3.2. Soil Contamination
Total content of cadmium in individual samples collected at the site under investigation in the years 2018 and 2022 is reported in
Table 2.
Since there were no statistically significant differences in the total concentration of Cd in the selected sampling points after four years, the total Cd can be considered unchanged. The results differ from other studies which reported a reduction in total concentrations due to downward leaching of cadmium with aging [
45,
63]. In the studied soil, metal leaching probably occurred shortly after contamination, estimated by local authorities at about 15/30 days before the first sampling, and the process had essentially finished by the time of the first sampling in 2018. In fact, leaching tends to be particularly significant in the early days after a contamination event [
45].
Of the various approaches to evaluate the degree of heavy metal contamination, the geoaccumulation index (Igeo), was selected because it refers to the local background concentration, which is crucial to evaluate the pollution level of soil [
64].
The Igeo index defines seven classes of soil contamination [
60,
65,
66,
67] from non-polluted (Igeo < 0) to extremely polluted (Igeo ≥ 5).
Based on the Cd concentration values of the soil samples in the contaminated site, five different degrees of contamination were found: extremely polluted (Igeo > 5), heavily to extremely polluted (4 < Igeo < 5), “severe” (3 < Igeo < 4), “moderate to severe” (2 < Igeo < 3) and “moderate” (1 < Igeo < 2). The class defined as unpolluted to moderately polluted (0 < Igeo < 1) in the specific case can be considered unpolluted as the values are identical to the background value (
Table 2).
The Igeo index values for each sampling point are reported in
Figure 1.
Although this index is widely used in the scientific literature, it is not used in the European regulations on contaminated soils. In the case of Italy in particular, the level of cadmium concentration (defined by the acronym CSC in Italian legislation) that would trigger an investigation into potential contamination is 5 mg kg
−1 for public and residential green areas and 15 mg kg
−1 for industrial soils (CSC1 and CSC2 respectively in
Figure 1). However, the Igeo index provides an interesting evaluation of the pollution since the concentration in the contaminated site is compared with the background value of the affected area in order to identify the anthropic contribution to the total concentration of the metal. Anthropic inputs are usually also characterized by greater mobility of the metal and therefore greater environmental danger [
35,
68]. In the case study it can be noted that the samples with a total metal concentration lower than 1 are substantially identical to the US background reference soil. furthermore, according to Italian legislation, the samples S15, S6, S13, S14, and S10 are considered uncontaminated.
3.3. Changes in Cd Extractability with Aging
The Igeo index helps to define the extent of contamination but fails to provide a picture of the evolution of contamination in the case of heavy metals that remain in place, as in this case. Different tools therefore need to be used to evaluate the effects of soil aging.
Cadmium mobility and bioavailability in soils are determined by chemical forms of the metal, therefore, metal fractionation by sequential extraction procedures (SEPs) is considered as the basis to evaluate Cd aging in soil [
69]. There are many varieties of sequential extractions used and all the SEPs are subject to possible criticism [
70]. However, although the different fractions of metals are only operationally defined, SEPs can provide valuable data concerning the distribution of metals in different pools of bioavailability in soil [
71].
The SEP used (H
2O, KNO
3, EDTA) separates Cd according to the strength of the linkages with soil solid phases in soluble (H
2O), exchangeable fractions (KNO
3) characterized by non-specific adsorption reactions and specific sorbed and complexed fractions (EDTA) [
51,
52,
53,
54,
55]. Cd extractability is reported in
Table 3.
The results obtained showed Cd soluble forms only in the first sampling (2018) and only in some of the selected sampling points belonging to the most contaminated zones. The water extractable species include free ions and soluble Cd complexes which are the most mobile and potentially bioavailable species [
30]. The results obtained in 2018 showed that where the contamination was highest (Igeo > 5), the concentration of extractable Cd was rather high at around 1.5 mg kg
−1 which accounts for about 3% to 6% of the total Cd. However, after four years, also in these sampling points, the concentration of Cd in this fraction was below the detection limit.
As regards the extractability by KNO
3, the extractable amount was quite high at around 15% of the total in 2018. After four years, Cd extractability in KNO
3 decreased in all sampling points from about 30% to about 60% compared to the 2018 data. However, in the most contaminated samples, the extractability was still quite high: around 8% of the total concentration. Cd chemical forms extracted by KNO
3 include weakly-sorbed metal species, particularly those retained on the soil surface by relatively weak electrostatic interactions and those that can be released by ion-exchange processes [
53], thus can be considered potentially bioavailable to plant uptake.
EDTA was used because this complexing agent is able to extract Cd ions that are bound to organic matter [
70] (Gleyzes et al., 2002), and to oxide in soils [
72]. The EDTA-extractable forms are generally considered to be inner-sphere surface complexed forms [
73].
The Cd extractable quantity in EDTA was very high. In 2018 in the most contaminated sampling points, it was around 65% of the total. The extractability in EDTA has an inverse trend compared to the other two extractants with the passage of time. In fact, after four years the Cd extractable quantity increased at all sampling points, reaching approximately 70% of the total. In the absence of leaching, this trend is not surprising and indicates a modification in the chemical forms of the Cd due to aging which induces the passage of the metal from the more mobile forms to the less soluble and less exchangeable ones.
The results from SEP are in accordance with previous studies regarding the change of chemical forms over time [
73]. However the results obtained in this long-term experiment differ considerably from those obtained in the laboratory. In fact in laboratory tests most of Cd added to soils is found in the soluble and exchangeable fractions. In our case instead the highest extractability appeared in the EDTA fraction. Even when laboratory tests are conducted for longer times the exchangeable fractions remained the most dominant in all soils [
73], while in the field, after 4 years very strong bonds are formed between Cd and soil surfaces, as shown by the increase in amount extractable using EDTA. In the initial period after the contamination event, the metal is assumed to be subject to adsorption reactions on the negatively charged surfaces of soil minerals and organic matter through the formation of outer-sphere complexes. With aging, chemical bonds with soil surfaces can become more stable due to the gradual diffusion of the metal within the micropores and mesopores of the solid phase which can lead to formation of inner-sphere complexes, entrapment within the solid phases as well as precipitation reactions [
47,
73,
74].
The data obtained showed that the sum of the extractable fractions tends to remain constant in soil samples with a low Igeo index, and tends to decrease, albeit with some exceptions, in soils with a higher Igeo index.
3.4. Effects of Aging on Cd Plant Uptake
The uptake of Cd by plants requires the release of the metal from solid soil surfaces and its transfer in the soil solution. Results from the SEP provide useful indications of this process, however the soil extractants measure only the potential Cd bioavailability [
75]. The plant physiology and related rhizosphere can modify the relationship between Cd and its plant uptake [
76]. Plants can be used to better understand the metal bioavailability and toxicity in soil during the aging process.
The bioassay experiments carried out on the soil sampled at the two different times showed a definite aging effect on the biomass production. In both 2018 and 2022 the plants grown in the contaminated soils showed no visual signs of stress or phytotoxicity, however the biomass yields were somewhat lower than those grown in uncontaminated soil. The results of the shoot biomass production are reported in
Figure 2.
There was a significant reduction in biomass production compared to the non-contaminated soil. The plants grown in the soil sampled in 2018 show a reduction in yields up to around 53% (sample S1) compared to the yield in US soil. In the tests carried out on the soil sampled in 2022, the biomass production decreased up to about 40% compared to the yield in the US soil. The results obtained are in accordance with other studies which show the direct effect of cadmium on the yield of different plant species [
77,
78]. After four years the reduction of the mobile and bioavailable chemical forms of Cd, due to aging, promoted an increase of plants biomass with respect to those of 2018.
The Cd concentration in the above ground part of
B. Juncea grown in the control soil was below the detection limit. In contrast in the microcosms prepared with the soils collected in contaminated areas in 2018, the Cd in shoots ranged from about 0.24 mg kg
−1 (S10) to about 15.96 mg kg
−1 (S1). Plants grown in the soils collected after four years showed a trend of reduction in the uptake of Cd and the concentration in the shoots ranged from 0.18 (S10) to 11.74 mg kg
−1 (S1). The plant uptake in the two years is reported in
Table 4 together with the translocation factor (TF) calculated as the Cd concentration in shoots/Cd concentration in roots [
79,
80].
When evaluating the contamination of the site under examination, the persistence of cadmium in the soil needs to be differentiated from the persistence of the mobile, soluble or easily exchangeable forms. The results of the sequential extraction provide an image of the aging processes that reduced the mobility and bioavailability of cadmium. Further confirmation is also provided from the results of the biological tests. The bioassay also shows that the bioavailability of cadmium decreased with aging. In fact, the production of biomass increased with time and the Cd concentration in the plant decreased after four years.
Comparing the results of the chemical and biological tests, there is an agreement between the extractability in KNO
3 and the concentration of the metal in the plants. In fact, the reduced extractable quantity corresponds to a decrease in concentration in the plants (
Figure 3).
On the contrary, while the extractable fraction in EDTA increased over time, there was no increase in plant uptake, thus the extraction in EDTA often used to define the bioavailable forms of metals [
81] resulted in contrast with the data of the biological test.
Since the extractable quantity of EDTA increased over time, while the uptake by the plants was reduced after four years, the extraction with EDTA, in this case, does not provide reliable information regarding the actual uptake by plants. Therefore, the evaluation of bioavailability needs an understanding of the chemical processes involved not only in the soil, but also of the complex interactions at the soil-plant interface. The results obtained in this case study indicate that the extractability in EDTA tends to overestimate the bioavailable amount of cadmium, which is best represented by the extractable amount in KNO3. However, EDTA extraction provides interesting data that can be used to evaluate the possibility of using this complexing agent as a Cd mobilizer in the case of assisted phytoextraction.
This result is important in terms of soil remediation. In this study it is clear that Cd concentration in soil affected the metal uptake. However, it should be noted that even in the most contaminated soil samples, the effect of the soil-plant barrier [
82] limited the absorption of Cd by
B. juncea. Moreover, the translocation of the metal in the aerial part of the plants was low. In both 2018 and 2022, for plants growing in all the 15 soil samples, the translocation factor (TF) defined by the ratio between the Cd concentration in shoots and Cd concentration in roots was always smaller than 1, ranging from 0.36 to 0.82. These results are in accordance with previous studies [
83,
84], and confirm that roots act as a barrier against the translocation of the metal to the aerial parts of
B. juncea.
Evaluation of the bioassays results as a feasibility test for the possible use of phytoextraction, must be based on the total removal of the metal from the soil by the plants. The efficiency of Cd phytoextraction was thus calculated according to the following relation [
80,
85,
86]:
The data reported in
Table 5 show that the brassica plants remove only a very low fraction of the Cd present in the soil, less than 0.1% even in the most contaminated samples.
Even considering the total removal in relation to only the bioavailable fraction, as is often recommended in the evaluation of phytoextraction [
87,
88], the amount removed from the plants, would not reach, on average, more than 5% (data not reported). Clearly, no conclusions can be drawn from the evaluation of a single plant species, and different species should be tested.
Brassica juncea which is considered a plant with great potential for phytoremediation [
89] is a good indicator for Cd uptake even if it is not an accumulator species [
90,
91,
92,
93]. However, the data obtained strongly suggest that the technology could only be applied in the form of “assisted phytoremediation” by applying, for example, a complexing agent such as EDTA [
94]. However, the use of complexing agents can also cause further environmental risks, in fact the quantity of cadmium mobilized may be higher than the quantity absorbed by the plants, and being in soluble form, it could percolate along the soil profile [
95].
3.5. Effect of Ageing on Cd Bioaccessibility
In addition to the soil-plant-food chain route, incidental ingestion of soil is an important exposure pathway. Oral ingestion is often the critical exposure pathway for children [
96]. The negative effects of this pathway can be assessed in terms of bioaccessibility, which is defined as the fraction of a metal in soil which, after ingestion, becomes soluble in the stomach and is thus available for intestinal absorption [
32,
33,
97,
98]. An estimation of Cd bioaccessibility is therefore useful to better assess the human health risks from soil ingestion exposure. The data on bioaccessible Cd in the two sampling campaigns are reported in
Table 6.
In natural soils the bioaccessible fraction is generally very low [
99,
100] and also in this study in the unpolluted soil, the Cd bioaccessible concentration was very low (0.02 mg kg
−1). In contrast in the contaminated samples, the Cd concentration values in the bioaccessible extracts were often rather high in both sampling campaigns with values ranging from 40 to 60% of the total content similarly to what was found for this metal in previous studies [
99,
101]. The differences between the two concentrations were not statistically significant (
Table 6).
The amount of bioaccessible Cd was significantly related to the total metal content (R
2 = 0.988) in soil and less to the most available fractions such as water-soluble and exchangeable fractions. This is in agreement with a previous study where different levels of bioaccessible Cd in soils were mostly related to the total metal content [
33], and it is consistent with the fact that both total and bioaccessible quantities are based on acid extraction.
In other cases, bioaccessible metals were found to be correlated with the exchangeable fractions [
44,
102]. However, in the latter studies, the soils were spiked with heavy metals, which could be important in explaining the diversity of the results obtained in our study where the effects of aging may have changed the original chemical forms of the metal.
Soil oral ingestion together with inhalation of soil dust and dermal contact have been recognized as a critical exposure pathway [
103,
104]. Including metal bioaccessibility in soil risk assessment and monitoring strategies has increased in the last few years in relation to human health protection [
101]. The influence of aging on bioaccessibility has been well documented for organic contaminants [
105], while several aspects of the aging trend of the bioaccessible fractions of metals in soil over time still needs to be still clarified. The speciation of cadmium in soil largely regulates its dissolution in the gastrointestinal system which is also promoted by the low pH in the stomach. Evaluating the change with time of the bioaccessible fraction of the metal may thus be of great help for the risk assessment of soil in relation to the potential negative health effects [
37,
106]. Even when long incubation times are used in the laboratory, the conditions are not fully comparable with those of naturally aged field soils [
42,
45,
107]. To explain the changes in bioavailability and bioaccessibility of Cd it must be taken into account not only the fast distribution of the metals among the soil phases after the first days of contamination, but also the slow sorption and desorption processes in the long time. In addition to the fast reactions of adsorption of the first phase which are studied in laboratory experiments with spiked soils, in this field experiment also the very much slower continuing sorption reactions impacted Cd speciation is soil. Thus bioavailability and bioaccessibility were influenced by the Cd diffusive penetration into soil constituents, precipitation and slow chemisorption on reactive soil surfaces where the metal strongly bound may not be likely to be easily released. The study of the aging of cadmium under real field conditions is therefore an important step for the accurate assessment of soil contamination.