellular damage, and its increased concentration in plant tissues directly reflects the severity of pollution. Increase in malondialdehyde concentration is thus a biomarker of plant stress response [23]. In our work, there is a significant enhancement in the malondialdehyde content in the plants of used ponds as compared to the plants of unused ponds. The chemicals in the pond water lead to the stress response in plants.

5. Conclusion

Pond water serves as a regular water source for everyday domestic activities, especially in rural areas. This leads to the accumulation of detergents and toxic materials in the pond water. These pollutants are an important factor in the deterioration of the quality of the pond ecosystem. The aquatic flora and fauna are constantly exposed to these harmful chemicals, which leads to their cellular damage as well as enters the food chain. Constant exposure to toxic water pollutants leads to the retardation of the normal physiological functioning of the aquatic plants. This affects the survival of the aquatic fauna, which is dependent on the aquatic flora. This, in turn, leads to the loss of biodiversity of the aquatic ecosystem. Thus, public awareness should be generated against using pond water for domestic purposes. Water should be supplied to households for cleaning purposes so that pond water remains safe for the sustenance of plant and animal life.

Author Contributions

S.M.—conceptualization and design, supervision. A.C.—material preparation and data collection; writing – original draft. S.R.—material preparation and data collection; writing – original draft. S.N.—material preparation and data collection. S.P.—supervision, data analysis, preparation of final draft.

Funding

This research did not receive any specific grant from any funding agencies.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Most data generated during this study are included in this article. The rest of the data is available from the authors upon reasonable request.