Remediation of Pb and Cd Polluted Soils with Fulvic Acid

: Heavy metal pollution is among the important environmental problems in the world. Many techniques have already been used to remove the heavy metals such as lead (Pb) and cadmium (Cd). Among them, the phytoremediation method is an environmentally friendly and green technology. This study was carried out to determine the efﬁciency of fulvic acid (FA) application in removing Pb and Cd from polluted soil using Tagetes eracta L. and Zinnia elegans Jacq. ornamental plants. The results indicated that, FA application, number of ﬂower per plants, and plant fresh weight of Tagetes eracta plants and Zinnia elegans plants increased 187.5%, 104.5% and 155.5%, 57.7%, respectively with application of 7000 mg L − 1 FA at 100 mg kg − 1 Pb pollution condition, whereas 42.85%, 16.5%, and 44.4–36.1% with application of 7000 mg L − 1 FA at 30 mg kg ± 1 Cd pollution condition, respectively. With the FA application in the Zinnia elegans plant, the root part has accumulated 51.53% more Pb than the shoot part. For Cd, the shoot part accumulated 35.33% more Cd than the root. The effect of FA application on superoxide dismutase (SOD), peroxidase (POD) and, catalase (CAT) of the Tagetes eracta were decreased as 32.7%, 33.1%, and 35.1% for Pb, 21.2%, 25.1%, and 26,1%, for Cd, and 15.1%, 22.7%, and 37.7% for Pb, and 7.55%, 18.0%, and 18.8% for Cd were in Zinnia elegans respectively. In conclusion, Tagetes eracta and Zinnia elegans can not be recommended for remediation of Pb and Cd polluted area, but FA can be recommended for Pb and Cd stabilization in polluted soil.


Introduction
Heavy metals (HM) released to the environment as a result of the activities of industrial enterprises such as mining activities, paint, artificial fertilizer production, urban or industrial waste are important factors in the deterioration of the natural balance and the destruction of ecology. Mineral processing and mining activities in particular cause soil pollution by producing large amounts of metal-rich waste materials [1]. This heavy metal stress (HMS) caused by environmental pollutants. It limits the growth of plants and decreases the yield and quality of the product [2]. The ever-increasing concentrations of HM in the environment disrupt the ecosystem balance and increase economic loss with deterioration in human health [3]. HM, especially Cadmium (Cd) and Lead (Pb), have emerged as serious toxic environmental pollutants in recent years due to the excessive release of HM such as industrial manufacturing and agricultural input production and use [4]. High concentrations of Cd and Pb adversely affect plant growth by disrupting natural ecosystems, polluting the soil, and reducing fertility and plant nutrient uptake [5]. In addition, HMS in high concentrations in the soil can adversely affect the morphology, anatomy and physiology of the plants and cause phytotoxic, effects, leading to a decrease in the yield of the plants and their death at excessive concentrations [6,7].
HM in high concentrations in the plant growing environment affect plant production negatively by affecting the physiological processes of plants, causing imbalances in nutrient elegans is an annual flowering plant, ability to grow in toxic metals, having fast growth, high biomass, easy to cultivate and harvest and is mainly cultivated for the purpose of flower production [27]. Tagetes erecta (Marigold) is an ornamental plant belonging to the Asteraceae family. It is one of the easiest outdoor ornamental plants to grow in gardens and parks. Tagetes species are native to the North and South America continents. The flowers are bright yellow, brownish yellow or orange. Tagetes erecta (Mexican marigold) yellow multilayered blooming marigold. It is common to grow it as one of the most suitable summer ornamental plants for parks, gardens, balconies and terraces that receive sunlight for a long time. Marigold adapts to any soil, any climate. In this respect, it is easily grown all over the world [28]. The use of ornamental plants in urban areas not only cleans contaminated areas but also contributes to the beautification of the environment [29].
Heavy metal contamination, the persistence of these elements in the environment and the toxicological consequences on plants, wildlife and human populations have become an important global problem in the destruction of biodiversity, especially accelerated by the industrialization process and exponentially due to global climate change [11].
The aim of this study is to evaluated efficiency of fulvic acid on Pb and Cd uptake from Pb and Cd contaminated soil, and the phytoremediation capacity and the hyperaccumulator properties of Zinnia elegans, Tagetes erecta plant.

Materials and Methods
This study to evaluate efficiency of fulvic acid on Pb and Cd uptake from Pb and Cd contaminated soil, and the phytoremediation capacity was conducted using a randomized complete block design. Two pollutant (Cd and Pb), one chelating agent (Fulvic acid; FA), and five replication (Control, Cd, Cd + FA, Pb, Pb + FA) with the soilmixture (1:1:1 torf:soil:sand) prior to filling into 2 L pots). To create the heavy metal stress conditions, soil was mixed with lead (100 mg Pb kg −1 as Pb(NO 3 ) 2 ) and with cadmium (30 mg Cd kg −1 as 3CdSO 4 H 2 O). Fulvic acid (FA) solutions were prepared by dissolving 350 g Fulvofeed ® in one liter water (350,000 mg L −1 ). The commercial product Fulvofeed ® (Humintech GmbH, Grevenbroich, Germany) was used as the fulvic acid (FA) source. The content of Fulvofeed ® on dry basis was 90-95% fulvic acid, 5% humic acid, 2% potassium as K 2 O, 1% total organic nitrogen. The element content of Fulvofeed ® (%, mass) was C-32, H-2.8, N-1.3. The ash content was 22.9 %. The element content on ash-free basis (% wt) was C-49.1, H-5.63, N-1.29, O-44.38. The pH was between 9 and 10. The particle size of insoluble constituents was less than100 µm. FA solutions were further diluted to obtain a concentration of 7000 mg L −1 . FA solutions were applied 20 L ha −1 to the soil three times a week, starting the day before planting. These concentrations are the maximum permissible limits according to U.S. Environmental Protection Agency and World Health Organization [30,31]. To simulate Cd and Pb pollutant soil, the incubation process continued as a 8 week after Cd and Pb application soil mixture. During the incubation period, the water level was kept 30% below the soil field capacity and the ambient temperature was 29 degrees. At the end of the 8 week The soil used in the experiment is classified as Ustorthents [32] and consists of 35% sand, 35% silt, 30% clay, 1.2% organic matter. The pH of the soil was 5.61. Cation exchange capacity and the electrical conductivity of the soil were measured as 15.5 cmol(+) kg −1 and 1.15 dS m −1 , respectively. The macronutrient content of the soil was as follows: total N: 1.15%, P: 15.5 mg kg −1 , K: 2.2 cmol(+) kg −1 , Ca: 11.0 cmol(+) kg −1 , Mg: 2.25 cmol(+) kg −1 . The microelements were found as Zn: 2.30 mg kg −1 , Cu: 1.1 mg kg −1 , Fe: 0.5 mg kg −1 , Mn: 1.8 mg kg −1 . Cd 0.05 mg kg −1 , Pb 0.02 mg kg −1 . After harvest soil Cd and Pb contentration was 0.4 mg kg −1 for Pb, and 2.1 mg kg −1 for Tagetes eracta and 0.05 mg kg −1 for Pb and 1.1 mg kg −1 for Zinnia elegans.

Plant Growth Parameters
Harvest took place 90 days after seedling planting. Plant height (mm), plant fresh weight (gr), plant dry weight (gr), stem diameter (mm), plant flower number, flower stem length (mm), flower diameter (mm), flower stem thickness (mm), flower fresh weight (gr), flower dry weight (gr), and chlorophyll content (SPAD) were measured. At the end of the experiment, all plants were harvested. The fresh weights of the plants were measured with an electronic balance (CX-600), incubated at 68 • C for 48 h, then measured again for dry weight. Leaf chlorophyll readings of Zinnia elegans L. and Tagetes Erecta plants were determined with a portable chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Osaka, Japan).

Plant and Soil Analyses
For mineral analysis, samples from the roots and leaves of Zinnia elegans L. and Tagetes Erecta plants were dried in an oven at 68 • C for 48 h and then ground. The Kjeldahl method with a Vapodest 10 Rapid Kjeldahl Distillation Unit (Gerhardt, Konigswinter, Germany) was used to determine the total N. An inductively coupled plasma spectrophotometer (Optima 2100 DV, ICP/OES; Perkin-Elmer, Shelton, CT, USA) was used to determine tissue K, P, Mg, Na, Fe, Zn, B, Ca, S, Pb, Cd, Mn, B and Cl [33].
After 8 weeks of incubation with HM, soil samples from each plot were taken from a 0-to 10-cm depth to determine baseline soil properties, heavy metal pollution degree, and soil element fractions using sequential extraction.
Physical and chemical analyses were performed after soil samples were air dried, crumbled, and passed through a 2-mm sieve [34].
Soil pH was determined according to McLean [35]. The method of Rhoades (1996) [36] was used to measure electrical conductivity (EC). Kjeldahl method [37] and the sodium bicarbonate method of Olsen et al. [38] were used to determine the organic N and plantavailable P contents, respectively. Soil organic matter was determined using the Smith-Weldon method [39]. The exchangeable cations were determined according to Thomas [40]. Diethylenetriaminepentaacetic acid (DTPA) extraction methods were used to determine the microelements in the soils [41].
Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activities were determined based on the method given by Sahin et al. [42].

Statistical Analysis
A completely randomized factorial design was applied in this study. The data were subjected to ANOVA. The statistical software IBM SPSS Statistics 20 was used for all analyses. Results were considered to be significant at the p ≤ 0.05 level.

Yield and Yield Components of Plants
Our results showed that the highest number of flower perplants, plant height, stem diameter, flower diameter length, flower diameter, flower stem thickness, flower fresh weight, flower dry weight chlorophyll reading value, plant fresh weight and dry weight of Tagetes eracta and Zinnia elegans plants were obtained when FA was applied at a concentration of 7000 mg L −1 , whereas the lowest value were found control and follow without FA application both Pb pollution and Cd pollution treatment (Tables 1 and 2). These findings indicate that, FA application, number of flower perplants, plant height, stem diameter, flower stem thickness, flower fresh weight, flower dry weight, chlorophyll reading value, plant fresh weight and dry weight of Tagetes eracta plants increased 187.5%, 5.8%, 17.3%, 21.6%, 176.7%, 197.8%, 28.6%, 104.5% and 120.3% respectively with application of 7000 mg L −1 FA at 100 mg kg −1 Pb pollution condition, whereas flower perplants, flower stem thickness, flower fresh weight, flower dry weight, and plant fresh weight of Tagetes eracta plants increased 42.85%, 39.2%, 10.7%, 163.1% and 16.5%, with application of 7000 mg L −1 FA at 30 mg kg −1 Cd pollution condition, respectively (Table 1).

Efficiency of Fulvic Acid on Uptake of Macro and Micro Nutrient Content
Lead and cadmium pollution decreased all mineral elements contents in the shoots and roots. Fulvic acid application does not affect the N, P, K, Ca, Mg, Na, Cl, S, Fe, Mn, B, Cu and Zn element contents in the shoots and roots of Tagates eracta and Zinnia elegans plants (Tables 3-6).  Notes. Different letters within the columns show significant differences at p < 0.05 for heavy metal and FA applications; improvement factor (%); PP, plant extraction potential (kg ha −1 ); BCFT, bioconcentration factor (stem and root) relative to total soil element (B, Pb or Cd); TF, transfer factor; TLF, translocation factor.

Efficiency of Fulvic Acid on Uptake of Pb and Cd in Shoots and Roots Part of Plants
The application of FA significantly affected Pb, and Cd uptakes of the plants under Pb and Cd-contaminated soils. Pb and Cd concentrations in plant shoots and roots increased with the fulvic acid application. The effect of FA application on the uptake of shoot and root parts of the Tagetes eracta plant in Pb and Cd uptake amounts were determined as 178.52% and 176.40% for Pb, 290% and 266.96% for Cd, respectively, compared to the control. The effect of chelation application on the intake of shoot and root parts of Zinnia elegans plant in Pb and Cd uptake amounts were determined as 117.32% and 168.85% for Pb, 274.75% and 239.42% for Cd, respectively, compared to the control. With the FA application in the Tagetes eracta plant, the shoot part accumulated 6.75% more Pb and 23.53% Cd compared to the root part. With the FA application in the Zinnia elegans plant, the root part has accumulated 51.53% more Pb than the shoot part. For Cd, the shoot part accumulated 35.33% more Cd than the root. It was determined that FA application increased the Pb and Cd uptake in shoots and leaves at a much higher rate compared to the control. Thus, the highest Pb, and Cd uptakes were observed from plant shoots followed by roots (Tables 3-6).

Effects of Fulvic Acid Applied under Heavy Metal Stress on Enzymatic Activity
It is known that the enzyme activities of plants grown in soils contaminated with various heavy metals are affected by stress [50,51]. In this study, the application of FA significantly affected enzyme activity of Tagetes eracta and Zinnia elegans at Pb, and Cd pollution. The effect of FA application on Superoxide dismutase (SOD), peroxidase (POD) and, Catalase (CAT) of the Tagetes eracta were decreased as 32.7%, 33.1%, and 35.1% for Pb, 21.2%,25.1%, and 26,1%, for Cd, and Zinnia elegans SOD, POD and CAT activity were 15.1%, 22.7%, and 37.7% for Pb, and 7.55%,18.0%, and 18.8% for Cd, respectively (Table 7). Obtained results showed that, the activities of SOD, CAT and POD were reduced after treatment with FA. This result indicates that FA may serve directly as an antioxidant to eliminate excess ROS, or as a signal molecule to promote antioxidant production. In consistent with our results, Yildirim et al. [52] reported that FA treatment in garden cress under cadmium stress reduced SOD and CAT activity.

Effects FA on Pb and Cd Remediation Factors of Tagetes eracta and Zinnia Elegans Grown in Contaminated Soil
Translocation Factor (TLF): It is a ratio that indicates the ability of a plant to transport metals from its roots to the harvestable part of the plant. In other words, it is used to measure the amount of heavy metal transferred from one organ to another. The heavy metal concentration in the stem of the plant is calculated by proportioning to the root. TLF factor in plants is usually less than 1 FA application doesn't affected TLF (Figure 1). Hyperaccumulator plants are plants that can accumulate HM such as Pb, Cd, Hg, Se, in their body when compared with cultivated plants [53,54]. Translocation factors are used to evaluate the hyperaccumulatory properties of plants, revealing the rates of leaf and fruit transport from the root zone to the stem, from the stem to the stem. As the ideal value used for phytoextraction, BCF > 100 and TLF > 1 are expected to have accumulator plant characteristics [55]. Plants with bioconcentration and translocation factors >1 can be used as bioaccumulators, and those with bioconcentration values >2 are considered high accumulators. can be used as a phytostabilizer if bioconcentration factors >1 and translocation factors <1, and as phytoextractor if bioconcentration factors <1 and translocation factors >1 [56]. Data presented in Figure 1 shows the values of TLF factors of Pb and Cd. Tagetes eracta and Zinnia Elegans show low TLF to Pb and Cd and therefore, are not suitable in phytoremediation studies and It cannot be considered as a hyperaccumulator plant.

Conclusions
It was carried out to determine whether landscape plants such as Tagetes eracta and Zinnia elegans are accumulator plants in a cadmium and lead polluted area. FA application was made to increase the efficiency of HM in the soil and to increase their uptake by the plant. The results obtained showed that Tagetes eracta and Zinnia elegans planted the ability to accumulate Pb and Cd in their tissues (shoots and roots), but it was determined that it did not have hyperaccumulatory properties. Cd accumulation in plant shoots was higher than accumulation from Pb, but Pb accumulation in plant root was higher than Cd. BCFT root value for Cd and Pb in Tagetes eracta was higher than BCFT shoot value, in Zinnia elegans BCFT shoot concentration for Cd and Pb was lower than BCFT root concentration. With the FA application, the TF values of the plant decreased and causes lead and cadmium to be stabilized in the root zone of the plant, reducing its ability to transport from soil to the upper part of the plant in Tagetes eracta and Zinnia elegans plants. It is a result of the fact that the application of fulvic acid caused the Pb and Cd HM in the soil to stabilize instead of increasing the uptake ability of the plant, and the TLF factor was less than 1.
In many studies conducted to date, there are many results that increase or decrease the mobility of HM such as humic and fulvic acids. Many studies showing that HM in the soil are easily taken up by the plant or reduced their uptake have also shown that it affects plants by increasing or decreasing the accumulation of Pb and Cd in total or exchangeable form. While there are results in the direction that these types of chelating agents increase the mobility of Pb and Cd in the soil, contrary to these results, it has been determined that fulvic acid applications serve to reduce the uptake of the plant by immobilizing, that is, to stabilize the soil, and to eliminate the uptake of these HM by the plant. Contrary to the views that humic acid and fulvic acid act in the same direction, that is, increase the  Transfer Factor (TF). The ability of a species to transfer metal from the soil to the upper parts of the plant is estimated using TF. The TF of a metal is calculated as the ratio of the total metal concentration of the root and stem parts of the plant at harvest time to the total metal concentration in the soil at the time of harvest. FA application affected the transition and TF of Pb and Cd from soil to plants. With the FA application, the TF values of the plant decreased.
Heavy metal transfer factors of Tagetes eracta and Zinnia elegans can be seen in Figure 1. According to this figure, the lowest TF in Pb + FA and Cd + FA applications in Tagetes eracta plants are 8.3 and 13.80 respectively. It was calculated as 15.95 and 8.13 for Pb + FA and Cd + FA applications in Zinnia elegans plant, respectively. In this case, FA application causes lead and cadmium to be stabilized in the root zone of the plant, reducing its ability to transport from soil to the upper part of the plant in Tagetes eracta and Zinnia elegans plants (Figure 1). Similar result was obtained from Yıldırım et al. [52] in garden cress plants.

Conclusions
It was carried out to determine whether landscape plants such as Tagetes eracta and Zinnia elegans are accumulator plants in a cadmium and lead polluted area. FA application was made to increase the efficiency of HM in the soil and to increase their uptake by the plant. The results obtained showed that Tagetes eracta and Zinnia elegans planted the ability to accumulate Pb and Cd in their tissues (shoots and roots), but it was determined that it did not have hyperaccumulatory properties. Cd accumulation in plant shoots was higher than accumulation from Pb, but Pb accumulation in plant root was higher than Cd. BCFT root value for Cd and Pb in Tagetes eracta was higher than BCFT shoot value, in Zinnia elegans BCFT shoot concentration for Cd and Pb was lower than BCFT root concentration. With the FA application, the TF values of the plant decreased and causes lead and cadmium to be stabilized in the root zone of the plant, reducing its ability to transport from soil to the upper part of the plant in Tagetes eracta and Zinnia elegans plants. It is a result of the fact that the application of fulvic acid caused the Pb and Cd HM in the soil to stabilize instead of increasing the uptake ability of the plant, and the TLF factor was less than 1.
In many studies conducted to date, there are many results that increase or decrease the mobility of HM such as humic and fulvic acids. Many studies showing that HM in the soil are easily taken up by the plant or reduced their uptake have also shown that it affects plants by increasing or decreasing the accumulation of Pb and Cd in total or exchangeable form. While there are results in the direction that these types of chelating agents increase the mobility of Pb and Cd in the soil, contrary to these results, it has been determined that fulvic acid applications serve to reduce the uptake of the plant by immobilizing, that is, to stabilize the soil, and to eliminate the uptake of these HM by the plant. Contrary to the views that humic acid and fulvic acid act in the same direction, that is, increase the mobility of HM in the soil, it has been revealed that fulvic acid is an important stabilizer in remediation technology with its high negative charges and high caton adsorption capacity. In many previous studies, inhibitory effects of humic substances on HM in acidic soils have been reported.
On the contrary, the humic acid content of humic substance, which is very high relative to the fulvic acid content, increases the heavy metal availability. Therefore, it can be said that humic acids are effective for heavy metal bioremediation because humic acids can interact with metals to form metal-humic complexes. On the other hand, it has been shown that fulvic acids inhibit metal availability and can be used to reduce metal accumulation in polluted acidic soils. Many studies have shown that humic acid applications, like ethylenediamine tetra acetic acid applications, increase the mobility of HM in the soil and promote phytoremediation, especially in alkaline soils with high pH. In conclusion, the application of FA may be suggested as an effective treatment for reducing the Cd and Pb uptake of the plants by stabilizing Cd and Pb in soil and preventing translocation of Cd and Pb from the roots of plant to its shoot and leaves. Tagetes eracta and Zinnia elegans can not be recommended for remediaion of Pb and Cd polluted area, but FA can be recommended for Pb and Cd stabilization in polluted soil.