1. Introduction
In the agricultural soil, the overuse of fertilisers and waste release from industrial processes has resulted in a large number of contaminated sites over Europe requesting to be reclaimed, contributing to exacerbated land degradation problems [
1]. A recent survey reported that in the European Union, approximately 340,000 contaminated sites are present, most of them polluted by metal(loid)s [
2]. The awareness about the harmful effects produced by such contaminants on human health, through plant cultivation and animal breeding, is forcing the characterisation of different environmental restoration technologies, among which eco-sustainable ones are largely studied and successfully applied, especially in sites characterised by moderate and diffuse contamination [
3,
4]. In this regard, the phytomanagement is recognised as an effective approach to carry out a risk management strategy [
5,
6], being constituted by an array of gentle remediation options (GROs) technologies that can be applied as a part of integrated site risk management solutions. In this sense, the phytomanagement approach represents an application of the phytoremediation biotechnology, exploiting in a broader way the ecological benefits offered by plants. Specifically, phytomanagement relies on the choice of suitable plant species for the purpose of the management of the contaminated sites [
7]. Among polluted sites, agricultural areas represent a particular concern for safe food production [
8], forcing local administrations to restrict their exploitation with relevant loss of income for farmers. Therefore, particular attention is currently being paid to investigating the possibility of cultivating non-food crops on contaminated lands that, besides the capability to remediate soils, could satisfactorily grow and produce biomass and other bio-products for multiple profitable uses, avoiding the metal transfer to the food chain [
9].
Because of its biological characteristics, such as rapid growth, high biomass production, wide root system, high genetic variability, remarkable ability to adapt to different environmental conditions, and low susceptibility to disease and pests [
10,
11], hemp (
Cannabis sativa L.) is a plant species of notable interest for phytomanagement. In fact, because of the multiple non-food uses [
12], it can offer a good opportunity to integrate soil recovery with the cultivation of a commercially exploitable resource. Specifically, hemp plants involved in phytomanagement strategies can profitably produce fibres that could be commercialised as insulating or composite material, cellulose materials from stem (suitable for packaging industry), and seeds representing a source of oil for biofuel production. The hemp potential for phytotechnologies has been poorly explored so far; most studies have focused on tolerance and accumulation of metals [
13,
14,
15,
16,
17] and few reports deal with plant behaviour versus organic contaminants such as chrysene and benzopyrene [
18] and radio-compounds [
19]. Therefore, a better characterisation of the growth and physiological responses of hemp plants in metal-contaminated soil under controlled conditions is requested. In this context, the application of the chlorophyll
a fluorescence analysis, which represents a rapid and non-destructive technique to analyze the changes at the physiological and biochemical level in the photosynthetic apparatus under stress conditions, and the evaluation of the chlorophyll content, also performed with non destructive methods, are particularly useful. Specifically, OJIP fluorescence transient analysis, known as the JIP test [
20], has been developed for the quantification of several phenomenological and biophysical expressions together with the energy flux parameters of photosystem II (PS II), and may be used to assess metal stress on plants in vivo [
21]. This approach could be of relevance for a successful screening of plant materials for phytoremediation. In the present study, an ex situ pot experiment was performed with soil sampled from the “Valle del Sacco” area. It is a contaminated site recognised as a National Interest Site by the Italian Ministry of Environment (L. 248/2005) for its huge extension (7235 ha) and diffused contamination by metals and organics that prevents any food crop cultivation and grazing, which formerly characterised the area. This preliminary experiment, performed in a greenhouse, aimed to investigate the growth potential of hemp plants on moderately metal-contaminated soils, evaluating the presence of the pollutants in the biomass for possible exploitation as a bio-resource.
3. Results and Discussion
As reported in
Table 1, the analyses of agronomic parameters revealed a higher content of organic matter (SOM), total N, available K, and exchangeable Mg in C-soil. Both samples showed sandy loam ground, slightly alkaline pH, and a similar cationic exchange capacity (CEC). Metal (loid) concentrations of As, V, and Pb were below the threshold fixed by the Italian Law (for sites devoted to green areas as for Decree Law n.152/06 [
23]) in C-soil and beyond the legal limit in the soil representing a moderate contamination (MC).
In
Table 2, some biometric parameters regarding hemp plants grown in pots filled with soils of the two sampled points are shown. Plant height and stem diameter were not affected by the different substrate composition, while leaf area was higher in plants grown in C-soil when compared with those grown in MC-soil. Anyway, the growth of plants (i.e., plant height, stem diameter, above ground, and root dry mass) can be considered as satisfactory with regards to data reported in field trials on different cultivars of fiber hemp [
10,
27], despite the notable differences in the growth conditions.
Biomass production data are reported in
Table 3. The results revealed that the growth of hemp plants in MC-soil was reduced compared with that observed for plants cultivated in C-soil. In particular, the main differences were found at the root and leaf level. This latter result was consistent with previous reported data on leaf area in
Table 2. Interestingly, stem and inflorescence biomass was not affected by the different soil characteristics. The calculation of the total amount of water loss for transpiration by plants revealed a higher total transpiration over the cultivation period in C-soil grown plants.
Taken together, results on growth performances highlighted a good adaptation of hemp plants to the soil sampled in the contaminated area. In this regard, it can be hypothesised that growth was supported mainly by the high SOM and nutrient content, especially with regards to C-soil grown plants. Moreover, it can be underlined that plant growth was not negatively affected by the presence of metal(loid)s in the soil, confirming previous data reported by several authors [
14,
15,
16]. Notably, the presence of vanadium in the soil was also not deleterious for hemp plants, while in the literature, such metal is reported to be extremely toxic for plant growth at concentrations even lower than those tested in the present work [
28,
29].
To investigate the responses of hemp plants grown in the two different soils at the photosynthetic level, measurements of leaf chlorophyll content and chlorophyll fluorescence were performed (
Table 4).
Leaf chlorophyll content is one of the most important factors in determining photosynthetic potential and primary production [
30]. Our results showed that a higher leaf total chlorophyll content was found in plants grown in C-soil compared with those grown in MC-soil. These data are in accordance with the higher leaf biomass and area found in plants grown in C-soil (
Table 2 and
Table 3). The analysis of chlorophyll fluorescence was focused on two parameters—the quantum yield of primary photochemistry (F
v/F
m) and performance index (PI
ABS) of PSII. Among the chlorophyll fluorescence parameters, F
v/F
m is recognized as a good indicator for photo-inhibitory or photo-oxidative effects on PSII [
31]. However, the most widely used parameter from the chlorophyll fluorescence OJIP transient is the PI
ABS, which provides quantitative information about the general state of plants and their vitality. PI
ABS is the product of three independent characteristics—the concentration of reaction centers per chlorophyll, a parameter related to primary photochemistry, and a parameter related to electron transport [
20]. PI
ABS reflects the functionality of both PSI and II and produces quantitative information of the plant performance, especially under stress conditions [
20,
32]. Contrarily to chlorophyll content, the results showed that no differences between plants cultivated in both types of soil were observed concerning the F
v/F
m and PI
ABS parameters. Therefore, these data confirmed the good physiological status of hemp plants grown in both soil conditions, as also visually observed for the lack of damage symptoms such as chlorosis or necrosis. Our results are in line with those reported by other authors [
33,
34], who found a reduction of chlorophyll fluorescence parameters such as F
v/F
m, only under elevated metal concentrations, highlighting the ability of hemp plants to tolerate metal stress and to grow in contaminated soil.
In
Table 5, the concentration of the analysed metal(loid)s in the hemp plant organs is reported. Notably, no detection of the most toxic metal(loid) elements, namely, As, V, and Pb, was observed in the above ground organs of plants (stem, leaves, and inflorescence). Conversely, as expected for an essential micronutrient, Zn was found in all organs, especially in the inflorescence, followed by leaves and stem. Contrarily to above ground organs, the presence of metal(loid)s was detected at the root level. In this case, differences in plants were found for As, Pb, and V, with their concentrations being higher in plants cultivated in C-soil compared with MC-soil.
The higher capability of C-soil-grown plants to accumulate the metal (loid) elements was in accordance with the higher growth performance highlighted by biomass production, leaf area, chlorophyll content, and leaf transpiration (
Table 2,
Table 3 and
Table 4), associated with a better nutritional status (
Table 1). Moreover, the hypotheses of a more recalcitrant soil pool in MC-soil or a more active excluder mechanism in MC-soil-grown plants cannot be ruled out.
The low ability of hemp plants to accumulate toxic metals from soils is consistent with previously reported works showing the preferential metal allocation in the root apparatus [
14,
17,
35], even if a low metal translocation to shoots was reported in these investigations. Accordingly, a large variability for this trait, because of genetic and environmental factors, was reviewed [
36]. In this regard, because of the remarkably higher Cd accumulation in the roots compared with the shoots, hemp was defined as a Cd-excluder plant [
16]. Preliminary observations of this work suggest that hemp plants can restrict the uptake of other metal(loid) elements, namely, As, Pb, and V. Further investigations are needed to better evaluate this aspect.
The calculation of the bioconcentration factor (BCF) (
Table 6) revealed that this index was higher in the roots of C-soil grown plants than MC-soil grown plants for all the metal(loid)s analysed, except for Zn, as a result of the different concentrations found in the plants and soils types, respectively. Anyway, the values of BCF found for hemp plants grown in both types of soils are to be considered very low following the literature on the matter [
37]. According to the present data, low BCF for roots in hemp plants treated with Pb was also found [
38,
39,
40]. A similar low BCF for As was also reported in hemp plants collected in a survey on a metal-contaminated site [
40].
Overall, results on metal(loid) accumulation evidenced that under our experimental conditions, hemp plants were able to exclude toxic metals—namely, As, Pb, and V—from entering the vascular tissues and being transported in the above ground organs. In fact, even though a high transpiration associated to a considerable biomass production was observed, metal loading by the rooting system was reduced and translocation to aerial parts was negligible. This aspect opens up intriguing questions about the possible toxic metal excluder mechanisms activated by hemp plants to cope with the metal presence in the soil to be addressed in future investigations. Contrarily, Zn was taken up and transported in stem, leaf, and inflorescence, where it is requested for physiological functions associated to its role as a micronutrient. Regarding the metal absorption capability of the hemp plants assayed in this trial, it should be taken into due account that the sub-alkaline condition of both sampled soils (
Table 1) could have negatively affected the mobility of metals in the circulating soil solution, which is generally favored by a sub-acidic soil pH [
41].