Phytotoxicity test of cadmium and lead on Sunflower (Helianthus annuus L.) as first step in phytoremediation

Phytoremediation is a remediation process by using a plant to eliminate, accumulate, or absorb contaminated material by utilizing hyperaccumulator plants, such as sunflower (Helianthus annuus L.). This method was chosen because it is eco-friendly, effective, low cost, and easy to be implemented. The aim of this study is to determine maximum concentration which Helianthus annuus L. can survive. This research was carried out for five weeks starting in August – September 2022 which began with preparation by seeding of sunflower seeds on garden soil, then continued with range finding test using variations of sunflowers and media composition. The contaminated soil was obtained from Tamangapa Landfill Makassar. The pH, temperature, and moisture of each variation media composition were also monitored. Based on the results, all variations of Helianthus annuus L. can survive without any apparent negative side effects in 100% contaminated soil media with cadmium and lead concentration at 2.233 mg/kg and 166 mg/kg, respectively.


Introduction
A massive increase in human population have created numerous waste disposal and soil contamination problems, particularly in the last few decades.Waste disposal will be placed in landfill and will decompose, oxidize, and producing leachate.Metal ions released by leachate will be distributed into the soil through surface or subsurface movements and contaminating soil with heavy metals such as cadmium and lead [1,2].Particularly, heavy metals in the soil may enter the food chain though plants and have potential threat to human and animal health.When large amounts of heavy metals are consumed, it can lead to acute or chronic toxicity [3].
Cadmium (Cd) is a toxic non-essential transition metal that is harmful to both humans and animals.It occurs naturally in the environment as a pollutant from agricultural and industrial sources.Some of the effects of cadmium exposure include impaired kidney, liver, pancreas, lung, and cardiovascular system functions.Cadmium can accumulate in the body around 25-30 years, before it can be eliminated.Cd toxicity in plants may cause a reduction in chlorophyll content, photosynthesis rate, stomatal behavior, transpiration rate, and relative water content, leaf necrosis, enzyme inhibition, IOP Publishing doi:10.1088/1755-1315/1250/1/012008 2 and an alteration in antioxidant defense system and nitrogen metabolism which ultimately reducing plant growth and development [4].
Lead (Pb) is a major anthropogenic pollutant that has accumulated in various terrestrial and aquatic ecosystems since the industrial revolution.It is a highly toxic metal with numerous studies on its effects on human health [5,6].Lead toxicity in humans can cause symptoms such as anemia, headache, abdominal pain, loss of appetite, fatigue, and vertigo.Constant exposure will result in impaired kidney function, liver function, respiratory tract dysfunction, digestive system dysfunction, brain dysfunction, and some even death [7].Furthermore, Pb toxicity in plants potentially lead in leaf chlorosis, impaired photosynthesis, respiration in mitochondria, impaired growth, and an imbalance in the accumulation of P, K, Ca, Mg, Fe, and Mn elements [8].
Sunflower (Helianthus annuus L.) such as big smile and sunspot, have the ability to remove heavy metals such as lead (Pb), cadmium (Cd), nickel (Ni), and zinc (Zn) effectively.This plant was chosen because it grows quickly, can tolerate high concentrations of heavy metals, easy to harvest and it is classified as a hyperaccumulator plant [9].The previous studies have shown that sunflower has a high tolerance to heavy metals.Therefore, this study aims to determine the maximum concentration at which Helianthus annuus L. has ability to survive without any negative side effects.Since this study is a part of a research project aimed at uptake and accumulation of Cd and Pb using phytoremediation method, the results will be used for further research.

Seed Preparation
The sunflower seeds are obtained through online purchases with variety of big smile and sunspot.The soil used in this study was originally from garden soil without fertilizer from the flower market.The soil used for planting the crops was previously dried and weighted before being place in each about 2 kg plastic polybag.After being put in polybag, the soil was watered for a nursery.During this process, sunflower seeds are soaked overnight to help the sprouts grow faster.In addition, gloves and a small spoon are also required materials for this process.This process lasted four weeks until the plants reached their ideal size and morphology.

Range Finding Test
After seed preparation for four weeks, plants at the same age and height were used in the Range Finding Test (RFT) stage.This was conducted in the hope that the initial conditions of the plants used in the study would be the same [10].This study used two varieties of sunflowers, namely big smile and sunspot, and varied the planting media using the ratio of garden soil and contaminated soil.As shown in Figure 1, the contaminated soil was taken from residential area near Tamangapa Landfill in Makassar City.The results show that the soil is contaminated with Cd and Pb of 2.233 mg/kg and 166 mg/kg, respectively.The range finding test was conducted for 7 days and during the process, pH, temperature, and moisture of all samples were also monitored.The variety of treatment used in range finding test can be seen in Table 1.

Seeding Stage
The seeding period lasted four weeks or 28 days, until the plants reached their ideal size and morphology.
Visual observations including growth rate and the number of leaves were made at this stage.The garden soil used has brownish sandy characteristics.The growth rate was observed by measuring the plant's height from the growing medium to the highest shoot and the number of leaves.Figure 2 shows the sunflower growth at day-3 while Figure 3 shows the sunflower at day-28.Observations of sunflower height and the number of leaves during seedling stage were shown in Figure 4 and Figure 5. Based on observations of sunflower plant height at the seeding stage, the plant heights of big smile and sunspot were 8 cm and 10 cm at 4 weeks, respectively.Based on observation, the number of leaves at the seeding stage of big smile and sunspot were 10 and 13 leaves at 4 weeks, respectively.As reported by Hapsari and Herlina [11] the sunflower height and number of leaves at 4 weeks ranged from 9 cm to 11 cm and 10 leaves to 12 leaves, respectively.

Range Finding Test
Range Finding Test (RFT) is used to determine the maximum concentration of contaminants that can be accumulated by plants without causing damage or disrupting growth [12].This stage carried out for 7 days where plants that can adapt and survive are used for further research in phytoremediation [13].Before any plant can be used in phytoremediation, it should be tested first to know the tolerance of the plant to Cd and Pb exposure in range finding test.Figure 6 and figure 7 showed that big smile sunflower grew well without any symptoms in all media with a height up to 9.4 cm and 13 leaves.Based on the results, big smile and sunspot sunflower grew well with no signs of growth disturbance in a 100% contaminated soil media.The big smile sunflower has a height of 9 cm and 12 leaves, whereas the sunspot sunflower has a height of 11.2 cm and 15 leaves, so the sunspot sunflower was chosen for the further research.The maximum concentration of cadmium and lead that Helianthus annuus L. can tolerate were 2.233 mg/kg and 166 mg/kg respectively.This indicated that Helianthus annuus L. can be considered as a hyperaccumulator because of its high tolerance to heavy metals.In accordance with Alaboudi et al. [14] Helianthus annuus L. has ability to survive Cd and Pb concentration up to 71.30 mg/kg and 107.70 mg/kg respectively.Sunflowers grow well in various soils, but well-drained soil with a high water capacity and a neutral pH of 6.5 to 7.5 is ideal for cultivation.This plant can thrive in sufficient soil moisture conditions that are neither too dry nor too wet.Adequate soil moisture can encourage root growth during the emergence and vegetative stages, resulting in a good response to nutrient uptake through roots [15].However, Jaenudin et al. [16] report a different result, a sandy to clay soil that is not acidic or salty, with a pH ranging from 5.7 to 8.1 is ideal for the growth of this plant.
In general, the results showed that the moisture ranged from 70 -90%.The average pH ranged from 6.3 -7.2 while the temperature ranged from 28 -32 o C throughout the five weeks which is normal for a tropical region.Figure 12 to figure 14 shows the physical parameters observation during this study.

Conclusion
The results showed that Helianthus annuus L. can survive without evident symptoms of toxicity in 100% contaminated soil media.This study demonstrated that Helianthus annuus L. has ability to survive at Cd and Pb concentration up to 2.233 mg/kg and 166 mg/kg, respectively.It was also showed that Helianthus annuus L. can be considered as a hyperaccumulator for contaminated soil because of its high tolerance to heavy metals.

Figure 4 .Figure 5 .
Figure 4. Growth rate of sunflower during seeding stage

Figure 6 . 6 Figure 7 .
Figure 6.Growth rate of big smile during RFT

Table 1 .
Variation of Range Finding Test