**3. Materials and methods**

The other Mediterranean region considered was the Mediterranean area of Australia. Climatic conditions are similar to the ones describe previously, although the properties of the soils present in this area differ slightly, and include, for example, soils with lower pH values. Adequate representative soils were sampled from the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Ginninderra Experiment Station (Australian Capital Territory —ACT), sampling those representative of the Mediterranean region [26] and that were

Regarding the protection and conservation of soils, it is important to consider that many Mediterranean countries, including Spain (representative of the European Mediterranean) and

More specifically, in Spain, and according to the Spanish Royal Decree 9/2005 [27], any soil must be considered as potentially contaminated (or contaminated) when concentrations (or concentrations 100 times) above the corresponding baseline value are determined in them. In agricultural soils, the baseline value for the different elements is established taking into account the upper limit of the normal range of concentrations, which covers the natural variability of the metal in soil associated with background levels at regional level. This normal range of concentrations considers diffuse or nonpoint pollution (e.g. fertilisation and atmospheric deposition) but does not include point pollution due to local human activities (e.g. industries) [17, 28–30]. These values are useful to identify the current contents of heavy metals and to assess the degree of contamination by human activities [30]. Regarding the establishment of these values, Micó et al. [30] and Sánchez et al. [31] established the baseline values for different heavy metals in agricultural soils under vegetable crops of the Valencian Mediterranean region. The baseline for Cu was 65.9 mg/kg, and it is similar to those established in other Spanish Mediterranean regions [32, 33] and in other European Mediterranean regions [34, 35].

On the other hand, Australian guidelines for metal contaminant concentrations in soil and soil amendments are established at a state level (e.g. [36–38]) and are based on European regula-

dedicated to agriculture.

66 Soil Contamination - Current Consequences and Further Solutions

**Figure 1.** Distribution of the Mediterranean climate in the world [22].

Australia, use soil quality standards to characterise contaminated soils.

#### **3.1. Sampling and soil characterisation**

Four agricultural plots from the Spanish Mediterranean region and three agricultural plots from the Australian Mediterranean region, all having different soil properties, were selected and sampled.

On one hand, the selection of the Spanish soils was performed considering the information and databases of previous studies [42, 43]. These classify them as representative of the European Mediterranean agricultural area. More specifically, the types of soils represented were two Calcaric Fluvisols with different soil properties (Sollana and Peníscola), a Gleyic Fluvisol (Nules) and a Salic Fluvisol (Rojales), according to the World Reference Base for Soil Resources [44]. The soils selected covered a wide range of the different types of soils devoted to vegetable crops in the European Mediterranean region [45].

On the other hand, the selection of the Australian soils was carried out considering the information of the Commonwealth Scientific and Industrial Research Organisation (CSIRO). The types of soils represented were a Chromic Luvisol (Soil 1), an Eutric Planosol (Soil 2) and a Pellic Vertisol (Soil 3), according to the World Reference Base for Soil Resources [44].

Soil properties were determined according to the official laboratory methods of the Spanish Ministry of Agriculture, Fishery and Food [46] for the soils of the Spanish Mediterranean Region, and to the official soil chemical methods for Australasia [47] for the soils of the Australian Mediterranean Region.

#### **3.2. Experimental design**

Three different sets of experiments were carried out and compared, each one including two different ecotoxicological assays (described later): one set of experiments with European Mediterranean soils and lettuce; another set with Australian Mediterranean soils and lettuce; the last set with European Mediterranean soils and tomato.

The sampled agricultural soils indicated previously were spiked with a Cu contaminant solution to achieve six different total Cu concentrations, the control (no Cu addition) and five different doses (65.9, 659.0, 1977.0, 3295.0 and 6590.0 mgCu/kg). These ranges of doses were selected and established after considering previous studies also carried out in Mediterranean agricultural soils [39, 48, 49].

Two different ecotoxicological assays were conducted in the contaminated soils: one to evaluate the effect of Cu over biomass production (28 days); and the other to analyse the absorption and accumulation of Cu in roots and stem and leaves for lettuce, or in roots, stem and leaves and fruit for tomato (3 months).

For the first assay, biomass production was assessed following the OECD test 208 [50], where 300 g of contaminated soils was placed in pots (10 cm in diameter) and ten lettuce or five tomato seeds were then seeded to 1 cm soil depth. Each treatment was replicated three times (three pots per Cu dose and three per control), and all pots were placed in a glasshouse. Experimental conditions were controlled and maintained according to the requirements specified in the biomass assay procedure [50].

For the accumulation assay, 1.2 kg of contaminated soils were placed in 25 cm diameter pots and ten lettuce or five tomato seeds were seeded to 1 cm soil depth, although only one of the germinated seeds was selected to grow until maturity. As for the biomass assay, each treatment was replicated three times (three pots per Cu dose and three per control) and all pots were placed in a glasshouse. Again, experimental conditions were controlled and maintained according to the requirements specified in the biomass assay procedure [50].

#### **3.3. Biomass data analysis**

Weight values obtained in the biomass assay were used to establish the EC50 and EC10 effective concentrations. Previous to this, homogeneity of variance and normality of weight data was checked using the Kolmogorov-Smirnov test and these were log-transformed when appropriate in order to stabilise variances. Dose-response data were fitted to a log-logistic curve according to Eq. (1) [51] for each of the soils tested in order to establish the EC50 and EC10. TRAP© version 1.22 (Toxicity Relationship Analysis Program, United States Environmental Protection Agency) was used for this purpose [52–54].

$$\mathcal{Y} = \frac{\mathcal{Y}\_0}{1 + e^{b(x - M)}} \tag{1}$$

where *y* = biomass (lettuce/tomato shoot weight of plants) produced (mg), *x* = log10(added Cu) (mg/kg), *y*0 = biomass produced with non-added Cu (control) (mg), and *M* and *b* are parameters to be fitted, where *M* = log10(EC50) and *b* is a slope parameter that indicates the inhibition rate. The concentration of Cu considered in the control dose was the initial Cu content of the soil assayed. The distribution of residuals, relationship between these and the fitted values and the adjusted coefficient of determination (*R*<sup>2</sup> adj.) were examined in order to determine the model's adequacy. The EC10 was also calculated as described above.

#### **3.4. Cu content in soils and plants**

The sampled agricultural soils indicated previously were spiked with a Cu contaminant solution to achieve six different total Cu concentrations, the control (no Cu addition) and five different doses (65.9, 659.0, 1977.0, 3295.0 and 6590.0 mgCu/kg). These ranges of doses were selected and established after considering previous studies also carried out in Mediterranean

Two different ecotoxicological assays were conducted in the contaminated soils: one to evaluate the effect of Cu over biomass production (28 days); and the other to analyse the absorption and accumulation of Cu in roots and stem and leaves for lettuce, or in roots, stem

For the first assay, biomass production was assessed following the OECD test 208 [50], where 300 g of contaminated soils was placed in pots (10 cm in diameter) and ten lettuce or five tomato seeds were then seeded to 1 cm soil depth. Each treatment was replicated three times (three pots per Cu dose and three per control), and all pots were placed in a glasshouse. Experimental conditions were controlled and maintained according to the requirements specified in the

For the accumulation assay, 1.2 kg of contaminated soils were placed in 25 cm diameter pots and ten lettuce or five tomato seeds were seeded to 1 cm soil depth, although only one of the germinated seeds was selected to grow until maturity. As for the biomass assay, each treatment was replicated three times (three pots per Cu dose and three per control) and all pots were placed in a glasshouse. Again, experimental conditions were controlled and maintained

Weight values obtained in the biomass assay were used to establish the EC50 and EC10 effective concentrations. Previous to this, homogeneity of variance and normality of weight data was checked using the Kolmogorov-Smirnov test and these were log-transformed when appropriate in order to stabilise variances. Dose-response data were fitted to a log-logistic curve according to Eq. (1) [51] for each of the soils tested in order to establish the EC50 and EC10. TRAP© version 1.22 (Toxicity Relationship Analysis Program, United States Environmental

> 0 ( ) 1 *bx M*

where *y* = biomass (lettuce/tomato shoot weight of plants) produced (mg), *x* = log10(added Cu) (mg/kg), *y*0 = biomass produced with non-added Cu (control) (mg), and *M* and *b* are parameters to be fitted, where *M* = log10(EC50) and *b* is a slope parameter that indicates the inhibition rate. The concentration of Cu considered in the control dose was the initial Cu content of the soil assayed. The distribution of residuals, relationship between these and the fitted values and the

adj.) were examined in order to determine the model's

*<sup>y</sup> <sup>y</sup> <sup>e</sup>* - <sup>=</sup> <sup>+</sup>

according to the requirements specified in the biomass assay procedure [50].

Protection Agency) was used for this purpose [52–54].

adequacy. The EC10 was also calculated as described above.

adjusted coefficient of determination (*R*<sup>2</sup>

agricultural soils [39, 48, 49].

biomass assay procedure [50].

**3.3. Biomass data analysis**

and leaves and fruit for tomato (3 months).

68 Soil Contamination - Current Consequences and Further Solutions

Stem and leaves, and root samples of the accumulation assay were grounded and 0.5 sieved prior to their analysis. Total Cu concentration in soils, stem and leaves, and roots was determined using the USEPA 3052 method [55]. Copper content in soils and plants was analysed by a Microwave Plasma Atomic Emission Spectrometer (MP-AES). The precision and the accuracy of the analysis were evaluated calculating the relative standard deviation (RSD) and the recovery of metal of external standards provided by the commercial house (Agilent) and different Certified Reference Materials (CRM). RSD values (from 4 to 9%) were smaller than 10% and were considered satisfactory [56]. Recoveries ranged from 83 to 111% and were within 80–120% interval proposed as satisfactory by [56].

In order to compare the Cu concentrations obtained in stem and leaves with the maximum Cu content in foodstuffs (10 mg/kg in fresh weight basis for lettuce) established by the identified legislation [57], different conversion factors were applied. These were calculated by assessing their moisture content through a gravimetric method [47]. Furthermore, and considering this maximum value, the critical limit that refers to the concentration of Cu in soil that results in the maximum concentration allowed in vegetable crops was defined when possible.

Moreover, to assess the accumulation and distribution of Cu in lettuce and tomato plants, and therefore their phytoremediation potential, three different concentration factors (CFs) were calculated. In this case study, the ratio between the heavy metal concentrations in root (mg/kg dry weight) and in soil; the ratio between the concentrations in stem and leaves and in root; and the ratio between the concentrations in fruits and in stem and leaves were calculated for each soil and dose.

It is important to point out that, in this study, the total Cu in soil that is bioavailable has been considered to be very similar to the total Cu concentration in soil. Although not realistic for aged contaminated soils, spiked soils realistically reflect the conditions in terms of contamination that can take place in agricultural soils as a result of different contamination processes. More specifically, they realistically reflect contamination processes and conditions associated with an excessive Cu-based pesticide and fungicide application, or due to spills [58] or intensive extractive activities nearby [59], where Cu is artificially added and is very bioavailable. In such cases, the values of total and bioavailable Cu content are very similar, so both concentrations can be used to analyse this type of contamination [39, 60].

#### **3.5. Statistical analysis**

(1)

After checking the distribution and homogeneity of variance, mean biomass produced for the different doses and soils was compared applying two-way ANOVAs and Turkey test, in order to elucidate differences amongst soils and doses. The influence of soil properties on biomass production and in the accumulation of Cu in the edible part of the plant was assessed by correlation analyses. Correlations were derived between each of the effective concentrations (EC50 and EC10) calculated and the soil properties of the different soils sampled, and between the soil properties and the concentrations in plants at the different doses assayed. The correlation coefficients considered were Pearson´s since the data had a normal distribution. All these statistical analyses were conducted using SPSS© version 19.3.

#### **4. Results**

**Table 1** summarises the main properties of the seven soils assayed (Rojales, Sollana, Nules, Peníscola, Soil 1, Soil 2 and Soil 3). As it can be observed, a wide range of different soil properties was covered with the selected soils, enabling this way to analyse the influence of the different properties over the dynamics of Cu in soils and its transference to the plant.


EC, electrical conductivity; SOM, soil organic matter content; CCE, calcium carbonate equivalent content; CEC, cation exchange capacity

**Table 1.** Properties of the seven soils assayed [39–41].


– not assayed.

a Effective concentrations of added Cu that caused a 10% reduction in the biomass produced. b Effective concentrations of added Cu that caused a 50% reduction in the biomass produced. Percentage of variance accounted for by the log-logistic model.

**Table 2.** Toxicity threshold values (EC10 and EC50, mg/kg) for Cu added to soil derived from the lettuce and tomato biomass tests in the seven soils assayed [39–41].

**Table 2** shows and sums up toxicity threshold values (EC10 and EC50) calculated for each soil and crop.

Copper Contamination in Mediterranean Agricultural Soils: Soil Quality Standards and Adequate Soil... http://dx.doi.org/10.5772/64771 71


All the results are expressed in mg/kg in dry weight basis [39].

– no biomass produced.

lation coefficients considered were Pearson´s since the data had a normal distribution. All these

**Table 1** summarises the main properties of the seven soils assayed (Rojales, Sollana, Nules, Peníscola, Soil 1, Soil 2 and Soil 3). As it can be observed, a wide range of different soil properties was covered with the selected soils, enabling this way to analyse the influence of the different

**Soil pH EC (dS/m) SOM (%) CCE (%) CEC (cmol(+)/kg) Sand (%) Silt (%) Clay (%) Initial Cu (mg/kg)**

EC, electrical conductivity; SOM, soil organic matter content; CCE, calcium carbonate equivalent content; CEC, cation

**EC10a EC50b R2**

Effective concentrations of added Cu that caused a 10% reduction in the biomass produced.

Effective concentrations of added Cu that caused a 50% reduction in the biomass produced.

Percentage of variance accounted for by the log-logistic model.

biomass tests in the seven soils assayed [39–41].

**soil Lettuce Tomato Lettuce Tomato Lettuce Tomato** Rojales 8.8 ± 0.9 32.9 ± 0.3 177 ± 2.1 500.7 ± 0.1 89 93 Sollana 46.2 ± 1.3 393.5 ± 0.2 680 ± 3.4 1223.8 ± 0.2 88 81 Nules 159 ± 3.4 491.4 ± 0.6 753 ± 2.9 1696.5 ± 0.4 97 50 Peníscola – 358.4 ± 0.2 – 663.8 ± 0.2 – 98 Soil 1 49.0 ± 1.7 – 104.0 ± 2.0 – 90 – Soil 2 106.9 ± 2.0 – 236.4 ± 2.4 – 94 – Soil 3 443.1 ± 2.6 – 728.9 ± 2.9 – 93 –

**Table 2.** Toxicity threshold values (EC10 and EC50, mg/kg) for Cu added to soil derived from the lettuce and tomato

**Table 2** shows and sums up toxicity threshold values (EC10 and EC50) calculated for each soil

 **adj. (%) c**

properties over the dynamics of Cu in soils and its transference to the plant.

Rojales 7.66 0.90 1.6 52 14.5 28 38 33 12.4 Sollana 7.48 2.38 3.8 53 27.6 12 41 47 30.9 Nules 7.72 3.26 8.7 39 37.1 19 34 48 58.5 Peníscola 7.72 1.86 2.7 45 16.8 49 25 25 17.4 Soil 1 5.36 1.10 3.7 0 4.2 10 10 80 7.6 Soil 2 5.67 1.34 4.6 0 13.1 26 36 38 17.6 Soil 3 7.41 2.05 3.5 0 36.5 42 43 15 15.5

statistical analyses were conducted using SPSS© version 19.3.

70 Soil Contamination - Current Consequences and Further Solutions

**4. Results**

exchange capacity

– not assayed.

and crop.

a

b

**Table 1.** Properties of the seven soils assayed [39–41].

a The conversion factors that have to be applied in order to calculate the content of metal in crop in fresh weight basis are the following: 11.2 for Rojales, 17.3 for Sollana and 17.6 for Nules. b Concentration factor.

**Table 3.** Mean copper content in the edible parts of lettuces (mg/kg in dry weight basis), and mean total contents of copper in the European Mediterranean soils assayed.


All the results are expressed in mg/kg in dry weight basis [41].

– no biomass produced.

CFs-r: concentration factor, between soil and root; CFr-l: concentration factor, between root and leaf. a The conversion factors that have to be applied in order to calculate the content of metal in plant in fresh weight basis are the following: 8.2 for Soil 1, 8.8 for Soil 2, 9.9 for Soil 3.

**Table 4.** Mean copper content in the Australian Mediterranean soils assayed and mean copper content in roots and the edible part of lettuce.

**Tables 3**–**5** show the results obtained in terms of Cu concentration in soils and in the different parts of the plants analysed, indicated previously.


All the results are expressed in mg/kg in dry weight basis [40].

– no biomass produced.

CFs-p: concentration factor, between soil and plant; CFp-f: concentration factor, between plant and fruit.

a The conversion factors that have to be applied in order to calculate the content of metal in plant in fresh weight basis are the following: 11.6 for Rojales, 10.2 for Sollana, 10.5 for Nules and 9.9 for Peníscola.

b The conversion factors that have to be applied in order to calculate the content of metal in fruit in fresh weight basis are the following: 16.7 for Rojales, 15.6 for Sollana, 14.8 for Nules and 18.5 for Peníscola.

**Table 5.** Mean copper content in the European Mediterranean soils assayed (mg/kg in dry weight basis), in plant (mg/kg in dry weight basis), and in the edible part of tomato (ripe fruit).

Regarding the definition of the critical limits, these could only be established for the European Mediterranean soils cropped with lettuce. For the Australian agricultural soils cropped with lettuce, the establishment of these limits was not possible due to the important toxic effect observed. On the other hand, forthe European Mediterranean soils cropped with tomato, these limits couldnotbe calculateddue to the facttheCucontentinfruitkept constant,independently of the Cu dose assayed and type of soil. The results obtained are shown in **Table 6**.

Finally, regarding the statistical analysis, and as explained previously, different correlation analysis were carried out in order to determine which soil properties influence the dynamic of Cu in soil and were more significant in terms of biomass production and of Cu absorption. For further details regarding these analyses, please consult [39–41].


**Table 6.** Critical limit for the soil studied.

**Tables 3**–**5** show the results obtained in terms of Cu concentration in soils and in the different

0.01 (control) 12.4 ± 1.7 23.8 ± 2.5 7.8 ± 1.9 1.92 0.33 30.9 ± 4.3 21.7 ± 2.2 8.2 ± 2.0 0.70 0.38

65.9 64.1 ± 8.9 28.8 ± 3.0 7.3 ± 1.7 0.45 0.25 79.1 ± 11.0 27.6 ± 2.9 8.1 ± 2.0 0.35 0.29

659.0 612.5 ± 84.9 31.9 ± 3.3 – 0.05 – 673.8 ± 93.4 26.3 ± 2.6 8.6 ± 2.0 0.04 0.33

1977.0 1879.9 ± 260.7 63.5 ± 6.6 – 0.03 – 2003.7 ± 277.8 27.8 ± 2.8 7.6 ± 1.8 0.01 0.27

3295.0 3670.0 ± 480.7 242.5 ± 25.1 – 0.07 – 2915.8 ± 404.3 28.5 ± 2.5 9.1±2.2 0.01 0.32

6590.0 6404.5 ± 888.1 641.3 ± 66.4 – 0.10 – 7080.0 ± 922.8 688.5 ± 71.2 – 0.10 –

0.01 (control) 58.1 ± 8.0 17.6 ± 1.8 6.6 ± 1.6 0.30 0.38 17.4 ± 2.4 20.4 ± 2.2 7.6 ± 1.8 1.17 0.37

65.9 108.5 ± 15.0 18.9 ± 2.0 8.8 ± 2.1 0.17 0.46 76.2 ± 10.6 23.7 ± 2.4 7.7 ± 1.9 0.31 0.32

659.0 683.2 ± 94.7 22.0 ± 2.3 6.8 ± 1.6 0.03 0.31 538.3 ± 74.6 31.4 ± 2.8 7.4 ± 1.8 0.06 0.24

1977.0 2023.1 ± 280.5 26.8 ± 2.8 7.8 ± 1.8 0.01 0.29 1658.2 ± 229.9 394.1 ± 40.8 8.3 ± 2.0 0.24 0.02

3295.0 2856.6 ± 396.1 44.4 ± 4.6 7.7±1.9 0.02 0.17 3185.6 ± 441.7 1187.5±122.9 9.9 ± 2.4 0.37 0.01

6590.0 6077.8 ± 842.7 1229.2 ± 127.2 8.7±2.1 0.20 0.01 6476.7 ± 897.9 – – – –

The conversion factors that have to be applied in order to calculate the content of metal in plant in fresh weight basis

The conversion factors that have to be applied in order to calculate the content of metal in fruit in fresh weight basis

**Table 5.** Mean copper content in the European Mediterranean soils assayed (mg/kg in dry weight basis), in plant

CFs-p: concentration factor, between soil and plant; CFp-f: concentration factor, between plant and fruit.

are the following: 11.6 for Rojales, 10.2 for Sollana, 10.5 for Nules and 9.9 for Peníscola.

are the following: 16.7 for Rojales, 15.6 for Sollana, 14.8 for Nules and 18.5 for Peníscola.

(mg/kg in dry weight basis), and in the edible part of tomato (ripe fruit).

**In soil In planta In fruitb CFs-p CFp-f In soil In planta In fruitb CFs-p CFp-f**

**In soil In planta In fruitb CFs-p CFp-f In soil In planta In fruitb CFs-p CFp-f**

parts of the plants analysed, indicated previously.

72 Soil Contamination - Current Consequences and Further Solutions

**Rojales Sollana**

**Nules Peníscola**

All the results are expressed in mg/kg in dry weight basis [40].

**Total content of Cu (mg/kg)**

**Dose Cu**

**(mg/kg)**

**Dose Cu**

**(mg/kg)**

– no biomass produced.

a

b

#### **4.1. European and Australian agricultural soils cropped with lettuce**

As detailed previously, agricultural soils from two different Mediterranean areas of the world were considered. Different biomass assays having the same experimental design and crop were carried out in these areas, enabling to compare the results obtained and to draw different conclusions regarding the behaviour of Cu in soils and plants.

The analysis of the toxicity threshold values obtained for the Spanish and Australian agricultural soils and lettuce showed that biomass production is greatly influenced by Cu and that similar soil properties are relevant when analysing the effect of Cu and its mobility and bioavailabity. As it can be observed in **Table 2**, the range of toxicity thresholds established covered similar ranges in both Mediterranean areas, being of 8–753 mgCu/kg in the Spanish Region, and of 49–728 mgCu/kg in the Australian Region. In both cases, the maximum threshold value was obtained for the soil having the highest pH and clay content, independently of the soil type. Therefore, these two soil properties seem to be very relevant when analysing Cu mobility and availability in soils. The difference between the maximum thresholds obtained in each region can be linked to the fact that the soil of the Spanish region had a higher SOM content and a basic pH, which increases the retention capacity of soil.

The comparison of the results obtained in both areas also pointed out the relevance of pH when analysing the mobility and availability of Cu in agricultural soils, even in soils with medium clay contents. For the all soils assayed in the Spanish Mediterranean Region, whose pH values varied slightly and were all between 7 and 8, no biomass was produced after the fifth dose, while no biomass was produced after the second, third and fourth dose in the different soils

of the Australian Region, increasing the toxic effect of Cu as pH decreased. In these latter soils, pH values varied amongst 5–7.5. The most important toxic effect was observed for one of the Australian soils assayed that had a low pH value (5.6) but a medium content of clay (38%).

Therefore, according to the results obtained, two different approaches have to be made when assessing Cu-contaminated agricultural soils, depending on the pH of these. In acidic soils (pH below 7), pH is the most relevant soil property and strongly influences the bioavailability of Cu, in spite of the contents and values obtained for other soils properties. Toxic effect of Cu increases as pH values decreased, and soil properties that we would expect to have some retention capacity are ineffective or have very little effect due to the influence of pH on their reactivity. In fact, at acid pH, the reactivity of SOM and clay is low or even null. Conversely, for basic soils (pH values exceeding 7), other properties have a more relevant effect, being clay/ sand content, SOM and salinity the most relevant ones. Clay and SOM retain Cu by adsorption reactions, while salinity and sand content make Cu more bioavailable and increase the toxic effect.

Analysis of the transfer of Cu from soil to plant showed that it varied between these two areas. However, it is important to point out that comparison of results was difficult due to the important toxic effect observed in the Australian agricultural soils. No biomass was produced after earlier doses in the case of these soils, which made it complicated to compare absorption values and rates. In both areas, Cu content in the edible part of the plant increased as Cu concentration in soils also did, but no clear absorption pattern could be identified due to the limited data obtained in the Australian assays. However, the correlation analyses carried out between Cu contents and soils properties showed similarities between them and with the results obtained for biomass production. In this case, pH, salinity and sand content are the most determinant soil properties which enhance Cu transference from soil to lettuce, while SOM and clay content reduce this metals' transference to lettuce.

Concerning the critical limits, as commented previously, these could only be calculated for the European Mediterranean soils. When compared to with the Spanish soil quality standard, the results varied significantly. The critical value calculated for the non-saline soils (Sollana and Nules) was above 100 times the baseline value for Cu, being higher in the soil with the highest organic matter and clay content (Nules), whereas it was below in the soil with high salinity and low organic matter content (Rojales). It is important to point out that these values have to be interpreted carefully and considering they are only theoretical, especially the ones for Sollana and Nules. For these soils, no biomass would be produced if these concentrations were reached, as it has been proved in the assays carried out, where no biomass production was observed when the dose of Cu was 6590 mg/kg.

#### **4.2. Lettuce and tomato cropped in different European Mediterranean**

Within the same region, two different crops in different agricultural soils were assayed in order to analyse their different responses and behaviours to Cu in soil, in terms of biomass production and Cu absorption, and to evaluate the influence of soil properties on the mobility and availability of this metal to plants.

Toxicity threshold values obtained varied significantly between crops for the different soils assayed. For lettuce, as commented previously, effective concentration calculated varied between 8 and 753 mgCu/kg, while for tomato these concentrations varied between 33 and 1697 mgCu/kg. A more detailed analysis of these results indicate that, for EC10, the values obtained for tomato are nearly twice the maximum value obtained for lettuce, except for one soil; and for EC50, the lowest value obtained for tomato is very similar to the maximum concentration obtained for lettuce. This clearly indicates the different response of these two crops to the different Cu concentrations in soils, showing that tomato is more tolerant than lettuce to Cucontaminated soils. In fact, according to [61] lettuce can be considered an accumulator crop, while tomato can be considered a non-accumulator crop.

of the Australian Region, increasing the toxic effect of Cu as pH decreased. In these latter soils, pH values varied amongst 5–7.5. The most important toxic effect was observed for one of the Australian soils assayed that had a low pH value (5.6) but a medium content of clay (38%).

Therefore, according to the results obtained, two different approaches have to be made when assessing Cu-contaminated agricultural soils, depending on the pH of these. In acidic soils (pH below 7), pH is the most relevant soil property and strongly influences the bioavailability of Cu, in spite of the contents and values obtained for other soils properties. Toxic effect of Cu increases as pH values decreased, and soil properties that we would expect to have some retention capacity are ineffective or have very little effect due to the influence of pH on their reactivity. In fact, at acid pH, the reactivity of SOM and clay is low or even null. Conversely, for basic soils (pH values exceeding 7), other properties have a more relevant effect, being clay/ sand content, SOM and salinity the most relevant ones. Clay and SOM retain Cu by adsorption reactions, while salinity and sand content make Cu more bioavailable and increase the toxic

Analysis of the transfer of Cu from soil to plant showed that it varied between these two areas. However, it is important to point out that comparison of results was difficult due to the important toxic effect observed in the Australian agricultural soils. No biomass was produced after earlier doses in the case of these soils, which made it complicated to compare absorption values and rates. In both areas, Cu content in the edible part of the plant increased as Cu concentration in soils also did, but no clear absorption pattern could be identified due to the limited data obtained in the Australian assays. However, the correlation analyses carried out between Cu contents and soils properties showed similarities between them and with the results obtained for biomass production. In this case, pH, salinity and sand content are the most determinant soil properties which enhance Cu transference from soil to lettuce, while

Concerning the critical limits, as commented previously, these could only be calculated for the European Mediterranean soils. When compared to with the Spanish soil quality standard, the results varied significantly. The critical value calculated for the non-saline soils (Sollana and Nules) was above 100 times the baseline value for Cu, being higher in the soil with the highest organic matter and clay content (Nules), whereas it was below in the soil with high salinity and low organic matter content (Rojales). It is important to point out that these values have to be interpreted carefully and considering they are only theoretical, especially the ones for Sollana and Nules. For these soils, no biomass would be produced if these concentrations were reached, as it has been proved in the assays carried out, where no biomass production was

Within the same region, two different crops in different agricultural soils were assayed in order to analyse their different responses and behaviours to Cu in soil, in terms of biomass production and Cu absorption, and to evaluate the influence of soil properties on the mobility and

SOM and clay content reduce this metals' transference to lettuce.

**4.2. Lettuce and tomato cropped in different European Mediterranean**

observed when the dose of Cu was 6590 mg/kg.

74 Soil Contamination - Current Consequences and Further Solutions

availability of this metal to plants.

effect.

The analysis of the influence of soil properties on the effect of Cu on plant biomass production led to similar results/conclusions in both assays. SOM, clay content and CEC are the most relevant properties affecting Cu soils dynamic [39, 40].

Regarding the metal accumulation in the plant, the concentrations determined both in tomato and lettuce shoots were also very similar, although this latter tends to accumulate slightly higher concentrations. The most important conclusion drawn is that in the case of tomato, low translocation rates to the edible part of the plant are observed, even in soils with high Cu concentrations, while Cu translocation and accumulation in the edible part of lettuce increase as soil Cu concentration increases. The results observed for tomato were particularly interesting, since Cu concentration in fruits kept low and constant, independently of the Cu concentration in soils and shoots. This indicates that these plants tend to accumulate Cu in shoots and roots, with very low translocation of it to fruit, pointing out its phytoremediation potential.

In both cases (lettuce and tomato), the increase in Cu concentration determined in plant was not proportional to the increase in Cu concentrations in soil, due to the fact that Cu accumulation in plant is limited. Since Cu concentration in tomato fruits kept constant, the critical limit of contaminant in soil for this crop could not be calculated and therefore cannot be compared with the critical limits calculated for lettuce.

The analysis of the influence of soil properties on the transfer and bioaccumulation of Cu in these crops also led to similar results/conclusions. Both salinity and sand content arised as soil characteristics that enhance the transfer of Cu from soil to plant; while SOM and clay content have the opposite effect.

Furthermore, it is important to point out that the maximum metal content in the edible part of the plant established by the identified legislations [19, 20] was not exceeded in any of the dose and soils assayed for tomato and by only one soil in the case of lettuce. This soil was the one having the highest salinity content, and therefore, it seems logical to observe this, due to the fact that, as explained previously, this soil property facilitates the transfer of Cu from soil to plant.

Finally, it is important to highlight that for both tomato and lettuce, and considering the results obtained for the effect of Cu and its interaction with soil properties on plant biomass production and metal bioaccumulation in plant, the soil quality standard established by the Spanish legislation is not valid from either approach. Toxicity threshold values calculated for both crops showed that this soils quality standard was too indulgent, and it indicated this approach as the most restrictive when establishing soil quality standards. Conversely, the critical limit calculated for lettuce (**Table 6**) and the results obtained for the accumulation of Cu in the edible part of the plant show that the soil quality standard established by the Spanish legislation was too restrictive, since this content would not be exceeded in any of the soils assayed. Only one critical limit established showed that this soil quality standard was too permissive and corresponded to the one calculated for the saline soil.

Therefore, the results obtained show that soil quality standards should be established considering the influence of the different soil properties and should be particular for each case and scenario.

Lastly, and since the baseline value considered and used in all the assays carried out is similar to those established in other Spanish Mediterranean regions [32, 33] and in other European Mediterranean regions [34, 35], it is important to highlight that the results obtained in this work could be used as guidance for all the European Mediterranean Region in order to propose adequate soil quality standards; and adequate and valuable phytoremediation strategies that could be applied to Cu-contaminated soils of this region.
