Preliminary phytotoxicity of Mercury in conventional gold mining wastewater on Typha latifolia and Pistia stratiotes

Small-scale mining, or conventional mining, is quite common in Indonesia. Almost all of these artisanal mining activities disregard environmental safety around the mining areas. The current widely used gold extraction method for gold exploitation is the amalgamation method, which utilizes mercury (Hg). The use of this method can result in environmental pollution from mercury. Based on the impact of mercury that can contaminate the environment, it is necessary to treat gold mining wastewater before disposing of it into the environment. One alternative that can be used to treat gold mining wastewater is to use plants (phytotreatment). The aims of study was to observe the tolerance of Hg to Typha latifolia and Pistia stratiotes plants, The methods was call the Range Finding Test (RFT). The study was conducted using synthetic Hg at concentrations of 0.3, 0.7, 1, 3, and 6 mg/L and a control (0 mg/L) in a batch reactor. The reactors were added with soil up to 25% as a growing medium. The physical observations of plants were carried out for 28 days. Typha latifolia and Pistia stratiotes could both grow and thrive at a Hg content of 6 mg/L, according to physical observations of the plants. In the first seven days, Pistia stratiotes plants exhibited a stress reaction in the form of yellowing and falling leaves. Both plants had vigorous, extended root development in all concentrations. Based on these findings, phytotreatment can be utilized as an alternate therapy to lower the Hg levels in gold mine wastewater.


Introduction
Small-scale mining, often referred to as artisanal mining, is quite prevalent in Indonesia.However, almost all of these artisanal mining activities disregard the environmental safety surrounding the mining sites.The current methods of gold extraction widely used for industrial-scale gold exploitation are the amalgamation method and the cyanidation method.The amalgamation method involves using mercury for gold extraction, while the cyanidation method has been used for over a hundred years since gold production was developed.Nevertheless, environmentally unfriendly gold mining activities can lead to significant problems such as environmental pollution, especially from heavy metal waste like mercury (Hg).Hence, the government enacted Law Number 11 of 2017 concerning the Minamata Convention, followed by Presidential Regulation Number 21 of 2019, to reduce the use of mercury.The potential impact of mercury is the generation of toxic effects and its accumulation in the environment, and this heavy metal can have detrimental effects on health, such as respiratory, digestive, and kidney disorders.
Gold mine effluent must be treated before being released into the environment since mercury has the potential to damage the ecosystem.Examining plants to lower the amount of the heavy element mercury IOP Publishing doi:10.1088/1755-1315/1263/1/012049 2 in wastewater from gold mines is the strategy employed to accomplish this goal.Phytotreatment is an effective and environmentally responsible way to deal with environmental pollution, lessen the negative effects that pollution has on the ecosystem, and rehabilitate contaminated areas [1].Typha latifolia and Pistia stratiotes are two plants that can be used to eliminate the heavy element mercury.Pistia stratiotes have been demonstrated in numerous studies to be capable of removing radionuclides, organic compounds, and heavy metals like arsenic (Fe, Zn, Cu, Cr, and Cd) from water [2].Typha latifolia was planted on soil that had heavy metal concentrations of lead (Pb) and cadmium (Cd) added to it.After a particular amount of time, the amount of heavy metals in the soil and plant leaves was measured.The study's findings demonstrated that Typha latifolia was capable of amassing considerable levels of lead (Pb) and cadmium (Cd) in its leaves [3].
Based on the environmental contamination caused by mercury, it is necessary to treat the wastewater from gold mines before disposing of it into the environment.The method used to achieve this goal is to stuudy plants to reduce the concentration of heavy metal mercury in gold mine wastewater using phytotreatment methods.It is hoped that this research will determine the ability of the cat's tail plant (Typha latifolia) and water lettuce (Pistia stratiotes) to tolerate the concentration of heavy metal mercury in gold mine wastewater.

Preparation of synthesis heavy metal Mercury (Hg) solution
The first step in making a synthetic mercury heavy metal solution is carried out by weighing 0.135 grams of HgCl2 where the weight is obtained from the following calculation: ➢ HgCl2 has a molecular mass of 271.49➢ Hg has a molecular mass of 200.59 Next, the HgCl2 is dissolved in distilled water and 1 ml of HNO3 into a volumetric flask until the volume reaches 100 ml.In the synthesis of this synthetic Hg, a solution concentration of 1000 ppm or 1000 mg/L will be obtained [4].Then, dilution is performed to achieve the desired concentration, with the following calculation: Based on the calculation, 0.3 ml of the solution containing 1000 mg Hg/L is required.Then, distilled water is added to the volumetric flask until the volume reaches 1000 ml, resulting in a solution concentration of 0.3 mg Hg/L.This procedure is also carried out for other concentrations of synthetic Hg.

Preliminary stage
A Preliminary stage, such as propagation and acclimation, is required for this study.The propagation stage of plant breeding aims to increase plant populations while preserving their key traits.It also helps ensure the survival of the species.The acclimatization phase is used to assess the degree of these plants' environmental adaptation [5].
a. Propagation Typha latifolia and Pistia stratiotes were propagated by first adding water to the reactor, and then placing each test plant inside the reactor.The goal of this stage of propagation is to increase the quantity of Typha latiofolia and Pistia stratiotes plants that will be employed in this study.
b. Acclimation Each mature plant from the propagation stage was taken and placed in the acclimatization reactor to complete the preliminary stage for Typha latifolia and water lily plants.Obtaining Typha latifolia and Pistia stratiotes plants that have adapted to the media is the goal of this stage since they will be used in the range finding test and phytoremediation test.For research on Typha latifolia and Pistia stratiotes, only specimens that meet the requirements for plant analysis will be used.

Range finding test (RFT)
In this range finding test, variations in concentration were conducted to determine the critical concentration limit.The concentration variations in the gold mining wastewater can be obtained by diluting the wastewater and then testing it at the treatment plants.USEPA Guidelines Part 850.4400 states that the range finding test involves 5 concentrations, with the concentration range following a geometric series, including concentrations of 0 mg/L, 0.3 mg/L, 0.7 mg/L, 1 mg/L, 3 mg/L, and 6 mg/L.The plants used in this stage are the ones obtained from the previous acclimatization stage.The criteria for selecting plants in the RFT are the same as the criteria used in the acclimatization stage.The range finding test is conducted for 7 days, following the USEPA (1996) guidelines [6].However, if no changes occur in the plants within 7 days, the test period is extended by 24 hours.If the extended RFT period still does not cause any changes in the plants, it is further extended up to 14 days.The following is a description of the RFT reactor used.

Range finding test of Mercury
The Range Finding Test (RFT) in phytotreatment is conducted to determine the tolerance of the selected plants to the specific pollutants and to establish the concentration of pollutants that can be tolerated by the plants.In this study, a heavy metal RFT (Hg) was performed on Pistia stratiotes and Typha latifolia.Physical observations of the plants are crucial in phytoremediation as they provide information about the plants' ability to absorb and accumulate pollutants.Physical observations include plant growth, leaf count, offshoot count, and changes in biomass.
The toxic effects of the heavy metal Hg on plants include growth retardation inhibition, and premature aging.Hg heavy metal has been found to inhibit protein synthesis in plant leaves and reduce photosynthetic activity due to its strong affinity for sulfhydryl or thiol groups involved in enzymatic reactions.Generally, methylated organic forms of Hg are more toxic and easily accumulate due to their lipophilic properties compared to inorganic forms of Hg, leading to biomagnification of methyl Hg in the food chain.The accumulation levels in aquatic biota are much higher than the concentrations of Hg found in the water column [7].The RFT observations are carried out through physical plant assessments, including leaves, roots, offshoots, and plant moisture content.

(a) Pistia stratiotes
The Pistia stratiotes plant, also known as water lettuce or water cabbage, originates from Africa but is currently found in nearly all tropical and subtropical freshwater habitats.The observation of the Pistia stratiotes plant can be clearly seen in Figure 2. The construction of batch reactors at each mercury concentration (P1, P2, P3, P4, P5, P6) reveals varying responses from the plants, particularly in terms of their physical characteristics, as shown in Figure 2. Leaves are plant organs that undergo photosynthesis to produce food and energy.The process of photosynthesis helps improve the quality of air and water by absorbing carbon dioxide (CO2) and releasing oxygen (O2).In the observation of heavy metal mercury (Hg) in the leaves of the Pistia stratiotes plant during the RFT, two observations were conducted: the percentage of yellowing leaves and leaf width.Heavy metal mercury in its ion form (Hg 2+ ) can bind to proteins found in plant vessels, namely xylem and phloem vessels, causing leaf stomata to close and potentially obstruct the physical flow of water in the plant, disrupting mitochondrial activity, and inducing oxidative stress.This disturbance can affect the lipid biomembranes and cellular metabolism of the plant [8].
The yellowing of leaves in the early days may be caused by oxidative stress generated by Hg 2+ ions.Oxidative stress occurs when the amount of free radicals (molecules with excess electrons that are unstable) in a cell exceeds the cell's ability to neutralize them.Free radicals can damage cell membranes and affect other cellular functions.When heavy metals contaminate the soil and water at levels higher than optimal, plants experience toxicity resulting in stress conditions.This stress response includes oxidative stress, which can lead to decreased strength and growth inhibition [9].
The rate of leaf growth indicates the successful metabolic activity of plants in terms of leaf growth rate.Among the six observed reactors, reactor P6 (control) exhibited the highest rate of leaf and root growth.The data trends show that as the mercury concentration in the reactor increases, the growth ratio decreases.This data trend is supported by Hutagalung's statement that the concentration of Hg in the environment can affect the physical and chemical factors of water, such as temperature and pH.The yellowing of leaves at the beginning of the experiment is caused by the plant's stress response to the heavy metal Hg.Heavy metals like Hg can damage cell membranes and disrupt plant metabolism.This can result in tissue damage in the leaves and lead to leaf yellowing [10].
According to Bradl's statement, plants growing in mercury-contaminated media will experience metabolic disturbances.These metabolic disruptions have an impact on the growth of plant cells, leading to inhibited organ growth [7].This can be observed from the root growth ratio in reactors P1 to P5, where the higher the concentration of the heavy metal Hg, the smaller the root growth ratio.Reactors P1 to P6 exhibit a trend of vigorous and elongated root growth, which is due to plants attempting to absorb nutrients from the soil, including Hg 2+ ions.The diagram shows variations in the number of offspring in each reactor, but overall, the reactors experienced an increase in offspring.The reactor with the highest number of offspring is reactor P5 (6 mg/L), which had the highest concentration of Hg in this experiment.The high number of offspring in P5 is marked by the abundance of leaves that died during days 1-7, which were then hydrolyzed and transformed into a source of N and P for the plants.According to Septyani, fallen and decomposed leaves can become a source of nutrients for plants.Additionally, the addition of soil to the reactor enriched the nutrient content [11].According to the study by Triharto et al., the total N content in the surface soil is 0.2%, and the potential P content is 0.01%.This indicates that the soil added to the reactor also contains sufficient concentrations of N and P for plant growth [12].

(b) Typha latifolia
The Typha latifolia plant has the ability to grow in fairly extreme environmental conditions.According to the research conducted by Adams [8], Typha latifolia is a plant that exhibits a high tolerance to heavy metal contaminants and produces a significant amount of biomass.This plant can thrive in acidic water conditions (pH 3.9-4.3).Typha latifolia, also known as cattail, is a type of large grass-like plant that inhabits marshes.It is a perennial, erect, robust plant, reaching a height of 1.5-3 meters, with round stems.Its linear leaves are somewhat pointed, measuring 8-22 cm x 6-16 mm, and are divided into numerous compartments and grow in sheaths [13].Based on the observations conducted, the percentage of yellowing leaves continuously increased from day 1 to day 28 of the observation Typha latifolia period.The initial observation showed no yellowing leaves, indicating that Typha latifolia plants did not exhibit a stress response to the entering contaminants.
Observations of the leaf growth ratio in Typha latifolia plants yielded relatively similar results to other reactors, thus not providing a distinction between the reactor with mercury addition and the reactor without mercury.The morphology of Typha latifolia stems resembles leaves in shape and color, but structurally, the stems of Typha latifolia have harder and more solid characteristics.During the observation, the stems of Typha latifolia did not show a significant response as a distinguishing factor or as a response factor in plant observations regarding mercury pollution.
Typha latifolia is highly suitable for treatment using constructed wetland systems.Typha spp. is a swamp plant that effectively absorbs nutrients.This plant stores biomass in its leaves, stems, and roots [14].Physically, the response of Typha latifolia plants shows a positive reaction to the addition of the heavy metal mercury.Among the reactors used in this experiment, reactor T5 (6 mg/l) with the highest mercury concentration exhibited the highest root growth compared to other reactors.As the root growth of Typha latifolia plants increases, the phytoremediation process also becomes more efficient.According to Adams et al., Typha latifolia is a plant that has a high tolerance for heavy metal contaminants and produces a significant amount of biomass in its root system [9].In the graph, it can be observed that the offspring of Typha latifolia plants exhibit synchronized growth responses in each reactor from day 14 to day 28 of the experiment.The above figure illustrates the pH increase in the water of Typha latifolia and Pistia stratiotes reactors over the treatment period from day 1 to day 28.Each reactor has a different acidity level.The reactor with a high concentration of mercury (Hg 2+ ) is the mercury (II) ion, or mercury (II) dication.This ion tends to be acidic due to its ability to accept electron pairs from other compounds or ions.According to Khasanah et al., (2018), plants contribute to the pH increase due to their photosynthesis activity in the tested plants.The photosynthesis process converts CO2 into C6H12O6, which requires hydrogen and energy.Hydrogen is obtained from H + derived from wastewater and air, thus the uptake of H + will raise the pH [15].In the context of phytoremediation, EC50 (effective concentration 50%) refers to the concentration of pollutant compounds required to achieve the desired effect or reduce the toxicity level by 50%.In phytoremediation, EC50 is often used to measure the effectiveness of plants or microorganisms in removing pollutants from the environment.The observation of Pistia stratiotes solely focuses on the leaf response to mercury toxicity, which is physically observed through the yellowing of leaves over a susceptible time period from day 1 to day 7. Therefore, the plant's response to mercury toxicity is demonstrated in the EC50 graph in Figure 7. Figure 7 shows the EC50 at a concentration of 4 mg/liter.This means that the water lettuce plant experiences a 50% leaf yellowing toxicity effect at a mercury concentration of 4 mg/liter.The linear equation indicates an R2 value of 0.6.The toxic effect of Hg 2+ (mercury ion) according to Zhang and Tyerman's (1999) study on mercury phytotoxicity in cucumber and wheat plants explains that mercury (Hg 2+ ) can bind to water channel proteins in plants, which subsequently causes stomata (pores on leaves) to close and physically impede water flow within the plants.This disrupts mitochondrial activity and triggers oxidative stress by promoting the formation of reactive oxygen species (ROS) [9].

Conclusion
Typha latifolia and Pistia stratiotes were able to develop and adapt to a mercury concentration of 6 mg/L.During the first week of observation, Pistia stratiotes developed yellow and weathered leaves as a stress response.Typha latifolia did not exhibit any signs of stress.This was shown by the fact that there was no discernible difference between the control reactor and the reactor with heavy metal mercury contamination.The pH in the reactor decreases as the concentration of the heavy metal mercury increases too.Based on these findings, both plants have the potency to be used in phytotreatment of Hg lower concentration levels in gold mine wastewater.

Figure 2 .
Figure 2. Observation of Pistia stratiotes on various concentrations of Mercury.

Figure 4 .
Figure 4. Observation of on various concentrations of Mercury.

Figure 5 .
Figure 5. Number of shoots during observation in Typha latifolia.
The accumulation effect of mercury only occurs in Pistia stratiotes and is not observed in Typha latifolia plants because Typha plants do not show any mercury toxicity effects.In the RFT experiment, plants' responses to various mercury (Hg) concentration treatments indicate a tendency towards phytoremediation plant toxicity effects, and the response of the plant leaves is subsequently observed in the EC50.