تأثیر EDTA و BTC بر گیاه‌پالایی سرب خاک توسط گیاه همیشه ‌بهار (Calendula officinalis)

Background and objectives: Soil contamination with heavy metals, particularly lead (Pb), is considered one of the most serious environmental issues, posing significant negative impacts on the health of humans, plants, and other living organisms. In this context, bioremediation technologies, particularly phytoremediation, have attracted increasing attention as effective, economical, and environmentally friendly approaches for reducing heavy metal concentrations in soil. The marigold medicinal plant (Calendula officinalis) is regarded as a suitable candidate for this process due to its characteristics such as rapid growth, adaptability to diverse environmental conditions, and high capacity for elemental uptake. Furthermore, chelating agents such as EDTA and BTC can significantly enhance the uptake of metals by increasing their solubility and bioavailability. The effect of these compounds on the bioavailability of lead in soil and its translocation to various plant organs can play a decisive role in the success of phytoremediation. However, a simultaneous evaluation of their effects on metal uptake efficiency, physiological and biochemical responses of the plant and the potential environmental consequences of their application, particularly under controlled greenhouse conditions, requires more comprehensive studies. Accordingly, the present study was designed and conducted to investigate and compare the efficiency of marigold medicinal plant in the phytoremediation of lead-contaminated soil under the influence of the chelating agents EDTA and BTC in greenhouse conditions.
Materials and methods: This study was conducted as a factorial experiment based on a completely randomized design with three replications and 32 treatments. The experiment was carried out under greenhouse conditions during the 2019-2020 growing season at the Agricultural Research Institute of Zabol University, located in Zahak County. Three factors were investigated: type of chelating agent (EDTA and BTC), chelating agent concentration at four levels (0, 0.5, 1, and 1.5%), and soil lead concentration at four levels (0, 150, 300, and 450 mg/kg), with lead supplied in the form of lead nitrate. The lead-contaminated soil was incubated for two weeks and then transferred to one-kilogram pots. After planting marigold medicinal plant, the chelating agents were applied to the soil, and the pots were regularly irrigated.
This research was conducted as a factorial experiment based on a Completely Randomized Design (CRD) with three replications, encompassing 32 treatments and 96 experimental units. The study was executed under greenhouse conditions during the agricultural year 2019-2020 at the Agricultural Research Institute of Zabol University, located in Zahak County. The three factors investigated were the type of chelating agent (EDTA and BTC), chelating agent concentration at four levels (0, 0.5, 1, and 1.5%), and soil lead (Pb) level at four levels (0, 150, 300, and 450 mg/kg), with lead supplied from lead nitrate (from Pb (NO3)2). For contamination, soil samples were first passed through a 2 mm sieve, weighed into 2 kg units, and then prepared for treatment. Specific amounts of lead and the chelating agents were applied as a solution using sprayers and thoroughly mixed with the soil. These contaminated samples were incubated for two weeks in the greenhouse at 25 C and maintained at a moisture level approximating field capacity. To ensure the stabilization of the heavy metal and better simulate natural contamination conditions, the soil was exposed to alternating wetting and drying cycles. Following the stabilization period, the contaminated and saturated soil was air-dried, compacted, and transferred to 1 kg pots. Calendula was planted in early December, and throughout the growth period, irrigation was meticulously controlled at field capacity using distilled water; this involved weighing the pots every two days to replenish the exact amount of water lost. Sampling was performed two months after planting, and the shoot and root parts were harvested separately. The evaluated parameters included photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) and the concentration of lead in plant tissues and soil. Analysis of variance (ANOVA) was performed using the Proc GLM procedure, and mean comparisons were conducted using Duncan’s multiple range test at the 5% significance level. All statistical analyses were performed using SAS software, version 9.4.
Results: The results of evaluating the effects of chelating agents and lead contamination on the photosynthetic pigments of Calendula officinalis showed that these factors had significant impacts on chlorophyll a, chlorophyll b, and carotenoid contents. As lead concentration increased, the levels of all three pigments decreased. This reduction was more pronounced for chlorophyll a in the absence of chelating agents. The application of chelating agents, particularly EDTA, especially under lead-free conditions and at a concentration of 1.5%, resulted in a significant increase in chlorophyll a. The highest chlorophyll a content (6.38 mg kg-1) was observed in the combined treatment of 1.5% EDTA and no lead. Moreover, EDTA partially mitigated the adverse effects of moderate and high levels of lead contamination (150 and 450 mg kg-1 Pb). Regarding chlorophyll b, the highest value (12.01 mg kg-1) was recorded in the control treatment (no chelator, no lead). Increasing lead concentration, especially at 450 mg kg-1, led to a decline in chlorophyll b across all treatments. This decline was more substantial in treatments with high chelator concentrations, particularly BTC, indicating a potential negative interaction between certain chelating agents and lead in chlorophyll b biosynthesis. For carotenoids, the application of chelators, especially 1% EDTA, resulted in a relative improvement in this pigment. The highest carotenoid content (2.90 mg kg-1) was observed in the treatment without lead and with 1% EDTA. However, at a higher EDTA concentration (1.5%) and in the presence of lead, a further reduction in pigment levels was observed, suggesting a possible adverse interaction between these two factors. The study also demonstrated that the type and concentration of chelating agents play a crucial role in lead uptake, translocation, and distribution within the plant. Treatments with 1.5% EDTA under high lead contamination (450 mg kg-1 Pb) led to the highest accumulation of lead in both the aerial parts (30.88 mg kg-1) and roots (62.39 mg kg-1). Compared to BTC, EDTA was more effective in facilitating lead transfer from soil to plant. In contrast, BTC significantly reduced residual lead in the soil (by up to 69.5% compared to the control), athough lead uptake by the plant was lower, likely due to the greater stability of BTC –lead complexes. Overall, increasing the concentration of chelating agents, particularly EDTA, was generally associated with enhanced lead accumulation in the plant, whereas treatments without chelators showed the highest levels of residual lead in the soil.
Conclusion: The findings of this study highlight the critical role of chelator type and concentration in enhancing the efficiency of phytoremediation under heavy metal contamination, particularly lead. The use of an appropriate chelating agent, such as EDTA, at controlled concentrations and within defined timeframes, can not only mitigate the adverse effects of contamination on the plant’s physiological and biochemical parameters but also enhance lead uptake and translocation, thereby reducing its residual levels in the soil. Therefore, the precise selection of the chelator type and dosage based on the severity of lead contamination is crucial, especially for preserving the health and performance of medicinal and ornamental plants. The success of this approach also depends on effective and sustainable environmental management to prevent unintended consequences, such as excessive accumulation of heavy metals in the consumable parts of the plant.