Crop suitability assessment in remediation of Zn contaminated soil

Chemosphere 246 (2020) 125706

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Crop suitability assessment in remediation of Zn contaminated soil Chiara Grassi a, Stefano Cecchi b, Ada Baldi a, Camillo A. Zanchi a, Simone Orlandini a, Andrea Pardini a, Marco Napoli a, * a b

Department of Agriculture, Food, Environment and Forestry (DAGRI), University of Florence, Piazzale delle Cascine 18, 50144, Firenze, Italy Institute for Bioeconomy (IBE) of National Research Council (CNR), via Madonna del Piano, 10, 50019, Sesto Fiorentino, Italy

h i g h l i g h t s
Spinacea oleracea uptakes Zn up to 1133.1 mg kg-1 in aboveground biomass.
Hordeum vulgaris was able to translocate efficiently Zn from roots to aerial tissues.
Zn level in the soil influenced the time requirement to reach plant maturity.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 August 2019 Received in revised form 13 December 2019 Accepted 17 December 2019 Available online xxx

Zinc (Zn) is naturally present in soils and constitutes an essential micronutrient for plants. Mining, industrial, as well as various agricultural activities all contribute to increasing the Zn concentrations in soils to levels that are toxic for plants. The aim of this study was to evaluate the capacity of field crops to remove Zn from contaminated soils. The experimental design included 28 treatments, comprising seven field crops (Hordeum vulgare L., Ricinus communis L., Phaseolus vulgaris L., Brassica juncea Czem., Sorgum vulgare L., Spinacea oleracea L., Solanum lycopersicum L.) and four Zn levels (0, 500, 1000, 1500 mg kg1) applied to soils. The dry weight (DW) of the aboveground biomass of R. communis and S. lycopersicum increased significantly as the Zn concentration in the soil increased, whereas the DW significantly decreased in P. vulgaris, B. juncea and S. vulgare. Results indicated that S. oleracea was the most efficient in concentrating Zn in the aboveground tissues, followed in decreasing order by H. vulgare, S. lycopersicum, R. communis, S. vulgare, P. vulgaris, and B. juncea. H. vulgare resulted the most efficient in accumulating Zn both in fruit and in leaves and stems, whereas S. lycopersicum resulted the most efficient in accumulating Zn in roots. The BAF and TF values indicated that H. vulgare and S. oleracea resulted being suitable for Zn phytoextraction, whereas the remaining crops being suitable for Zn phytostabilization. These results highlight the phytoremediation potential of the seven analysed crops. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: T Cutright Keywords: Zinc soil Phytoremediation Zinc translocation Bioaccumulation factor

1. Introduction Zn is classified as essential microelement, indispensable in small concentration for several physiological and metabolic processes in humans, animals and plants (Ali et al., 2013; Sarwar et al., 2017; Tang et al., 2009). In plants, Zn is involved in many physiological and metabolic processes, including phytohormone and enzyme activities, protein and carbohydrate metabolism, gene regulation

* Corresponding author. E-mail addresses: chiara.grassi@unifi.it (C. Grassi), [email protected] (S. Cecchi), ada.baldi@unifi.it (A. Baldi), camillo.zanchi@unifi.it (C.A. Zanchi), simone.orlandini@unifi.it (S. Orlandini), andrea.pardini@unifi.it (A. Pardini), marco.napoli@unifi.it (M. Napoli). https://doi.org/10.1016/j.chemosphere.2019.125706 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

and photosynthesis (Alloway, 2004; Barrameda-Medina et al., 2014; Broadley et al., 2007; Duan et al., 2016; Marschner, 1995; Noulas et al., 2018; Vaillant et al., 2005). Vegetable species show differential tolerance levels to either Zn deficiency or toxicity, depends on soil characteristics and climatic factors, plant species, time of exposure and the concentration of metal in the soil (Alloway, 2004). Zn concentrations in leaves, lower to 10e20 mg kg1 can indicate zinc deficiency in many plant species, whereas levels between 30 and 100 mg kg1 are considered normal, while values from 100 to 500 mg kg1, are considered the upper toxic levels range in various plants (Kabata-Pendias and Pendias, 2001). Zn deficiency in plants can lead to maturation delays, root apex necrosis, stunted growth, small and irregular leaf shape and chlorosis (Alloway, 2004; Broadley et al., 2007; Kabata-

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Pendias and Pendias, 2001; Noulas et al., 2018). Zea mays, Oryza sativa, Citrus spp. and Sorgum vulgare, are reported to be very sensitive to Zn deficiency, while Asparagus officinalis, Daucus carota, Secale cereale, Triticum spp and Pisum sativum are considered not particularly susceptible to the shortage of this micronutrient (Alloway, 2004; Broadley et al., 2007; Noulas et al., 2018). Plants such as Lactuca sativa, Brassica spp. and Beta vulgaris are reported as being highly susceptible to excess soil Zn (Chaney, 1993; Chaney et al., 1997). In contrast, Silene vulgaris, Arabidopsis halleri, and Erica andevalensis (Barrameda-Medina et al., 2014; Broadley et al., 2007) and many species of the Poaceae and Chenopodiaceae families are able to grow in soil or nutrient solution containing high Zn concentrations (Bhargava et al., 2007; Meharg et al., 1993). The Zn concentration and availability in soil depends on the type and physico-chemical properties of soil (Alloway, 2004; Noulas et al., 2018). Due to the intensification of industrialization and agricultural activities, Zn content in soils has increased, becoming one of the major soil problems worldwide (Ali et al., 2013; Ashraf et al., 2019; Chen et al., 2012; Mahar et al., 2016; Sarwar et al., 2017; Yadav, 2010). In particular, the Zn concentrations in soils has increased as a consequence of human activities such as metalliferous mining, galvanizing processes, fuel combustion, manufacturing and industrial processes (Alloway, 2004; Barrameda-Medina et al., 2014). Even the agricultural sector is responsible for increased Zn pollution, because of the continuous and excessive distribution of zinc-containing agrochemical compounds and sewage sludge on crops (Alloway, 2004; BarramedaMedina et al., 2014; Broadley et al., 2007; Mahar et al., 2016; Noulas et al., 2018; Sarwar et al., 2017; Yadav, 2010; Yadav et al., 2009). Globally, the average Zn concentration in soil ranges from 50 to 55 mg kg1 (Alloway, 2004; Noulas et al., 2018), while in Italy, the Zn content on the soil surface ranges from 16 to 157 mg kg1 (Kabata-Pendias and Pendias, 2001). Various authors reported that Zn contents in agricultural soils can vary from 10 to 300 mg kg1, with a value above 150 mg kg1 considered high and potentially toxic (Noulas et al., 2018; Venterea et al., 2005; Yadav, 2010). The Italian law set maximum limits for zinc in soils as follows: 150 mg kg1 for green public areas and residential areas, and 1500 mg kg1 in soils destined for industrial and commercial uses, respectively (D.Lgs 152, 2006). For agricultural soils, no limit for Zn concentration has been defined by Italian law. However, a maximum level of 300 mg kg1 was established for Zn content in soils over which sewage sludge is distributed (D.Lgs 99, 1992). The use of remediation techniques to remove the soil heavy metal, such as Zn, are dependent on the chemical-physical properties of the soils, the dimension of areas to be remediated and the cost necessary to implement them (Luo et al., 2005). Phytoremediation is an eco-friendly and low expansive reclaiming method, consisting of the use of plant species, able to absorb and accumulate metals in vegetative tissues (Ali et al., 2013; Lasat, 2003; Luo et al., 2005). Phytostabilization is a type of phytoremediation technology aiming to reduce the toxicity through reducing their mobility and bioavailability (Bolan et al., 2011). Phytostabilization involves plants, such as Festuca rubra, which is capable to retained a higher amount of chemical elements in the roots than in the leaves i.e. these are plants that restrict the transport of contaminants from roots to the leaves (Gaji
c et al., 2016). On the opposite, phytoextration is a type of phytoremediation technology aiming to remove contaminants from the soil by using plants able to uptake, translocate and accumulate contaminants from the plant root into above-ground plant tissue. The efficiency of this methodology is directly related to the capacity of plants to accumulate metal and produce biomass (McGrath and Zhao, 2003; Napoli et al., 2019; Sarwar et al., 2017). Earlier

phytoextraction studies highlighted the importance of using hyperaccumulator plants, which are plants that are able to grow in substrates containing very high concentrations of metals, absorbing and accumulating them in vegetative tissues (Rascio and NavariIzzo, 2011). Among these, Thalapsi and Eleocharis acicularisis are well known as Zn hyperaccumulator plants (Lasat, 2003; Sarwar et al., 2017; Sekara et al., 2005). However, even Zn hyperaccumulators display significantly reduced growth when exposed to high Zn concentrations (Knight et al., 1997; Rengel, 2000; Shen et al., 1997; Talukdar and Aarts, 2008). On the other hand, several studies evidenced the effective utilization of moderate accumulator plants, that are able to produce high biomass and that can be easily cultivated using established agronomic practices (Ebbs et al., 1997; Meers et al., 2005; Sekara et al., 2005; Solhi et al., 2005; Zhuang et al., 2007). This study can increase the knowledge about the growth of seven field crops Hordeum vulgare, L. (barley); Ricinus communis, L. (castor bean); Phaseolus vulgaris, L. (pea); Brassica juncea, Czem. (Indian mustard); Sorgum vulgare, L. (sorghum); Spinacea oleracea, L. (spinach); Solanum lycopersicum, L.(tomato) in the presence of excess soil Zn. The specific aims were to investigate the biomass production of these seven crops grown in Zn spiked soils, and to evaluate the accumulation and distribution of Zn in different plant parts (roots, stems and leaves, fruits). This study should provide insight for using these plants to remediate Zn-contaminated sites. 2. Materials and methods 2.1. Reagents Zinc sulphate eptaidrate (purity grade  99.0% w/w) (SigmaAldrich, Saint Louis, Missouri, USA) was used to spike the soils used for the respective experiments, thereby simulating artificial contamination. A 10% (v/v) nitric acid (HNO3) solution was prepared by diluting super-pure HNO3 for trace metal analysis (Carlo Erba, Rodano, Milan, Italy) with distilled water. A 5% (v/v) solution of hydrochloridric acid (HCl) was prepared by diluting HCl for analytic analysis (Carlo Erba, Rodano, Milan, Italy) with distilled water. The diethylenetriaminepentaacetic acid (DTPA, purity grade  99.0% w/w, Sigma-Aldrich, Saint Louis, Missouri, USA) was used for determining the exchangeable Zn concentration in soil. The argon used for inductively coupled plasma (ICP) spectroscopy was characterized by a purity grade greater than 99.99%. 2.2. Experimental set-up The experiments were carried out for two consecutive cropping seasons, from January 2008 to December 2009, at an experimental site of the University of Florence, located in San Casciano Val di Pesa, Italy (WGS84; 43.6639 N; 11.1469 E; 256 m a.s.l.). A SM3830 meteorological station (SIAP comp., Italy), for measuring air temperature and rainfall amount, was present on the site. The climate was sub-Mediterranean, with a bimodal rainfall regime characterized by two maxima in November and February and a minimum in July (Napoli et al., 2019). According to Napoli et al. (2017), the area experienced an average annual rainfall of about 854 mm and an average annual temperature of approximately 14.9  C. During the study period, the climate was drier than the long-term average Fig. S1(Supplementary Fig. 1). In fact, in 2008 and 2009, the annual rainfall amount was 17.6 and 21.4% below the long-term mean, respectively. While the temperature measured in 2008 was consistent with the long-term average, in 2009, the average annual temperature exceeded the long-term average by about 0.7  C. In particular, in 2009, the monthly average temperature in April, August and November was higher than the long-term monthly

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average temperature by about 1.6, 2.1 and 1.9  C, respectively. The soil used in this experiment was sampled (0e0.15 m layer) from a cultivated field at the experimental site of the University of Florence (Napoli et al., 2015) (Supplementary Table 1). The soil contained 87.2 and 21.6 mg kg1 of total and DTPA-exchangeable Zn which was considered as background levels. The soil was air-dried, crushed, sieved to 5 mm, and finally homogenized with a concrete mixer. Then, a total of 560 pots (30 cm diameter; 30 cm depth) were filled with 18 kg of the homogenized soil. The treatments comprised four levels of Zinc (Zn) added to the soil and seven field crops. Each treatment was replicated 20 times. Four levels of Zn were obtained by adding solutions containing 0, 39.6, 79.1, 118.7 g of zinc sulphate eptaidrate corresponding to 0 (control), 500, 1000, 1500 mg kg1 of total Zn added to soil, respectively. The 500 and 1500 mg kg1 of Zn levels were selected by considering the phytotoxicity threshold reported by (Alloway, 2004) and the maximum limits for zinc in commercial and industrial soils according to the Italian law (D.Lgs 99, 1992), respectively. To ensure an even concentration in the soils, the latter were mixed thoroughly. The average DTPA-exchangeable Zn in soil was found 21.6, 227.9, 364.3, and 460.6 mg kg1 of Zn for the doses 0e1500 mg kg1 of Zn added to soil. Hordeum vulgare, Ricinus communis, Phaseolus vulgaris, Brassica juncea, Sorgum vulgare, Spinacea oleracea, and Solanum lycopersicum were sown or transplanted into the pots on the dates shown in Supplementary Table 2. For each treatment, soil moisture content was monitored throughout the experiment by three sensors installed in three randomly chosen pots. Soil moisture sensors were connected to an automatic irrigation scheduling system, which then automatically drip-irrigated the pots of the same treatment according to the soil moisture. 2.3. Sample preparation and spectrophotometric determination At the end of the biological cycle of each crop, the different plant parts (roots, stems and leaves, fruits or seeds) were collected separately, oven-dried at 105  C, weighted (DW, kg) and milled. Dried samples (1 g) for each of the different plant parts for each crop were ashed at 550  C for 12 h in a muffle furnace. The resulting greyish-white ashes, after cooling, were treated with 5 mL of the HNO3 solution, and the mixture was slowly heated to dissolve the residues. The solution was filtered, using a Whatman 42 filter paper, transferred to a 10 ml volumetric flask and then made up to volume with distilled water. The blank consisted of the 10% dilute HNO3 used for extraction. Zn determinations were performed by using PerkinElmer Optima 7300 DV ICP-OES (PerkinElmer, Inc. Shelton, CT, USA) with the following instrumental conditions: forward power 1450 W; argon plasma flow rate 15 L min1; auxiliary argon flow rate 0.3 L min1; argon nebulizer flow rate 0.6 L min1; axial plasma view; wavelengths 213.855 nm. All containers and test tubes were poly-propylene (De Leonardis et al., 2000), and were previously cleaned with the HCl solution. Quality Control (QC) for Zn measurements included triplicate analysis for each sample. Moreover, six of every 30 samples analysed were known QC samples (blank, 1, 5, 10, 50, and 100 mg of Zn L1).

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Zn concentrations and dry matter production data were analysed for all crops, as well as each crop parts (stems and leaves, fruits or seeds, roots) to evaluate the effects of Zn soil treatment on plant dry matter accumulation and Zn storage capacity. The uptake of Zn (mg kg1 DW) was calculated on the basis of the plant material DW at harvest. The bioaccumulation factor (BAF) and the translocation factor (TF) were calculated to investigate the Zn absorption and/or phytoremediation potential of the studied crops. The BAF (Equation (1)) indicates the plant capacity for metal uptake and accumulation (Asensio et al., 2018; Liu et al., 2016; Singh et al., 2017; Zhuang et al., 2013).

h BAF ¼

Znplant tissues

i (1)

½Znsoil 

where [Znplant tissues] and [Znsoil] were the Zn concentration in plant tissues and DTPA-exchangeable Zn in soil, respectively. The TF (Equation (2)) was used to evaluate plant capacity to transfer Zn from roots to aerial tissues and, hence, the potential to accumulate Zn in aerial parts (Lam et al., 2017; Sun et al., 2008).

h BAF ¼

Znplant tissues

i

½Znsoil 

(2)

Plants with BAF < 1 and TF < 1, BAF <1 and TF >1 as well as BAF >1 ans TF < 1 are considered as excluders suitable for phytostabilization whereas plants with BAF>1 and TF > 1 are considered as accumulators suitable for phytoextraction (Gaji
c et al., 2018; Lam et al., 2017; Ma et al., 2001). A split plot design was set up, where the main factor was the year of trial and the second factor was the Zn level. The year of trial was considered as random effect factor, while the four Zn levels were considered as fixed effect factors. The analysis of variance (ANOVA) was performed on the dependent variables (cumulated GDU; DW; Zn concentration in plant parts) by using the R statistical software package (R Core Team, 2017). ANOVA was performed for each crop separately. Differences between means were compared for significance by means of the Tuckey honest significant difference (Tuckey HSD) test (p < 0.05) (R Core Team, 2017). The Pearson r correlation was used for determination of relationship between the Zn levels and the dependent variables. Zn concentration and DW values for the different crop and plant tissues, respectively, were presented using box and whiskers plots. The box represented the interquartile range, with the upper and lower ends of the box representing the 75th and 25th quartiles, respectively. A horizontal line inside the box indicated the median. The whiskers extended to the values within 1.5 times the interquartile range above the 75th and within 1.5 times the interquartile range below the 25th percentile. Values beyond the whiskers range were considered as outliers and represented as a black point. 3. Results and discussion

2.4. Data analysis

3.1. Effect of soil Zn concentration on the duration of the plant growth period

The phenological development of the plants was monitored twice a week following the BBCH scale (Meier, 2001). The growing degree units (GDU) were computed to relate the air temperature to the phenological development of crop. For each crop, starting from either the seeding or transplanting date, the GDU was calculated as the accumulation of daily average temperature exceeding a base temperature of 0  C (Parthasarathi et al., 2013).

No inhibitory effects of Zn on seed germination and early seedling growth were found, regardless of the plant species and the Zn concentration in the soil. For none of the studied species was there a significant inter-annual difference in cumulated GDUs to move to the next developmental stage (Supplementary Table 2). In contrast, the four Zn levels resulted in significant differences in cumulated GDUs within the same crop. A significant decrease

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(p < 0.05) in the GDUs values required to reach the physiological maturity was detected with the increase in Zn levels in soil for all the crops, with the exception of the Brassica juncea, in which GDUs were found to increase as the Zn levels in soil increase. These results suggested that the GDUs necessary to fulfill the growth requirements decreased as the Zn content in the soil was increased. To the contrary, Brun et al. (2003) and Jin et al. (2015) suggested that high metal concentration delayed the time to reach phenological maturity for herbaceous plants. Stapper and Harris (1989) found that the DW accumulation increased as the vegetative growth period increased. However, the crops in this study showed different correlation between the DW and the GDUs required to reach maturity. No correlation between the DW and the GDUs was found for Hordeum vulgare (r ¼ 0.02), Brassica juncea (r ¼ 0.42) and Spinacea oleracea (r ¼ 0.33). A significant positive correlation (p < 0.05) between the DW and the GDUs was found for common Phaseolus vulgaris (r ¼ 0.75), whilst significant negative correlations (p < 0.05) were found for Ricinus communis (r ¼ 0.96), Sorgum vulgare (r ¼ 0.72) and Solanum lycopersicum (r ¼ 0.83), respectively.

3.2. Effect of soil Zn concentration on DW and Zn accumulation in different plant parts 3.2.1. Hordeum vulgare Results indicated that both the 500 and 1000 mg kg1 Zn treatments significantly (p < 0.05) enhanced the dry matter production in aerial tissues of Hordeum vulgare, with respect to the control treatment (Fig. 1). On the contrary, a significant (p < 0.05) reduction in the DW of both fruit and leaves and stems, with respect to the control, was observed by adding 1500 mg kg1 Zn to the soil. No significant (p > 0.05) effects on root DW were observed by adding both 500 and 1000 mg kg1 Zn to the soil, while a significant (p < 0.05) DW reduction of roots was observed in the 1500 mg kg1 Zn treatment. Moreover, Kherbani et al. (2015) found

that the Hordeum vulgare, did not present signs of stress after 4 weeks of culture in soil contaminated by Zn. Furthermore, in a hydroponic experiment containing 100 mM of ZnSO4, conducted by Ebbs and Kochian (1998), a shoot biomass reduction of about 29%, was observed, with respect to the control, indicating a tolerance of Hordeum vulgare, towards high Zn concentrations in solutions. On the contrary, Boawn and Rasmussen (1971), testing Zn accumulation in Hordeum vulgare shoots after a growing period of 35 days, measured a biomass decrease of about 76% in a Zn contaminated soil of 500 mg kg1. Over both years of the present study, results showed that the Zn concentration in all tissues significantly increased with the increase in soil Zn concentration. The highest Zn concentrations were measured in leaves and stems, ranging from 5.3 to 7.3 times more Zn than that measured in the roots, respectively, and from 1.3 to 2.3 times more Zn than that recovered in the fruits, respectively.

3.2.2. Ricinus communis The DW of the fruits and roots in Ricinus communis did not increase significantly (p > 0.05) with increasing soil Zn concentrations. As shown in Fig. 2, no relationship was observed between the DW of the fruit and root tissues with the soil Zn concentration. Results indicated that the DW of the leaves and stems increased significantly (p < 0.05) as the Zn concentration in the soil increased to 1500 mg kg1. Yadav et al. (2009) found that Jatropha curcas, a Euphorbiaceous species, related to Ricinus communis, was able to survive on Zn contaminated soils up to 3000 mg kg1. At the same time, these authors reported that soil Zn enhanced the growth of Jatropha curcas up to the concentration of 1000 mg kg1, while the plant growth was stunted at higher Zn concentrations. Wang et al. (2016) found that the biomass of Ricinus communis increased along with increasing soil Zn concentrations up to 400 mg kg1, while Zn concentrations higher than 600 mg kg1 reduced plant growth with respect to the control. The concentration of Zn in all crop tissues increased significantly (p < 0.05) as the

Fig. 1. Box and whisker plots of the dry weight (top) and Zn concentration (bottom) measured in different tissues of Hordeum vulgare for 0 (white box), 500 (light-grey box), 1000 (grey box), and 1500 mg kg1 Zn treatments (dark-grey box). Means are reported over the respective box and whiskers. Lowercase letters indicate different means between treatments (p < 0.05) according to the post hoc Tuckey test.

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Fig. 2. Box and whisker plots of the dry weight (top) and Zn concentration (bottom) measured in different tissues of Ricinus communis for 0 (white box), 500 (light-grey box), 1000 (grey box), and 1500 mg kg1 Zn treatments (dark-grey box). Lowercase letters indicate different means between treatments (p < 0.05) according to the post hoc Tuckey test.

Zn concentration in the soil increased. Results indicated that Zn concentrations in Ricinus communis decreased in the sequence as follows: roots > leaves and stems > fruits. It was also observed that the fruits of the plants grown in soils with the highest Zn level contained up to 2.3 times more Zn than those grown in the control. Previous authors have reported on the ability of Ricinus communis to grow in Zn polluted soils and to accumulate Zn (Kiran and Prasad, 2017). Wang et al. (2016) found a positive correlation between Zn concentrations in the soils and the Zn accumulated in the plant tissues. The results of the present study were consistent with Wang et al. (2016) who found Zn concentrations varying from 167 to 713 mg kg1 and from 138 to 420 mg kg1 in the shoots and in the roots of Ricinus communis, respectively. 3.2.3. Phaseolus vulgaris Results indicated that the biomass production in all tissues decreased significantly (p < 0.05) as the soil Zn concentration increased (Fig. 3). Similar results were found in a soil-less experiment by S
anchez et al. (1999), wherein the DW of Phaseolus vulgaris plants was shown to decrease significantly (p < 0.05) with increasing concentrations of Zn in the solution. At the same time, the Zn concentration in all tissues was found to increase signifi
nchez et al. cantly with increasing Zn in the soil. As observed by Sa (1999), the concentrations in the fruits, leaves and stems, and roots of Phaseolus vulgaris, respectively, were directly correlated to the Zn concentration. The Zn concentration in fruits was 13e65 times higher than that measured in experimental plots by Zhou et al. (2016), who reported average concentration of Zn in Phaseolus vulgaris fruits of about 5.67 mg kg1. The highest Zn concentration was measured in the leaves and stems, ranging from 5.3 to 7.3 times more Zn than that measured in roots, respectively, and from 1.3 to 2.3 times more Zn than that recovered in the fruits, respectively. The Zn distribution between tissues was consistent with Sekara et al. (2005), who found the Zn concentrations Phaseolus vulgaris grown in experimental plots decreasing in the sequence as follows:
nchez et al. (1999) stems > pods > leaves > roots. In contrast, Sa

found that in the Zn concentration in roots higher than that recovered in the aerial tissues. 3.2.4. Brassica juncea According to our results, DW production in the Brassica juncea was negatively affected (p < 0.05) by the soil Zn concentration (Fig. 4). The highest DW was measured in the control, while in Zn spiked soils, the DW of Brassica juncea declined significantly (p < 0.05) compared to the control plants, indicating the negative effects of Zn on plant growth. No significant variation in DW of fruits, was recorded between plants grown in soils treated with 500 and 1000 mg kg1 Zn. Results were consistent with Ebbs and Kochian (1998), who found severe toxicity and nutrient deficiency in plants of Brassica juncea grown in soils treated with 3100 mg kg1 Zn. Prasad et al. (1999) observed that 0.05 mM of Zn promoted Brassica juncea seedling growth in vitro culture, while causing a significant reduction in growth at concentrations exceeding 5 mM. Various authors (Barrameda-Medina et al., 2014; Ebbs and Kochian, 1997; Mourato et al., 2015) reported Brassica species being accumulator plants with high tolerance to heavy metal in soils. The Zn concentration in Brassica juncea tissues increased significantly (p < 0.05) as the soil Zn concentration increased. Among the considered plant parts, the results indicated that roots, leaves and stems had the highest Zn accumulation especially in presence of 1500 mg kg1 Zn. No significant (p > 0.05) differences in Zn concentration were found between leaves and stems and roots. However, the Zn distribution between aboveground tissues was found to decrease significantly (p < 0.05) from leaves and stems to fruits. Similar results were found by Ebbs and Kochian (1998), who measured about 250 mg kg1 Zn in shoot tissues of Brassica juncea plants grown in soils spiked with 3100 mg kg1 Zn, and Singh et al. (2012), who measured 48 and 64 mg kg1 of Zn in stem and leaf tissues, respectively, of plants grown in open field plots with soil containing 146.4 mg kg1 Zn. Results indicated that roots accumulated greater amounts of Zn, thus confirming studies of Ebbs and Kochian (1997) performed

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Fig. 3. Box and whisker plots of the dry weight (top) and Zn concentration (bottom) measured in different tissues of Phaseolus vulgaris for 0 (white box), 500 (light-grey box), 1000 (grey box), and 1500 mg kg1 Zn treatments (dark-grey box). Lowercase letters indicate different means between treatments (p < 0.05) according to the post hoc Tuckey test.

Fig. 4. Box and whisker plots of the dry weight (top) and Zn concentration (bottom) measured in different tissues of Brassica juncea for 0 (white box), 500 (light-grey box), 1000 (grey box), and 1500 mg kg1 Zn treatments (dark-grey box). Lowercase letters indicate different means between treatments (p < 0.05) according to the post hoc Tuckey test.

under hydroponic conditions. On the other hand, these authors, by analyzing plants approximately a month after sowing, found that Zn concentrations in roots exceeded that in the leaves. To the contrary, the present results on fully developed Brassica juncea plants indicated non-significant (p > 0.05) differences between Zn content measured in the roots with that in the leaves and stems. Barrameda-Medina et al. (2014) described the increase of Zn in

leaves and stems as a result of the tolerance mechanism of Brassica oleracea grown hydroponically in Zn-spiked solution. Zn phytotoxicity in hydroponic culture. Barrameda-Medina et al. (2014) showed that Zn toxicity induced the plants to increase the production of organic acids (malate and citrate) to promote translocation of the metal into the leaves and stems, and then to sequestrate Zn in vacuoles.

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3.2.5. Sorgum vulgare Results indicated that in both years of experimentation the vegetative growth of Sorgum vulgare was not significantly affected (p < 0.05) by soil Zn concentrations up to 1500 mg kg1 (Fig. 5). Furthermore, results indicated that the 1000 and 1500 mg kg1 Zn treatments significantly (p < 0.05) enhanced the DW production in roots with respect to the control treatment. However, a significant decrease (p < 0.05) in the DW of the fruits was detected with the increase of the concentration of soil Zn. These results were consistent with those obtained by Zhuang et al. (2009) in field trial, where no visible symptoms of Zn toxicity were observed in Sorgum vulgare plants grown in agricultural soils with a Zn contamination of about 834 mg kg1. On the contrary, Boawn and Rasmussen (1971) found a biomass reduction of approximately 80% in the shoots with respect to the control, in soil spiked with 500 mg kg1 Zn. In both years of experimentation, a significant increase (p < 0.05) in the accumulation of Zn in roots and leaves and stems was detected with the increase in Zn soil content. Instead, the Zn concentration in the fruits was significantly (p < 0.05) lower than that measured in the leaves and stems and in the roots. These results were consistent with those of Angelova et al. (2009) where analogous Zn concentrations were noted in fruit and root tissues of different Sorgum vulgare varieties grown in experimental plot with soil containing 33.9 and 1903.8 mg kg1 Zn, respectively. Similarly, Kos et al. (2003) reported an average concentration of Zn of about 101 mg kg1 in the shoots of Sorgum vulgare plants grown in soil containing 800 mg kg1 Zn. 3.2.6. Spinacea oleracea The growth performance of Spinacea oleracea in terms of biomass, in relation to Zn, is shown in Fig. 6. Spinacea oleracea roots resulted too fragile to be collected from the soil. So it was not possible to recover enough root biomass for weight determination and performing chemical analysis. Results indicated that Zn addition to soil significantly (p < 0.05) affected DW production in Spinacea oleracea The highest DW was measured at 500 mg kg1 Zn,

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followed by 1000 and 1500 mg kg1 and then the control. To the contrary, other authors found DW significantly reduced DW in Spinacea oleracea grown in Zn spiked soil. For example, in a soil treated with 750 mg kg1 Zn, shoot and root DW decreased by 6 and 14%, respectively, as compared to control treatment (Alia et al., 2015) Similarly, Boawn and Rasmussen (1971) found that in soils treated with 750 mg kg1 Zn, shoot and root DW decreased by 6 and 14%, respectively, as compared to control treatment. The leaves and stems of Spinacea oleracea showed a high Zn amount as the soil Zn concentration increased. In contrast, no significant differences (p > 0.05) in Zn concentrations between Spinacea oleracea cultivars growing on 237 mg kg1 Zn-spiked soil were observed with respect to the control treatment (Alexander et al., 2006). Furthermore, the Zn concentration in the leaves and stems in the present study were higher than that found by previous authors in field and plot trials (Afolayan and Jide, 2012; Singh et al., 2012; Zhou et al., 2016). 3.2.7. Solanum lycopersicum The DW for the fruits and for the leaves and stems measured in Solanum lycopersicum plants grown in Zn-spiked soil was significantly higher (p < 0.05) than that measured in plant grown in control (Fig. 7). The highest DW for the fruits and for the leaves and stems were measured in Solanum lycopersicum plants grown in soils contaminated with 1000 and 1500 mg kg1 Zn, respectively. The 500 mg kg1 Zn level increased the root biomass with respect to the control, while for the two upper Zn level levels no significant differences were observed, respectively. These results indicated that Zn level did not limit Solanum lycopersicum growth under the experimental conditions. However, caution should be used while interpreting these results. In fact, as Zn phytotoxicity is highly influenced by soil pH (Berry and Wallace, 2006), different results could be obtained in different soils. Mohammad and Moheman (2019) and Vijayarengan and Mahalakshmi (2013) found that the root and shoot biomass of Solanum lycopersicum decreased as the Zn concentrations in the soil exceeded 100 mg kg1. Additionally, Cherif et al. (2011), using growth chambers, observed a significant

Fig. 5. Box and whisker plots of the dry weight (top) and Zn concentration (bottom) measured in different tissues of Sorgum vulgare for 0 (white box), 500 (light-grey box), 1000 (grey box), and 1500 mg kg1 Zn treatments (dark-grey box). Lowercase letters indicate different means between treatments (p < 0.05) according to the post hoc Tuckey test.

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Fig. 6. Box and whisker plots of the dry weight (top) and Zn concentration (bottom) measured in different tissues of Spinacea oleracea for 0 (white box), 500 (light-grey box), 1000 (grey box), and 1500 mg kg1 Zn treatments (dark-grey box). Lowercase letters indicate different means between treatments (p < 0.05) according to the post hoc Tuckey test.

Fig. 7. Box and whisker plots of the dry weight (top) and Zn concentration (bottom) measured in different tissues of Solanum lycopersicum for 0 (white box), 500 (light-grey box), 1000 (grey box), and 1500 mg kg1 Zn treatments (dark-grey box). Lowercase letters indicate different means between treatments (p < 0.05) according to the post hoc Tuckey test.

reduction in biomass production of Solanum lycopersicum seedlings after 7 days of metal exposure at concentrations of 100 mmol L1 or more. Furthermore, Boawn and Rasmussen (1971) measured a biomass decrease of about 24% in Solanum lycopersicum shoots grown for 35 days in soil contaminated with 500 mg kg1 of Zn. The highest Zn concentration was measured in roots, followed by leaves and stems and fruits, respectively. These results were consistent

with those of Zhou et al. (2016), showing that in soil containing 820 mg kg1 Zn, the edible parts of Solanum lycopersicum plants showed a Zn content of about 1.42 mg kg1. Singh et al. (2012), testing Solanum lycopersicum in a soil contaminated with 146.39 mg kg1 Zn, observed the highest Zn concentrations in leaves followed by roots, stem and fruits, with an average accumulation of about 115, 90, 73 and 29 mg kg1, respectively.

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3.3. Differences in Zn accumulation in aboveground tissues between crops For the four levels of Zn added to the soil, significant differences were found between the average quantities of Zn accumulated in the aboveground tissues of the seven crops. The order of accumulation in the control are reported as follows [in parenthesis Zn accumulated in the aboveground tissues of crops as mean and standard deviation in mg of Zn per plant; letters indicate different means (p < 0.05) according to the post hoc Tuckey test]: Sorgum vulgare (2.77 ± 0.74 a) > Ricinus communis (1.87 ± 0.39 b) > Solanum lycopersicum (1.41 ± 0.6 c) > Hordeum vulgare (1.07 ± 0.45 d) > Phaseolus vulgaris (0.74 ± 0.16 e)  Brassica juncea (0.65 ± 0.15 e)  Spinacea oleracea (0.51 ± 0.28 e). For the treatment containing 500 mg kg1 Zn, the order of accumulation was as follows: Solanum lycopersicum (8.49 ± 1.85 a) > Sorgum vulgare (6.11 ± 1.05 b)  Ricinus communis (5.64 ± 0.91 b) > Hordeum vulgare (4.67 ± 1.18 c)  Spinacea oleracea (4.02 ± 0.93 c) > Phaseolus vulgaris (1.03 ± 0.19 d)  Brassica juncea (0.96 ± 0.18 d). Then, for the treatment containing 1000 mg kg1 Zn, the order of accumulation was: Solanum lycopersicum (10.21 ± 1.52 a) > Hordeum vulgare (8.61 ± 1.47 b) > Sorgum vulgare (6.47 ± 0.85 c) > Ricinus communis (5.78 ± 0.96 d)  Spinacea oleracea (5.46 ± 1.22 d) > Brassica juncea (1.03 ± 0.13 e)  Phaseolus vulgaris (0.96 ± 0.13 e). Finally, for the highest Zn level, Solanum lycopersicum accumulated the highest amount of Zn, which was almost the 39% more than that in Ricinus communis the second largest average value. The order of accumulation was: Solanum lycopersicum (15.06 ± 3.31 a) > Ricinus communis (10.83 ± 1.39 b)  Sorgum vulgare (10.79 ± 1.48 b) > Spinacea oleracea (8.56 ± 1.94 c) > Hordeum vulgare (4.98 ± 1.28 d) > Brassica juncea (1.14 ± 0.17 e)  Phaseolus vulgaris (1.11 ± 0.17 e). 3.4. Plant phytoremediation efficiency According to the BAF values (Supplementary Table 3), Hordeum vulgare plants demonstrated capacity for metal uptake and accumulation. Moreover, in all treatments, TF values indicated that Hordeum vulgare was able to efficiently translocate Zn from the roots to aerial tissues. Therefore, by considering both BAF and TF values, Hordeum vulgare can be considered as a Zn accumulator suitable for phytoextraction. Boawn and Rasmussen (1971) measured about 2110 mg kg1 Zn in shoot of Hordeum vulgare grown in soils contaminated with 500 mg kg1 Zn. On the contrary, Sekara et al. (2005) found insufficient Zn translocation from the roots to aerial tissues in Hordeum vulgare grown in field conditions. The estimated BAF value for Ricinus communis plants grown in spiked soil was less than 1, with the largest value calculated for the in soils contaminated with 500 mg kg1 Zn (0.77). On the contrary, the BAF value for plants grown in control was higher than 1. The TF value was less than 1, thus indicating that the roots of Ricinus communis retained more Zn than the aboveground tissues and suggesting the potential of this species in phytostabilization. Wang et al. (2016) found TF values varying from 1.02 to 1.59 and BAF values ranging from 0.65 to 1.48, respectively. According to the BAF values, Phaseolus vulgaris plants resulted being able to accumulate Zn in the soil with lowest level of DTPA-exchangeable Zn but not in the soil spiked with Zn. The TF values of Phaseolus vulgaris were higher than 1 for the control, while the values were less than 1 for the other soil Zn levels. These results indicated that Phaseolus vulgaris plants can be suitable for Zn phytoextraction in soil with low Zn concentration, while being suitable for phytostabilization at higher levels. Phaseolus vulgaris results in the present study were consistent with previous studies, classifying Phaseolus vulgaris and other members of the Leguminosae as “low accumulators”

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(Alexander et al., 2006; Kuboi et al., 1986). Similarly, Zhou et al. (2016), showed a BAF less than 0.25 on a soil containing 820 mg kg1 Zn, thus confirming Phaseolus vulgaris bening low Zn accumulator. Both the estimated BAF and TF values for Brassica juncea were less than 1 for the Zn spiked soil, whereas for the control the BAF and TF values were higher than 1. These results indicated that Brassica juncea can be potentially used for phytostabilization in high Zn polluted soil. In Sorgum vulgare the BAF and TF values of Zn were lower than 1 except BAF values of control which was higher than 1. This means that Zn was retained in the roots and did not able to effectively translocate Zn from the roots to aerial tissues. Therefore, according to these results Sorgum vulgare can be considered for Zn phytostabilization. These results contradicted those of Boawn and Rasmussen (1971) and Zhuang et al. (2009) showing that sorghum Sorgum vulgare was a Zn hyperaccumulator which is capable to efficiently translocate Zn towards the areal tissues. Results in this study were consistent with those by Angelova et al. (2009), who suggested a reduced movement of Zn through the vascular system. In Sorgum vulgare the BAF and TF values of Zn were lower than 1 except BAF values of control which was higher than 1. This means that Zn was retained in the roots and did not able to effectively translocate Zn from the roots to aerial tissues. Therefore, according to these results Sorgum vulgare can be considered for Zn phytostabilization. As Spinacea oleracea roots were not collected, TF value was not calculated and BAF value was computed for the sole aboveground tissues. The BAF value estimated for Spinacea oleracea was higher than 1 in all treatment, thus indicating the potential of this species in phytoextraction. The BAF values indicated Solanum lycopersicum being able to effectively accumulate Zn only in control and soil contaminated with 500 mg kg1 of Zn. However, in all treatments TF values were less than 1, thus indicating a reduced Zn translocation from the roots to aerial tissues. These results suggest Solanum lycopersicum being suitable for Zn phytostabilization. Result of other authors indicated BAF values higher than 1. For example, Boawn and Rasmussen (1971) reported Zn concentrations in aerial tissues of Solanum lycopersicum up to 514 mg kg1 when grown in soil contaminated by 500 mg kg1 Zn. Afolayan and Jide (2012) found an average Zn concentration of about 61.96 mg kg1 in Solanum lycopersicum grown in soil containing an average of 53 mg kg1 Zn. 4. Conclusions The Zn concentrations in soils has increased as a consequence of mining, manufacturing and industrial processes, and distribution of zinc-containing agrochemical compounds. Plant based remediation techniques represents a low cost and environmentally friendly strategy to recover Zn polluted soil. Moreover, several studies suggested that soil remediation can be achieved by using moderate accumulator herbaceous crop, that can produce large amount of biomass and that can be cultivated with established agronomic practices. This study was conducted to evaluate the phytoremediation potential of seven crops growing on Zn polluted soil. Results indicated that no Zn inhibitory effects on seed germination and early seedling growth were found in the studied crops. The DW of the aboveground biomass in Ricinus communis and Solanum lycopersicum increased significantly as the Zn concentration in the soil increased. On the contrary, the DW of the aboveground biomass in Phaseolus vulgaris, Brassica juncea and Sorgum vulgare decreased significantly as the Zn concentration in the soil increased. Over the two-year trial, Spinacea oleracea was the most efficient in concentrating Zn in the aboveground tissues (up to 1133.1 mg kg-1 of Zn), followed, in decreasing order, by Hordeum vulgare, Solanum lycopersicum, Ricinus communis, Sorgum vulgare, Phaseolus vulgaris, and Brassica juncea. Among the crops considered, Hordeum vulgare

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resulted the most efficient in accumulating Zn both in fruit and in leaves and stems, whereas Solanum lycopersicum resulted the most efficient in accumulating Zn in roots. Despite none of the seven crops can be identified as Zn hyperaccumulators, BAF and TF values indicated that these crops can be suitable for phytoremediation. In fact, Hordeum vulgare and Spinacea oleracea resulted being suitable for Zn phytoextraction, whereas the remaining crops being suitable for Zn phytostabilization. These results highlight the phytoremediation potential of the seven crops analysed. However, caution should be exerted when extrapolating the results of this pot study to the field. In particular, it could be necessary to support the growth of these field crops by scheduling more nutrients and irrigation. CRediT author statement Chiara Grassi: Data curation, Visualization, Writing - original draft. Stefano Cecchi: Conceptualization, Investigation, Resources. Ada Baldi: Writing - original draft. Camillo A. Zanchi: Conceptualization, Methodology, Funding acquisition, Supervision. Simone Orlandini: Funding acquisition, Supervision. Andrea Pardini: Visualization. Marco Napoli: Conceptualization, Investigation, Methodology, Formal analysis, Resources, Writing - review & editing. Acknowledgments The authors thanks Dr. Anne Whittaker Ph.D. (Department of Agricultural and Food Sciences, University of Bologna) for English judgments. We thank unknown referees for constructive reviews. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125706. References Afolayan, C., Jide, A.J., 2012. Heavy metal contamination of vegetables cultivated in home gardens in the Eastern Cape. South Afr. J. Sci. 108, 1e6. https://doi.org/ 10.4102/sajs.v108i9/10.696. Alexander, P.D., Alloway, B.J., Dourado, A.M., 2006. Genotypic variations in the accumulation of Cd, Cu, Pb and Zn exhibited by six commonly grown vegetables. Environ. Pollut. 144, 736e745. https://doi.org/10.1016/ J.ENVPOL.2006.03.001. Ali, H., Khan, E., Sajad, M.A., 2013. Phytoremediation of heavy metalsdconcepts and applications. Chemosphere 91, 869e881. https://doi.org/10.1016/ J.CHEMOSPHERE.2013.01.075. Alia, N., Sardar, K., Said, M., Salma, K., Sadia, A., Sadaf, S., Toqeer, A., Miklas, S., 2015. Toxicity and bioaccumulation of heavy metals in spinach (Spinacia oleracea) grown in a controlled environment. Int. J. Environ. Res. Public Health 12, 7400e7416. https://doi.org/10.3390/ijerph120707400. Alloway, B.J., 2004. Zinc in Soils and Crop Nutrition. IZA Publications, International Zinc Association, Brussels. Angelova, V.R., Babrikov, T.D., Ivanov, K.I., 2009. Bioaccumulation and distribution of lead, zinc, and cadmium in crops of solanaceae family. Commun. Soil Sci. Plant Anal. 40, 2248e2263. https://doi.org/10.1080/00103620902961227.
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