Assisted phytoremediation of a multi-contaminated soil: Investigation on arsenic and lead combined mobilization and removal

Journal of Environmental Management 203 (2017) 316e329

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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman

Research article

Assisted phytoremediation of a multi-contaminated soil: Investigation on arsenic and lead combined mobilization and removal Meri Barbafieri a, Francesca Pedron a, Gianniantonio Petruzzelli a, Irene Rosellini a, Elisabetta Franchi b, Roberto Bagatin b, Marco Vocciante c, * a b c

Institute of Ecosystem Study, National Council of Research, Via Moruzzi 1, 56124, Pisa, Italy Eni S.p.A., Renewable Energy & Environmental R&D, Via Maritano 26, 20097, S. Donato Milanese, MI, Italy  degli Studi di Genova, Via Dodecaneso 31, 16146, Genova, Italy DCCI, Dipartimento di Chimica e Chimica Industriale, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 February 2017 Received in revised form 5 May 2017 Accepted 31 July 2017

The removal of contaminants from an earthy matrix by phytoremediation requires the selection of appropriate plant species and a suitable strategy to be effective. In order to set up an assisted phytoremediation intervention related to a disused industrial site affected by an arsenic and lead complex contamination, an extensive experimental investigation on micro and mesocosm scale has been conducted. Particular attention was given to the choice of plant species: using crop plants (Lupinus albus, Helianthus annuus and Brassica juncea) a series of parallel test campaigns have been realized to investigate different scenarios for the reclamation. With regard to the arsenic contamination, which is certainly the most worrying, the possibility of employing a hyper-accumulator species (Pteris vittata) has also been investigated, highlighting advantages and difficulties associated with such an approach. The application of various mobilizing agents in different concentrations was tested, in order to maximize the extraction efficiency of plants in respect of both contaminants, showing the necessity of a chemically assisted approach to promote their uptake and translocation in the shoots. Phosphate addition appears to produce the desired results, positively affecting As phyto-extraction for both hyper-accumulator and crop plants, while minimizing its toxic effects at the investigated concentrations. With regard to Pb, although tests with EDDS have been encouraging, EDTA should be preferred at present due to lower uncertainties about its effectiveness. The performed tests also improved the addition of mobilizing agents, allowing the simultaneous removal of the two metals despite their great diversity (which in general discourages such approach), with significant saving of time and an obvious improvement of the overall process. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Arsenic Lead Phyto-extraction Mobilizing agents Plant uptake Bioavailability

1. Introduction Arsenic (As) is among the most toxic inorganic pollutants and its contamination is a very serious global problem. Apart from its own toxicity, which makes this contaminant particularly dangerous for human health in case of direct contact (Garelick et al., 2008), it can be assimilated from the food chain and drinking water resulting in

* Corresponding author. E-mail addresses: meri.barbafi[email protected] (M. Barbafieri), francesca.pedron@ ise.cnr.it (F. Pedron), [email protected] (G. Petruzzelli), irene. [email protected] (I. Rosellini), [email protected] (E. Franchi), roberto. [email protected] (R. Bagatin), [email protected] (M. Vocciante). http://dx.doi.org/10.1016/j.jenvman.2017.07.078 0301-4797/© 2017 Elsevier Ltd. All rights reserved.

bioaccumulation with occasional biomagnification (Duruibe et al., 2007), causing development of neurotoxicity, cardiovascular disndez et al., 2012; Hughes et al., eases and carcinogenic effects (Ferna 2011; Straif et al., 2009). In soil, arsenic may be present in a wide variety of forms, both organic and inorganic; in particular, As(III) and As(V), the most important and spread inorganic forms, are also the most toxic ones. Although less toxic, also lead tends to accumulate in living organisms, causing long-term phenomena of intoxication, since it is not an essential element (just as arsenic), and can cause loss of biological activity when above certain concentrations. For these reasons, lead is considered among the most harmful metals for human health due to its negative effects on kidneys liver, nervous

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system and brain (WHO, 2010). The metal can cause serious toxicity effects also in plants, with reduced root growth, damages to some enzymatic activities and disturbances to plant hormonal status (Gopal and Rizvi, 2008; De Souza et al., 2012; Sharma and Dubey, 2005). Nowadays, several remediation technologies exist (Khan et al., 2004); however, since there are no standard approaches to deal with specific contaminants, finding a proper solution might sometimes remain an open question. In case of potentially toxic metals, the most common solutions comprise either the excavation and landfilling, or the soil washing with stabilization/inertization; unfortunately, these technologies are extremely invasive, resulting in a low level of environmental and social sustainability (U.S. EPA, 2006). In some previous contributions, we presented the ElectroKinetic Remediation Technology (EKRT) as a possible alternative to conventional approaches; it is a promising in situ solution, potentially able to allow results similar to on-site and/or off-site interventions (Rosestolato et al., 2015), but with substantially higher levels of sustainability and acceptability (Vocciante et al., 2016a, 2017). However, alongside the EKRT, the phytoremediation is another remediation strategy for polluted soils that is receiving renewed and significant attention as a zero-impact and environmentally safe approach (Barbafieri et al., 2013). Although most metals are non-essential elements for plants, and As and Pb are both known to be generally highly phytotoxic, some plant species have shown a particular resistance to their toxicity, sometimes together with interesting hyper-accumulation capabilities in relation to these contaminants. The phyto-extraction of toxic metals has been originally based on the use of hyper-accumulators, i.e. plants that are able to gather a high concentration of metals or metalloids within their shoots (Brooks, 1998). However, the natural phyto-extraction has its limitations, since the use of hyper-accumulators is often hindered by their reduced production of biomass (relatively small shoot biomass with low growth rate and long life cycle) and because of their ability to accumulate only one specific element, which makes them impractical in the case of a multi-contaminated soil. To overcome these difficulties, an “assisted” approach (Bhargava et al., 2012) can be employed: plant species with a high biomass production (i.e. crops) are used in combination with chemical additives, which change the soil properties increasing the bioavailability of the contaminants (Blaylock et al., 1997; Cooper et al., 1999; Evangelou et al., 2007; Pedron et al., 2009). Crop plants may compensate for the difference in metal accumulation (which is up to three orders of magnitude higher in hyper-accumulator plants) through a high and rapid production of biomass, if they are chemically induced to collecting large quantities of metals in their upper parts. The use of specific chemicals, which increase the solubility (and thus the bioavailability) of metals in the soil, has proven to dramatically stimulate the potential of metal accumulation (Meers et al., 2008). The possibility to plan repeated growing cycles with harvesting of metal-rich biomass, and the minor level of specificity of these species towards contaminants, may make this approach more advantageous than the recourse to hyperaccumulator species. Different additives are commonly used, in particular chelating agents such as ethylene-diaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA), or weak acids like e.g. citric acid, to enhance the uptake from crop plants (Doumett et al., 2008; Pedron et al., 2014). However, these chemicals may persist in the soil, with residual toxic effects; besides, also the possible risk of metal leaching into the groundwater has to be considered (Karczewska et al., 2011; Lestan et al., 2008). Thus, it is advisable to monitor the movement of metals in soil through an appropriate control of water

317

movements (Vocciante et al., 2016b), in order to prevent a downward displacement and/or the contamination of groundwater resources with soluble metal species. Aim of this work is the identification of the best experimental conditions to perform an assisted intervention of phytoremediation in relation to a disused industrial site affected by a complex contamination comprising arsenic and lead. Several chelating agents have been assessed to promote the uptake and translocation of the contaminants in plant shoots, and numerous tests at the scale of micro and mesocosm have been set. The investigated crop species were Lupinus albus, Helianthus annuus, and Brassica juncea, and the addition of chemical species like phosphate (for removing As) and EDTA (for Pb), at different concentrations, was considered both separately and in a combined application. To improve lead mobilization, also the alternative use of ethylenediamine-N,N0 -disuccinic acid (EDDS) has been taken into account. Moreover, an assisted approach using a hyper-accumulator for As (Pteris vittata) has been tried, which has allowed to highlight the advantages and difficulties associated with such an approach compared to the use of selected crop plants. Discovered in 2001, P. vittata is a fern belonging to the pteride family (Ma et al., 2001) which results to be very interesting because of its characteristics and potential application outcomes. Several publications are present on role and interference of phosphate on As uptake and accumulation in hyper-accumulator (Danh et al., 2014). Even now, this species still continues to attract research both for basic studies on physiological and biochemical mechanisms that characterize hyper-accumulator plants, as well as to test and evaluate its practical application in the decontamination of matrices such as soil and water contaminated by arsenic (Tiwari et al., 2016). In this work, we also investigated the addition of phosphate to this species, since some contrasting results have been previously obtained. In some cases, an increase in the accumulation of As was highlighted, with a subsequent reduction of leaching (Cao et al., 2003), while other studies have shown no increase in the accumulation of As (Caille et al., 2003), or even a reduction if cultivated in different substrates like in hydroponic experiments (Wang et al., 2002). In addition to check whether phosphate treatments can further improve its phyto-extractive efficiency, the investigation on P. vittata also aimed to highlight possible differences compared to the use of crop plants and evaluate which is the best solution. Finally, subsequent regrowth tests were conducted for crop plants, outlining clear guidelines for a practical application in the field. In addition to clarify the actual efficacy of the selected species to treat the contaminants under consideration, the extensive investigation aimed primarily at affirming the real possibility of a concrete and feasible use of the phytoremediation, even in the case of complex contamination where pollutants are characterized by a significantly different behavior, thanks to the use of appropriate methods of addition of the right amendments.

2. Materials and methods 2.1. Site description and sampling The soil considered was collected from an industrial area in the middle of Italy (Tuscany), which has an As contamination arising from chemical plant activities. Soil samples were collected from the plough layer (0e20 cm), air-dried and sieved through a 2 mm sieve before laboratory analysis. Soil pH was determined using a glass electrode at a soil/water ratio of 1:2.5 (Thomas, 1996), cation exchange capacity (CEC) using barium chloride (pH ¼ 8.1) (Sumner

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and Miller, 1996), and texture (sand, silt, and clay) by the pipette method (Gee and Bauder, 1986). N was determined by the regular Kjeldahl method using a block digestor (Bremner, 1996), P by digestion with perchloric acid (Kuo, 1996), K by the ammonium acetate method (Helmke and Sparks, 1996), and organic matter by wet combustion (Nelson and Sommers, 1996). The contamination is mainly due to As and Pb and appears to be highly heterogeneous. On this basis, the area was subdivided in Thiessen polygons and the most significant soil samples were chosen to verify the feasibility of the phyto-extraction and determine which plant species, among those chosen, were the most suitable. On the considered soil samples (three for each selected polygon), total concentrations of As and Pb were determined, together with their speciation by sequential extraction.

2.4. Further investigation for the combined removal of arsenic and lead In view of the investigations conducted during this experimentation, it was decided to carry out further tests on a polygon in particular, number 409, which proved to be the most interesting for the application of phyto-extraction. The mesocosm tests were prepared as reported in Table 2. For these tests, the two types of treatment (K2HPO4 and EDTA) were added in succession during the same day, at a few hours of distance from each other. More details on this procedure can be found in the corresponding section of the results.

2.5. Tests of subsequent growth

2.2. Speciation and bioavailability of metals Given their different chemical nature, the potentially bioavailable concentrations of the two metals have been determined using two separate tests. The speciation of As, whose solubility in water is lower than the limit of detection, was performed by a sequential extraction with 0.05 M (NH4)2SO4 and 0.05 M K2HPO4, according to the first steps of the procedure suggested by Wenzel et al. (2001). A sequential extraction procedure with H2O, 1 M KNO3 and 1% EDTA (Pedron et al., 2009) was applied to determine Pb chemical forms in soil. The first two steps (H2O and KNO3) reflect the quantity readily available to the plants, whereas the third one indicates the maximum amount of lead that can be moved in the liquid phase from a soil by a treatment with EDTA, the additive chosen for the assisted phyto-extraction. For samples that showed the most significant values of lead contamination, another complexing agent of Pb, the EDDS, was tried in alternative to EDTA, due to its higher biodegradability with respect to the latter, which would make it preferable its use in real applications in case of proved effectiveness.

To evaluate the effect of treatments, a second growing cycle was carried out on the same soil (polygon 409). Details are reported in Table 3.

2.6. Mesocosms tests with Pteris vittata The campaign of mesocosms on P. vittata was conducted according to the modalities reported in Table 4. All soils have been tested with the exception of the 320, which was discarded for its low arsenic content as a not sufficient number of plants was available.

2.7. Lead and arsenic analysis Samples from the sequential extraction of soils and of arsenic concentration from plants were analyzed by ICP-OES (Varian AX Liberty) using a method for the generation of hydrides (Sparks, 1998). On the same samples, Pb concentrations were determined via atomic absorption spectrophotometry (Sparks, 1998) using flame AAS (Varian AA 240FS).

2.8. Quality assurance and quality control 2.3. Mesocosm tests Mesocosms were polypropylene containers filled with contaminated soil where selected species were grown. Each pot had a hole in the middle of the base where a plastic tube was inserted to collect the leachate in a plastic bottle. After 30 days from sowing, the treatment of soil with additives started according to an experimental design already used in Pedron et al. (2009). K2HPO4 for As and EDTA for Pb were respectively selected as mobilizing agents. Control mesocosms (CT) consisted of replicates for each species grown in untreated soil in the same conditions and were run simultaneously. The collection of any leached liquid was planned before starting the treatment as well as at the end of the test, when the plants are harvested. During the experimental campaigns, the seeds germination was assessed to be greater than 96%. After harvesting, in addition to a washing with deionized water, the sample collecting procedure included an ultrasonic treatment (Branson Sonifier 250 ultrasonic processor; Branson, Danbury, Conn.) on root samples in order to remove the soil particles possibly still present. In the end, the plant mineralization was achieved through grounding and digesting dry plant samples with an acid mixture (HNO3þH2O2). Details are reported in Table 1. The agronomic soil analysis has shown, even with their variability, a sufficient amount of nitrogen, phosphorus and potassium, thus no further inputs of fertilizer was added at this stage.

QA/QC were performed by testing two standard solutions (0.5 and 2 mg L1) every 10 samples. Certified reference materials, CRM ERM e CC141 for soil and CRM ERM - CD281 for plants, were used. In the case of Pb, the values obtained were 31.1 ± 1.2 mg kg1 and 1.69 ± 0.20 mg kg1, in good agreement with the certified values of 32.2 ± 1.4 mg kg1 and 1.67 ± 0.11 mg kg1, respectively. For As, the values were 7.3 ± 0.9 mg kg1 and 0.04 ± 0.01 mg kg1, in the same order, in agreement with the certified values of 7.5 ± 1.4 mg kg1 for CRM ERM e CC141 and 0.04 ± 0.01 mg kg1for CRM ERM e CD281. The detection limits for Pb and As were of 5 and 50 mg L1, respectively. The recovery of spiked samples (5%) ranged from 93% to 101% with a relative standard deviation (RSD) of 1.93% of the mean for Pb and from 92 to 101% with a RSD of 1.90 of the mean for As.

2.9. Statistical analysis All statistical analyses were performed using STATISTICA (v.6.0 by StatSoft, Inc. (StatSoft, 2017)). Effects of treatments were analyzed using one-way analysis of variance (ANOVA). Differences between means were compared and a post-hoc analysis of variance was performed using the Tukey's honestly significant difference test (P < 0.05).

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Table 1 First mesocosm campaign. Separate investigation on the removal of arsenic and lead. Entry

Quantity

Additional Information

Mesocosms [#]

111

Total duration [days]

60

Mobilizing solution [mL per mesocosm]

250

Plants growth conditions

e

Sample collection procedure

e

 Tests were conducted in greenhouse using 5 kg for each pot.  Each mesocosm was prepared using 12 seeds for L. albus, 9 seeds for H. annuus and 0.5 g for B. juncea.  For each plant species, trials were set up to have one control (in duplicate) and one treatment (in triplicate); 75 pots (5 per species, on each of the five soil samples investigated) for the As investigation, and 36 pots in the case of Pb (6 per species, on each of the two soil samples investigated).  For each pot, the collection of leached liquid (via a silicone tube that connected the base of the pot with a container on the ground) was planned before starting the treatment as well as at the end of the test, when plants were harvested.  The daily average volume of irrigation water was of 200 mL per pot.  In the first phase of growth of the plants, a daily irrigation was carried out by spraying the water on the ground surface to avoid disturbance to seeds.  Treatment began after about 30 days from sowing.  The mobilizing agents were 0.1 M K2HPO4 (0.82 g kg1soil) for As, and 2 mM EDTA (0.04 g kg1 soil) for Pb. In addition, for lead contamination also 10 mM EDDS was investigated.  To minimize the possible phytotoxic effects, the solutions were added by dividing the total doses during five days, supplying a daily volume of 50 mL per mesocosm, diluted in 150 mL of water to meet the crop's water needs.  In these first tests, no fertilizer was added to not introduce additional variables in the system. The greenhouse, located in the Research area of the CNR in Pisa, was maintained under control of temperature (24-19  C for day/nights period) and humidity (about 65%), in order to create the best conditions for germination and growth of plants.  The collection procedure started on average about ten days after the end of the treatments, leaving the plants in the pots until it was possible, based on their health status.  Plants were inspected to accurately separate the aerial part from the roots.  Samples were washed with deionized water; for the roots, a further washing within an ultrasonic bath for 10 min was performed in order to remove the soil particles possibly still present.  Samples were dried in a ventilated oven at 40  C until constant weight and their dry weight was gravimetrically determined.  At the end, plant samples were mineralized and analyzed, to determine the concentrations of accumulated metals: known weights of dried vegetal tissues were ground and then digested using acid-oxidant digestion (HNO3/H2O2 mixture 2.5:1 ratio), using an ETHOS-900 microwave system (MILESTONE S.r.l., Bergamo, Italy) with a pulsed-mode emission in Teflon vials. After digestion, plant samples were made up to 25 mL with milliQ water and then analyzed.  Analyses were conducted using IPC for arsenic and atomic absorption for lead.

Table 2 Mesocosm campaign for the combined removal of arsenic and lead. For the omitted information, refer to Table 1. Entry

Quantity Additional Information

New Mesocosms 15 (polygon 409) [#]

 For each plant species, trials have been set up to have one control (in duplicate) and one treatment (in triplicate).  The mobilizing agents (0.1 M K2HPO4 for As and 2 mM EDTA for Pb) were administered together, at a few hours of distance.  A NPK fertilization was also carried out: the solution was prepared by diluting 11 mL of liquid fertilizer (Gesal® universal fertilizer) with 989 mL of water and then by supplying 250 mL of this solution per mesocosm, for four times, distributed over a period of 2 weeks.

3. Results and discussion 3.1. Soil characteristics and metals bioavailability The main characteristics of the soil are reported in Table 5. While these values are the same in the whole area, the concentration of As and Pb varied greatly within the contaminated site. On this basis, the most significant soil samples were chosen, to verify the feasibility of the phyto-extraction. The mean total concentrations of As and Pb are reported in Table 6, together with their extractability by sequential extraction. The table shows the significant variability of arsenic concentration within the contaminated area that has been divided according to Thiessen polygons. As can be noted, there are polygons in which the total As concentration is around 25 mg kg1 and others in which it is approximately 100 times greater. When comparing the extractable quantities, the differences greatly reduced with

Table 3 Additional mesocosm campaign for the combined removal of arsenic and lead: second regrowth tests. For the omitted information, refer to Table 1. Entry

Quantity

Additional Information

2nd Regrowth Mesocosms (polygon 409) [#]

10

 Each mesocosm was prepared using 10 seeds for H. annuus and 0.8 g for B. juncea. L. albus was not further investigated.  For each plant species, trials were set up to have one control (in duplicate) and one treatment (in triplicate).  An NPK fertilization was also carried out by diluting the solution of a liquid fertilizer (Gesal® universal fertilizer) in water 1:100. The dose was of 250 mL of this solution per mesocosm, for four times, distributed over a period of 2 weeks.  The mobilizing agents were K2HPO4 for As at a concentration double (0.2 Me1.74 g kg1 soil) with respect of previous tests, and 2 mM EDTA for Pb. They were administered together, at a few hours of distance.

sulfate extracts, while remained significant in the second extraction step when phosphate was used as extractant. With respect to the total content, the extractable (and therefore potentially bioavailable) amount is rather low, with percentages ranging from less than 1% (polygon 117) to just over 3% (polygon 434). Regarding Pb, similar considerations can be drawn. The total content is highly variable from 34.7 (polygon 320) up to 3234 mg kg1 d.m. (polygon 443). The soluble amount (water extraction) and those exchangeable (KNO3 extraction) are very low. The extraction with EDTA dissolves considerable quantities of metal,

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Table 4 Mesocosm campaign for the removal of arsenic using Pteris vittata. For the omitted information, refer to Table 1. Entry

Quantity

Additional Information

Mesocosms [#]

20

Total duration [days]

270

Mobilizing solution [mL per mesocosm]

10

Plants growth conditions

e

 The transplant was carried out by mixing the contaminated soil with topsoil in the ratio 4:1 and 1.5 g per pot of a slow-release fertilizer (Baycote®, Bayer CropScience S.r.l. Milano, Italy) were added to improve growth conditions. The fertilizer contains NP-K at 17-6-9% respectively and soluble microelements: B 0.02, Cu 0.06, Fe 015, Mn 0.06, Mo 0.007, Zn 0.015%.  Tests were conducted in greenhouse using pots with a volume of 500 mL (c.a. 750 g).  In each pot, one P. vittata plant at fourth frond stage was transplanted.  The plants of P. vittata transplanted were five for each of the soil considered (soil 320 was not included in these tests).  Trials were set up to have two controls and one treatment (in triplicate).  The daily average volume of irrigation water was of 10 mL per pot.  Irrigation was carried out by spraying the water on ground surface.  Treatment began after about 120 days from the transplant.  Twigs with evident signs of distress of each test were pruned before starting the test.  The mobilizing agent was 0.1 M K2HPO4.  To minimize possible phytotoxic effects, the solution was added by dividing the total doses during five days, supplying a daily volume of 2 mL per mesocosm, diluted in 8 mL of water to meet the crop's water needs. The climatic chamber was maintained under controlled conditions of temperature (28-23  C for day/nights period), humidity (about 70%) and illumination (200 mmol m2 s1 for 14 h day1), in order to create the best conditions for germination and growth of plants.

proportional to the total content. The results from the extractability tests, showing very low amount of bioavailable metals, strongly support the need of using an assisted phyto-extraction to remove larger amounts of As and Pb. In regards to EDDS, it is known to form with Pb less stable complexes than EDTA, but this last forms also complexes with Ca (present in great abundance in the soil), so the concentrations of Pb extracted with the two complexing agents is quite similar (Doumett et al., 2008). This result makes the EDDS interesting to investigate, as it has a higher biodegradability with respect to EDTA. Anyway, the high variability of As and Pb concentrations in the site, clearly highlighted by the data in Table 6, should be taken into account during the design of the real remediation process, when it is necessary to estimate the contamination baseline for evaluating the objectives and procedures for implementing the technology (Ferrucci et al., 2017). 3.2. Mesocosm tests The results of As uptake from mesocosm test described in

Table 5 Soil characteristics. pH OM CEC [cmol(þ) kg1] Clay Silt Sand

8.26 3.53% 17.5 8.4% 18.0% 73.6%

Table 6 Total concentrations of arsenic and lead and extractable amounts in the contaminated soil samples. Data are reported as mean values [mg kg1 d.m.] and standard deviation.

Arsenic

Lead

Sample

443

117

409

434

320

Average St. dev. 0.05 M (NH4)2SO4 0.05 M K2HPO4 Extractable % Average St. dev. H2O 1 M KNO3 1% EDTA 10 mM EDDS Extractable %

2595 90 1.4 30.1 1.2 3234 194 1.1 1.8 168 123 5.3

581 37 0.3 1.8 0.4 50 12 0.1 0.9 6.8 e 15.6

1064 64 4.1 24.9 2.7 2150 101 0.9 1.9 123 92 5.9

878 30 3.5 23.6 3.1 512 79 0.3 1.7 55.6 e 11.3

25 8 0.1 0.3 1.4 35 13 0.1 1.1 7.4 e 24.8

Table 1 are reported in Table 7 for each plant species (aerial and root part). The results obtained show that there is no substantial influence of the treatment with phosphate on biomass production: no statistically significant differences between the biomass production of plants grown on untreated and K2HPO4- treated soils were detected. On the other hand, there was no evidence of adverse effects such as yellowing of the leaves and other symptoms of suffering by plants, even if the treatment increased the As uptake by plants. In general, the treatment with the K2HPO4 solution has favored the uptake of As by the plants in all soils examined, confirming what was observed in previous experiments (Tassi et al., 2004a, 2004b). This effect was seen both in the roots and in the aerial part, with increases ranging from 2 to 30 times; in all these tests, apart from those performed with soil 117, the best response was observed by H. annuus, with the highest concentrations of As in the shoots. The results obtained are in agreement with the sequential extraction tests, which showed (Table 6) a greater presence of soluble forms of arsenic in samples 409, 434 and 443 (these are also the soils with the highest As content). However, after the addition of phosphate, a good uptake by plants was found also in the case of soil sample 117. The total amount of arsenic removed by the plants, referred to as the total uptake (Jarrell and Beverly, 1981), was calculated by multiplying the concentration of the element for the corresponding biomass produced; the obtained values are shown in Fig. 1. The trend of the total uptake confirms that the treatment with phosphate dramatically improves the ability to extract arsenic from the soil by all three investigated species. Considering the overall contribution of the root and aerial part, the major values were obtained with B. juncea. However, the best response to treatment is observed in H. annuus, with a higher increasing of the extracted quantity compared to untreated samples, and a strong ability to displace almost all the pollutant in the aerial section, since the contribution of the aerial part is the most important in phytoextraction. The evaluation of plants for phyto-extraction can be derived from the ratio between the concentration of contaminants in the shoots and the total concentration in soil (Cshoot/Csoil). This relationship is often called phyto-extraction coefficient (PEC) or bioconcentration factor (BCF) and can be used to evaluate plants species in a short-growing period in terms of phytoremediation efficiency. When PEC is calculated for a remediation target based on the total concentration, a value higher than 0.6 is considered acceptable for arsenic phyto-extraction (Ampiah-Bonnet et al.,

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Table 7 Biomass and uptake of arsenic for the mesocosm tests described in Table 1. Data are reported as mean values and standard deviation. Note: the effect of treatment was examined separately for each plant species, for shoots and roots, and for each soil sample. Values in each row with the same letter were not significantly different from each other, according to the Tukey test (p < 0.05). Lupinus albus Biomass [mg d.m.] CT

Sample 443

Sample 117

Sample 409

Sample 434

Sample 320

Aerial Mean St. dev. Roots Mean St. dev. Aerial Mean St. dev. Roots Mean St. dev. Aerial Mean St. dev. Roots Mean St. dev. Aerial Mean St. dev. Roots Mean St. dev. Aerial Mean St. dev. Roots Mean St. dev.

1821a 490 521a 270 806a 449 205a 77 1215a 228 105a 34 711a 145 113a 21 3203a 458 425a 42

K2HPO4 0.1 M 2037a 685 411a 267 2176a 1516 343a 160 1520a 356 142a 57 1491b 380 280b 65 3837a 686 587b 72

Brassica juncea

As [mg kg CT 30a 4 444a 133 15a 2 23a 2 45a 6 310a 121 28a 3 678a 165 2a 0.3 4a 0.9

1

]

K2HPO4 0.1 M 153b 23 1858b 696 163b 31 249b 33 156b 25 1865b 909 149b 19 2766b 840 21b 3 41b 12

Biomass [mg d.m.] CT 3523a 415 112a 28 2614a 497 716b 147 2263a 326 302a 93 3416a 350 519a 85 2487a 299 618a 132

2007). The results obtained in this study show that, with regard to As, this value was initially above 0.60 only for H. annuus grown in the soil from polygon 320, with a value of 1.20. After the treatment with phosphate, also B. juncea and L. albus plants showed a PEC higher than 0.60 in the polygon 320, with values of 0.84 and 1.32 respectively, whereas in the same polygon the PEC of H. annuus raised up to 2.0. If the target of phyto-extraction is to remove only the “bioavailable fraction”, the PEC can be calculated as the ratio between the concentration of the metal in the aerial parts of the plants and the potential bioavailable concentration in the soil. This ratio has been defined bioavailability factor (BF) (Mandal et al., 2012) and the same value of 0.60 might also be indicatively assumed as acceptable when the remediation target is focused on the bioavailable fraction of arsenic in the contaminated soil (Pedron et al., 2015). Considering the bioavailable fraction as the amount extractable by phosphate extraction, in the present study the PEC value was always above 0.60, even in untreated soils. For L. albus it varied from 1.00 (polygon 443) to 6.67 (polygon 320), whereas it ranged from 1.27 (polygon434) to 26.6 (polygon320) for B. juncea. Also in this case H. annuus showed the highest values of BF ranging from 2.22 (polygon 117) to 100 (polygon 320). However, after adding the mobilizing agent, arsenic concentration increased considerably in plants and PEC values grow to a maximum of 70 for L. albus (polygon 117), 110 for B. juncea (polygon 320) and 167 for H. annuus (polygon 320). Another parameter that has to be taken into account is the ability of plants in the translocation of the contaminants from the roots to the shoots defined by translocation factor (TF): the ratio between the concentration of an element in shoots and in roots (Cshoot/Croot). While greatly increasing the concentration of arsenic absorbed by the plants, phosphate treatment did not introduce significant changes in this ratio: for H. annuus in untreated soils, the values ranged from 0.38 in polygon 443 to 0.85 in polygon 409, whereas TF values in the treated polygons ranged from 0.30 in polygon 443 to 0.89 in polygon 117.

K2HPO4 0.1 M 3785a 557 170a 53 2400a 571 412a 106 3181b 573 457a 176 3236a 415 516a 105 2322a 349 565a 151

Helianthus annuus

As [mg kg CT 69a 2 975a 331 39a 2 27a 8 54a 1 873a 15 30a 1 620a 19 8a 0.6 11a 2

1

]

K2HPO4 0.1 M 272b 8 1530a 649 111b 8 232b 82 149b 5 2262b 47 144b 9 1706b 67 33b 3 62a 16

As [mg kg1]

Biomass [mg d.m.] CT 1321a 330 113a 40 3952a 468 305a 51 1513a 195 113a 2 1709a 370 115a 3 1646a 206 201a 35

K2HPO4 0.1 M 2586b 808 209a 93 4585a 679 274a 57 1435a 231 111a 2 1733a 469 112a 4 2735b 428 277a 60

CT 165a 5 427a 116 4a 0.4 5a 0.5 125a 5 146a 17 97a 2 156a 3 30a 3 51a 5

K2HPO4 0.1 M 527b 22 1753b 597 135b 16 151b 17 355b 16 605b 90 226b 6 322b 8 50b 7 70b 8

Also for L. albus, TF values were substantially identical before and after treatment, ranging from 0.04 (polygon 434) to 0.65 (polygon 117) for untreated soils and from 0.05 (polygon 434) to 0.65 (polygon 117) after treatment with phosphate. Only B. juncea plants, which showed the greater variability between the polygons ranging from 0.07 in polygon 443 to 0.72 in polygon 320, it showed a slightly more pronounced variation following phosphate treatment, with values ranged from 0.06 (polygon 409) to 0.53 (polygon 320). Obviously, the value of the TF ratio is just indicative of the potential efficiency of phytoremediation, but it is important to note that, for arsenic, there are significant differences between plant species and some differences even for the same plant depending on the soil where it has grown. These aspects will have to be taken into account in planning remediation at field scale. In the same samples prepared for investigating the removal of arsenic, the behavior of the lead was also checked. However, no significant concentration changes were found in soils: as expected, the addition of phosphate did not substantially modify the amount of lead absorbed by plants. Indeed, under the experimental conditions adopted, phosphate does not form soluble compounds with lead as the PbHPO4 solubility product (Clever and Johnston, 1980) is not reached in the soil solution and thus phosphate addition did not increase Pb amount in the soil solution and did not change Pb bioavailability. This confirms the fundamental role played by the bioavailability processes in the soil, and that, to ensure a greater extraction efficiency by plants, it is necessary to resort to the addition of a specific additive for lead. Therefore, mesocosm tests were set up to evaluate the effect of the addition of EDTA on lead mobilization and removal. Soils from polygons 409 and 443 where selected due to their higher Pb contents. Since, as known, many criticisms have been raised on the use of EDTA due to its low degradability and long persistence in soil with a possible toxic effect on plant growth, contextually it was examined whether it would be possible to employ a different additive, the EDDS, which is characterized by an intrinsically lower toxicity. The results are reported in Table 8.

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Fig. 1. Total uptake of arsenic during the mesocosm tests described in Table 1.

Positive results were obtained with both complexing agents, which induced a very similar response in plants also in terms of total uptake (Fig. 2). The EDDS, although being slightly less effective, has the advantage of being more readily biodegradable; unfortunately, it is also more expensive. All these aspects should be considered in the evaluations during the design phase of the field trial. Unlike the case of arsenic, there are no PEC reference values for lead. The results showed that values are essentially the same for the three plant species tested and for all polygons, with an average value of 0.010 for untreated soils. The trend remained the same even after treatment with the mobilizing agent (EDTA), without differences among plant species and among polygons, showing a slight increase in the PEC mean value up to 0.018. Considering the extractable amount in EDTA as the potentially bioavailable fraction, similar results were also obtained in the case of BF, which remained very similar among the various species and among the polygons, and with an average value of 0.19 which raised to 0.30 after the treatment with the mobilizing agent (EDTA 2 mM). Also regarding TF values, substantially no differences between

the three plant species and little variability between different polygons were found. The mean value of TF was 0.20 and this value did not change as a result of the addition of the mobilizing agent. Thus in the case of lead, PEC and TF ratios appear less utilizable to plan the phytoremediation. Once verified the efficiency of the selected mobilized agents in the investigated soils, their addition in sequence had to be evaluated to determine the best strategy to use in the field. As mentioned, also the leachate collection was predisposed for additional investigations. However, in these as well as in subsequent tests, no appreciable production of leachate was observed. The main reason for this is due to the mode of irrigation, which was conducted in order to provide the plants with only the amount of water necessary to their welfare.

3.3. Further investigation for the combined removal of arsenic and lead The simultaneous phyto-extraction of arsenic and lead is not easy to realize because of their different chemical behavior in the

39b 2 312b 17 39b 2 302b 15 42b 3 310b 23 45b 4 310b 34 27a 1 163a 10 28a 2 131a 11 1410a 140 180b 20 1320ab 110 120a 11 1260a 80 150b 10 1250a 90 150b 12 30b 4 335b 52 37b 2 226b 14 37b 3 383b 37 38b 3 225b 19

1320a 70 110a 6 1510b 87 110a 7

EDDS 10 mM EDTA 2 mM CT EDDS 10 mM EDTA 2 mM

CT

EDTA 2 mM

EDDS 10 mM

20a 1 170a 13 23a 1 92a 6 3810a 320 150b 14 2200a 130 250a 16 3990a 380 110a 12 2310a 230 320a 35 3520a 270 110a 9 2260a 180 300a 26 32b 4 275b 40 31b 4 232b 34

CT EDDS 10 mM EDTA 2 mM CT EDDS 10 mM

Aerial

Roots Sample 409

Roots Sample 443

Aerial

Mean St. dev. Mean St. dev. Mean St. dev. Mean St. dev.

1750a 240 500a 75 1450ab 210 100a 16

19a 2 119a 12 21a 2 99a 12 1780a 320 480a 95 1520b 180 130a 17 1820a 260 520a 82 1220a 116 110a 12

EDTA 2 mM CT EDTA 2 mM CT

EDDS 10 mM

Pb [mg kg1]

35b 4 280b 34 32b 4 229b 35

Pb [mg kg1] Biomass [mg d.m.]

Helianthus annuus

Pb [mg kg1] Biomass [mg d.m.]

Brassica juncea

323

soil. On soil from polygon 409, where the concentrations of both contaminants are very high, further mesocosm experiments were carried out by performing both treatments (phosphate and EDTA) contextually, for the combined removal of the two pollutants. The addition of the two additives was carried out in succession in the same day, firstly phosphate and then EDTA, the latter being administrated after 6e7 h; the treatment was realized during 5 days. The choice of providing firstly the phosphate is due to the fact that, in addition of being a mobilizing agent for As, it acts also as a fertilizer and thus can be a support for plants subjected to stress due to the uptake of metals and the presence of EDTA, which may cause phytotoxic phenomena. Also in this case, despite the different conditions due to the joint addition of the two mobilizers, no formation of any soluble compounds of phosphate with lead was observed. The results obtained are reported in Table 9, whereas Fig. 3 shows the total uptake registered for the two contaminants. From these tests, B. juncea appears to be the best species to accumulate Pb, whereas H. annuus showed the best results for As. Data show that, with this timing, no antagonistic effects and no reductions in the effectiveness of EDTA in the mobilization of lead have been encountered. Finally, such a procedure can be easily adopted in field tests, with great savings in the time of treatment. 3.4. Tests on subsequent growth

Biomass [mg d.m.]

Lupinus albus

Table 8 Biomass and uptake of lead for the mesocosm tests described in Table 1. Data are reported as mean values and standard deviation. Note: the effect of treatment was examined separately for each plant species, for shoots and roots, and for each soil sample. Values in each row with the same letter were not significantly different from each other, according to the Tukey test (p < 0.05).

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After harvest, a further growing cycle was carried out on the same soils in order to investigate the effect of a second treatment with K2HPO4 and EDTA. Obtained data are reported in Table 10. In this case, the concentration of the phosphate added was increased to 0.2 M. In general, the biomass developed by plants during the second regrowth was lower than that harvested with the first crop. It should also be noted that, during the second regrowth, in the case of the treated pots, the plants showed signs of suffering in the early stages of growth. Regarding the concentrations of As and Pb, during the second regrowth, the two considered plant species have shown a different response. The lead concentration in the aerial part of B. juncea was slightly less than the one get with the first growth, with an average value of 83 mg kg1 as compared to 112 mg kg1. On the contrary, H. annuus showed an opposite trend, with a higher concentration of the metal which increased from 60.2 mg kg1 of the first growth to 114 mg kg1. In regards to As, a positive effect of phosphate 0.2 M was found in B. juncea (584 mg kg1 in the shoots as compared to 142 mg kg1) and H. annuus (716 mg kg1 compared to 314 mg kg1). The same trend was also found for the roots. The total uptake of both contaminant compounds during this second regrowth are illustrated in Fig. 4. The results from these mesocosm tests indicate that the application of phosphate with a double concentration represent an effective strategy, which can be used even in full field to facilitate the absorption of As during the reclamation. For example, a low concentration of phosphate could be used for the first crop, and a double dose supplied to the successive growths. 3.5. Mesocosm tests with Pteris vittata As anticipated, tests using P. vittata with soil samples taken from the contaminated site of interest had a threefold purpose: i) assess the growth capacity under these conditions; ii) check whether a treatment with phosphate can further increase the metal accumulation; iii) investigate differences with respect to the use of crop plants and evaluate what is the best solution. After the transplant, it was observed that some plants (20% of

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Fig. 2. Total uptake of lead during the mesocosm tests described in Table 1.

Table 9 Biomass and uptake of metals for the mesocosm tests described in Table 2. Data are reported as mean values and standard deviation. Note: the effect of treatment was examined separately for each plant species, for shoots and roots, and for each metal. Values in each row with the same letter were not significantly different from each other, according to the Tukey test (p < 0.05). Biomass [mg d.m.]

Lupinus albus

Aerial Roots

Brassica juncea

Aerial Roots

Helianthus annuus

Aerial Roots

Mean St. dev. Mean St. dev. Mean St. dev. Mean St. dev. Mean St. dev. Mean St. dev.

As [mg kg1]

Pb [mg kg1]

CT

K2HPO4 0.1 M þ EDTA 2 mM

CT

K2HPO4 0.1 M þ EDTA 2 mM

CT

K2HPO4 0.1 M þ EDTA 2 mM

2502a 223 187a 11 5260a 829 202a 72 3420a 639 226a 50

2255a 251 175a 13 4127a 812 430a 192 2440a 570 208a 58

39a 3 189a 22 58a 14 874a 208 107a 3 100a 20

107b 11 917b 132 142b 42 1966b 584 314b 11 897b 221

22a 1 99a 16 30a 4 206a 57 15a 3 126a 30

49b 3 155b 31 112b 18 387a 133 60b 15 275b 82

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325

Fig. 3. Total uptake of arsenic and lead during the mesocosm tests described in Table 2.

Table 10 Biomass and uptake of metals for the mesocosm tests described in Table 3. Data are reported as mean values and standard deviation. Note: the effect of treatment was examined separately for each plant species, for shoots and roots, and for each metal. Values in each row with the same letter were not significantly different from each other, according to the Tukey test (p < 0.05). As [mg kg1]

Biomass [mg d.m.]

Brassica juncea

Aerial Roots

Helianthus annuus

Aerial Roots

Mean St. dev. Mean St. dev. Mean St. dev. Mean St. dev.

Pb [mg kg1]

CT

K2HPO4 0.2 M þ EDTA 2 mM

CT

K2HPO4 0.2 M þ EDTA 2 mM

CT

K2HPO4 0.2 M þ EDTA 2 mM

1720a 210 73a 11 470a 100 29a 6

1560a 230 62a 15 490a 160 30a 5

57a 5 284a 26 172a 14 120a 20

584b 25 1830b 171 716b 30 1269b 138

17a 2.3 172a 13 33a 3 152a 21

83b 4 297b 16 114b 16 322b 28

the ferns) were not able to overcome the shock. This percentage is considered normal for the type of plantation, particularly delicate especially in its early stages of growth. After this first period, despite some plants showed signs of stunted growth (resulting in

drying and subsequent death), the percentage of plants lost did not increase. By analyzing the symptoms of dead plants, a possible reason was found to be the high content of As in the soils, because such phenomenon has already been observed by other authors

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Fig. 4. Total uptake of arsenic and lead during the 2nd growth mesocosm tests described in Table 3.

(Caille et al., 2003; Wang et al., 2002). However, the previous experiments on P. vittata described in literature differ from this investigation not only for the different types of soils, but also for the different modes of application of the phosphate. In particular, in the experimentation by Cao et al. (2003) and Caille et al. (2003), the phosphate was supplied by mixing it to the culture medium before the transplantation of the plants, while in our case the phosphate addition was performed only at the second month of growth, thus after a period of adaptation for the ferns. The results obtained in this study (Table 4) are reported in Table 11 and Fig. 5. Phosphate treatment showed its beneficial effects increasing the biomass production in all tested soils, in particularly in the soil from polygons 443 and 117 (respectively 16 and 9 times). The arsenic uptake also significantly increased in phosphate treated samples, except for the sample 117. Although performed with different soils and level of As contamination, the obtained findings are in general accordance with the majority of previous experiments. The As uptake increased in the fern, when phosphate was added to As contaminated soils in

Table 11 Biomass and uptake of arsenic on Pteris vittata obtained for the mesocosm tests described in Table 4. Data are reported as mean values and standard deviation. Note: values in each row with the same letter were not significantly different from each other, according to the Tukey test (p < 0.05). Biomass [mg d.m.] K2HPO4 0.1 M

CT Sample 443 Sample 117 Sample 409 Sample 434

Mean St. dev. Mean St. dev. Mean St. dev. Mean St. dev.

a

302 33 342a 13 174a 21 657a 25

b

4875 120 3103b 125 652b 26 1469b 101

As [mg kg1] CT

K2HPO4 0.1 M a

1661 198 1176a 78 3219a 475 2712a 177

4502b 318 1101a 70 4569b 602 4471b 105

which the fern was being grown (Tu and Ma, 2003; Cao et al., 2003; Fayiga and Ma, 2006; Mandal et al., 2012; Lessl et al., 2014). This seems to confirm that any different behavior than that observed is to be attributed to additional interferences related to

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327

consideration. All efforts should be done to increase the biomass production. The best fertilization program must be planned, according to plant species, specific soil and climatic conditions. The preparation of the seedbed must be very accurate to allow optimum seed-soil contact and a uniform depth of sowing. This activity should take place some days before planting, to allow a homogenization of the humidity within the surface layer, by means of irrigation, so as to optimize the phase of seed germination. The irrigation planning is essential for remediation. Therefore, it is necessary to consider both the amount of water needed (by monitoring the drainage, which can affect the stability of the soil) and the potential leaching of metals solubilized by the additives. Indeed, the recourse to solubilizing agents introduces an environmental risk due to their high mobility in the soil, which can result in a displacement of the contaminant along the soil profile. Alternative methods to induce the phyto-extraction should be considered to avoid introducing risks to the environment: for instance, positive effects have been obtained with the use of specific fertilizers such as phosphate for arsenic and thiosulfate for Mercury and arsenic (Franchi et al., 2016). A promising possibility to increase the biomass production consists in enriching the rhizosphere with bacteria that promote the plants growth (PGPB), in order to stimulate shoot initiation, delay the leaf senescence, and enhance the resistance to potentially toxic metal stress of the growing plants (Ullah et al., 2015). Fig. 5. Total uptake of arsenic on Pteris vittata obtained for the mesocosm tests described in Table 4.

the nature of the soil or contamination from other compounds. For instance, in the study by Shelmerdine et al. (2009), the fern has been grown in a sewage sludge amended soil, showing a decrease in As uptake in presence of high available phosphate concentrations; in this case, the same authors suggested that this behavior was ascribable to the presence of other potentially toxic metal(loid) s in the sludge. On the contrary, under the adopted experimental conditions, a comparison of the phyto-extraction potential for As of P. vittata and other crop plants pointed out that the fern is more effective, with a phyto-extraction capability about 13 times higher. The latter removed up to 20 mg of As (in soil 443), while the highest As removal in crop plants was about 1.5 mg for H. annuus, as reported in Fig. 1. However, P. vittata is a delicate species, with a difficult and slow growth. The inability to effectively increase its uptake with a mobilizing agent (since the plant autonomously adjusts the As uptake), together with the impossibility to simultaneously treat the contamination by lead, discourages its use compared to crop plants, which in fact have proven to be more versatile and able to compensate a smaller accumulation capacity with a rapid growth and the possibility to carry out more growing seasons. 3.6. Considerations for the field test One of the major limitations of phytoremediation consists in the quite long application times, since it is a technique related to vegetables cycles of growth. Based on results from the mesocosm tests, it is possible to assess that, with the adopted experimental conditions (chosen plants and tested additives), a significant part of the bioavailable fractions of As and Pb has been removed, without any problem of leaching arising from the addition of the mobilizing agents. However, to make a realistic estimation of the time required for a remediation, biomass production per hectare at field scale should be taken into

4. Conclusions The test results confirm the necessity to add specific mobilizing agents for improving the removal of arsenic and lead, in order to increase their bioavailability. In regards to the former, the addition of phosphate appears to produce the desired results, as it significantly contributes to the release of arsenate and arsenite ions within the soil solution, thus increasing their bioavailability for  ska and Karczewska, 2013; Niazi et al., 2017). plants (Lewin The concentration plays a primary role, as it is known that the sensitivity of plants to arsenic is closely linked to the absorption of phosphate: plants that are not initially resistant can increase their tolerance to arsenic by raising the internal levels of phosphorus, which causes a decrease in the accumulation of arsenate through a block of its transport. The results obtained seem to indicate that, for the investigated concentrations, the plants have grown in such conditions as to balance the internal phosphate-arsenate ratio, thus allowing an effective absorption of arsenic but minimizing its toxic effects. The treatment with phosphate positively affects the phytoextraction of As for both plant typologies (hyper-accumulator and not), but with different effects. If it is true that P. vittata further increases its potential, showing positive effects on both the biomass and the content of As, this gain is lost in the continuation of the tests. In any case, P. vittata has shown the highest phyto-extraction potential, but these ferns usually require specific environmental conditions. Their adaptability and growth rate make the results of their use in a real reclamation very uncertain, whereas the tested crop plants are well known and suitable for the Mediterranean climate conditions, and have the additional advantages of a fast growth and a lower selectivity towards absorbable metals. In regards to Pb, although results obtained with EDDS have been encouraging, at present we believe that the use of EDTA should be preferred, due to lower uncertainties about its effectiveness (which add to a lower cost). Further investigations on the phytotoxic effects of the two mobilizing agents could change the situation, especially if an advantage of EDDS in terms of environmental sustainability is confirmed.

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Having determined the need for the use of the two additives (precisely phosphate and EDTA), the best way to administer them has been investigated. Downstream of some additional microcosm tests, conducted in parallel to evaluate if and how different methods of adding the mobilizing agents could affect the growth of plants (data not shown to avoid further complicating the discussion), a procedure has been developed that involves their addition in succession (phosphate first and then EDTA, in the same day, after 6e7 h), with a total treatment diluted in 5 days. In this way, there is a time saving of about one week in the duration of the treatment. It is interesting to underline that EDTA manifests a good complexing capacity towards lead, even after the phosphate addition. Based on the results obtained, the same procedure could be applied in the field, if economically viable. Studies with these plant species have been carried out at mesocosm scale to better understand the trend of metals uptake, in a real contaminated soil. The experimental system has been designed to consider all factors that can influence metal phyto-availability, which is strictly dependent on the soil characteristics (Melo et al., 2011). The feasibility test provides decision-makers and stakeholders with the opportunity to evaluate which plant species should be used for the phytoremediation, by allowing a comparison of the results obtained with the hyper-accumulator species, with those achieved using crop plants. It must be considered that the hyperaccumulator species is specific for arsenic, and therefore is not able to interact with the lead. Using crop plants is instead possible to remove the bioavailable amounts of metals with the same plants, by providing a proper supply of additives (at different times, in the same day). Another very promising way is the use of PGPB that, by promoting the growth of plants, are able to increase the biomass produced, therefore the total uptake and, ultimately, the amount of metals actually removed from the soil. In any case, the implementation of such an approach must take into account the variables that come into play during a scale-up, including the strong heterogeneity of the site of interest, which could lead to significant differences in the behavior of plants. All these aspects need to be properly considered in order to optimize the efficiency of the remediation. Acknowledgements This research was supported by Eni S.p.A, Research & Technological Innovation Department, San Donato Milanese (Italy) and fully funded by Syndial S.p.A (contract number 3500038857). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jenvman.2017.07.078. References Ampiah-Bonnet, R.J., Tyson, J.F., Lanza, G.R., 2007. Phytoextraction of arsenic from soil by Leersia oryzoides. Int. J. Phytoremed. 9, 31e40. Barbafieri, M., Japenga, J., Romkens, P., Petruzzelli, G., Pedron, F., 2013. Protocols for applying phytotechnologies in metal-contaminated soils. In: Gupta, D.K. (Ed.), Plant-based Remediation Processes. Springer, Berlin, Heidelberg, pp. 19e37. Bhargava, A., Carmona, F.F., Bhargava, M., Srivastava, S., 2012. Approaches for enhanced phytoextraction of heavy metals. J. Environ. Manag. 105, 103e120. Blaylock, M.J., Salt, D.E., Dushenkow, S., Zakharova, O., Gussman, C., Kapulnik, Y., Ensley, B.D., Raskin, I., 1997. Enhanced accumulation of Pb in Indian Mustard by soil-applied chelating agents. Environ. Sci. Technol. 31, 860e865. Bremner, J.M., 1996. Nitrogenjtotal. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods, Soil Science Society of America Book Series. Soil Science Society of America Inc., Madison, pp. 1085e1121. Brooks, R.R., 1998. Plants that Hyperaccumulate Heavy Metals. CAB, New York. Caille, N., Swanwich, S., Zhao, F.J., McGrath, S.P., 2003. Arsenic hyperaccumulation

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