**6. Results**

Leaching of ash was made by mixing 10 g of ash with 200 ml of nitric acid (HNO3), concentrated to 65%. The mixture was heated to boiling point for 60 minutes and then cooled down to room temperature. After complete cooling, in the mixture were added, on ice, 200 ml of concentrated

For the extraction of heavy metals, an electrolytic cell was designed (Figure 5), with stainless steel electrodes and 200 ml of the filtrate obtained from the leaching of the ash as electrolyte. The electrolysis was carried out for 90 minutes with an electric intensity of 1.5 A, and a voltage of 11.4 V. The microscopic and gravimetric methods were used to assess the metal deposition

Determination of heavy metal concentration, in both plants and soil underneath, was done by Inductively Coupled Plasma - Atomic Emission Spectrometry method (ICP-AES). For analyze, the samples were mineralized in Berghof microwave digester, plants by mixture with 10 ml of nitric acid concentrated 65% and 2 ml of hydrogen peroxide, and soil in mixture 1:1 with nitric acid (according with Berghof method). The advantage of this method is the multiele‐ mental detection, which give the possibility, in one shot, to read a wide range of elements [46]. For this research, analyzes were conducted with Liberty 110 spectrometer of Varian brand. The minimal detection limits of device range according to the analyzed element and is 0.4 mg/ kg for Zn, Mn and Cu; 0.5 mg/kg for Cr and Co; 0.6 mg/kg for Sn, Ni and Pb. The concentrations values for analyzed metals were expressed in milligrams of metal per kilogram of dry soil or

The soil pH was determined with a portable pH-meter, WTW 3110 SET 2, with precision of 0.01 units. For pH analyzes, 5 g of each soil sample were mixed with 50 ml KCl 0.1N, F 1000,

The deposition on electrodes was evaluated by microscopy and quantitative assessed by Energy Dispersive X-Ray Fluorescence method (EDXRF) [47], using a PW4025 – MiniPal – Panalytical type EDXRF Spectrometer. The XRF determinations were conducted in Helium atmosphere, excited for 300 s, without any filter, at 16 kV voltage. The current intensity was automatically adjusted by the use of a 3.6 µm Mylar tissue [48]. The surface of electrodes was

The bioaccumulation factor (BF) for studied plants was calculated as the ratio between metal

*plant soil*

*<sup>C</sup>* <sup>=</sup> (1)

*C*

*BF*

Tt 0.0056 g/ml and homogenized for 15 minutes with a magnetic stirrer.

evaluated for heavy metal concentration before and after electrolysis.

concentration in plants and metal concentration in soil:

sulfuric acid (H2SO4). After half an hour of rest, the mixture was filtered.

on electrodes.

plants (mg/kg).

**5.3. Data analysis**

**5.2. Analytical methods**

320 Environmental Risk Assessment of Soil Contamination

#### **6.1. Heavy metal concentration in soil**

The soil samples consisted in the upper layer of soil, 0 – 20 cm, where the most of the roots can be found. The content of soil in macronutrients was about 13 g/kg for Ca, 3 g/kg for Mg, and 1 g/kg for P and K. The soil reaction had the value of 7.30±0.42. Heavy metal concentration in soil (Table 1) was compared to the normal values of agricultural soils and alert thresholds for industrial soils according with the Romanian regulations [49]. The average content of Cu, Sn and Pb in soil exceeded the alert threshold for agricultural soils, 100, 35, 50 mg/kg respectively, but in some sampling points the concentrations exceeded even the alert threshold for industrial soils, 250, 100, 250 mg/kg. For Zn, the mean concentration in soil was in normal limit for agricultural soil (300 mg/kg), but in some sampling points exceeded the alert threshold of 700 mg/kg. The Co concentration in soil had low values with a uniform distribution between the sampling points. None of the samples showed values of Co concentration higher than 30 mg/ kg, the alert threshold for agricultural soils. The mean value of Ni concentration did not exceed the alert threshold for agricultural soils (75 mg/kg), but the concentration was varying in a wide range from one sampling point to another and some results showed values of concen‐ tration close to the alert threshold for industrial soil (200mg/kg). The mean concentration of Mn in studied sample of soil was higher than alert threshold for agricultural soil (1500 mg/kg), and some of results showed values of Mn concentration that exceeded the alert threshold for industrial soils (2000 mg/kg). The mean concentration of Cr was just under the alert threshold for agricultural soils (100 mg/kg), but some sampling points showed values for Cr concentra‐ tion higher than the alert threshold for industrial soils (300 mg/kg).


<LD – below limit of detection

**Table 1.** Heavy metal concentration in soil underneath plants (mg/kg dry matter)

The wide variability of metal concentration in soil was according with the orientation and the distance against the pollution source. Some heavy metals showed a uniformity in metal distribution (e.g. Co and Sn), which is probably because of the geological origin of these metals, more than from the pollution source.

#### **6.2. Heavy metal bioaccumulation in plants**

Perennial grasses develop a large plant biomass in a relatively short time and are known as heavy metal tolerant biosystems, accumulating high levels of these elements. Also, perennial grasses have a high content of dry matter: *Lolium perenne* 36%*, Festuca pratensis* 33%*, Stipa capillata* 43%*, Agrostis alba* 42%*, Cynodon dactylon* 40%*, Luzula campestris* 50% and *Agrostis tenuis* 46%, compared to other species such as *Papaver rhoeas, Cirsium arvense* or *Artemisia vulgaris* which have only 12 – 36 % dry matter.

According to the perennial grasses tolerance for heavy metals and because of environmental and weather conditions favorable to their development, analyzed plant species have adapted to the toxic heavy metal concentration in soil and accumulated these elements to high levels [50]. In addition to these aspects, the concentration of metals in plants was influenced by plant age, topography and synergistic and antagonistic effects of the elements found in soil.

The heavy metal concentration in perennial grasses was widely different between the species for all studied metals (Table 2). Copper concentration range between 1.76 and 113.83 mg/kg, with the highest value for *F. pratensis*. In the same species was found the highest value for the tin concentration, 379 mg/kg, while the lower value of tin concentration was for *L. campestris*, 8 mg/kg. Zinc concentration range for studied species between 62 – 922 mg/kg, and lead concentration varies between not detectable level of concentration in most of studied species and 201 mg/kg. The maximum values of zinc and lead concentration were found for *L. perenne* species, 922 mg/kg and 201 mg/kg respectively.

The cobalt concentration was below limit of detection for three of studied species. The highest values of this element concentration were found for species *S. capillata* and *A. tenuis*. The accumulation of Co was influenced by the metal concentration in soil and by the soil moisture which lead to the leaching of some cobalt compound and increasing of cobalt availability for plants. The mean values of Ni concentration in studied species of plants varied widely from one species to another even inside of the same genus. The lowest value of Ni concentration was 3.88 mg/kg for *A. tenuis* and the highest was for *A. alba*, 60.23 mg/kg. The range of Mn concentration was between 165.9 mg/kg and 703.92, with the highest values of concentration for *L. perenne* species. The mean concentration of Cr in studied plants ranged between 10.04 mg/kg for *L. campestris* and 191.99 mg/kg for *S. capillata.*


<LD – below limit of detection

sampling points. None of the samples showed values of Co concentration higher than 30 mg/ kg, the alert threshold for agricultural soils. The mean value of Ni concentration did not exceed the alert threshold for agricultural soils (75 mg/kg), but the concentration was varying in a wide range from one sampling point to another and some results showed values of concen‐ tration close to the alert threshold for industrial soil (200mg/kg). The mean concentration of Mn in studied sample of soil was higher than alert threshold for agricultural soil (1500 mg/kg), and some of results showed values of Mn concentration that exceeded the alert threshold for industrial soils (2000 mg/kg). The mean concentration of Cr was just under the alert threshold for agricultural soils (100 mg/kg), but some sampling points showed values for Cr concentra‐

**Metal**

**Cu** 152.4±177.7 21.9-600.4 **Co** 15.9±4.3 7.1-23.54 **Zn** 194.3±231.7 42.6-870.3 **Ni** 53.4±48.6 11.9-185.4 **Sn** 65.7±30.7 24.6-125.41 **Mn** 1545.7±334.07 1159.9-2348.76 **Pb** 65.2±87.2 <LD-294.3 **Cr** 98.0±112.0 12.9-315.6

The wide variability of metal concentration in soil was according with the orientation and the distance against the pollution source. Some heavy metals showed a uniformity in metal distribution (e.g. Co and Sn), which is probably because of the geological origin of these metals,

Perennial grasses develop a large plant biomass in a relatively short time and are known as heavy metal tolerant biosystems, accumulating high levels of these elements. Also, perennial grasses have a high content of dry matter: *Lolium perenne* 36%*, Festuca pratensis* 33%*, Stipa capillata* 43%*, Agrostis alba* 42%*, Cynodon dactylon* 40%*, Luzula campestris* 50% and *Agrostis tenuis* 46%, compared to other species such as *Papaver rhoeas, Cirsium arvense* or *Artemisia*

According to the perennial grasses tolerance for heavy metals and because of environmental and weather conditions favorable to their development, analyzed plant species have adapted to the toxic heavy metal concentration in soil and accumulated these elements to high levels [50]. In addition to these aspects, the concentration of metals in plants was influenced by plant

age, topography and synergistic and antagonistic effects of the elements found in soil.

**Mean ±SD Range Mean ±SD Range**

**Soil concentration (mg/kg)**

tion higher than the alert threshold for industrial soils (300 mg/kg).

**Table 1.** Heavy metal concentration in soil underneath plants (mg/kg dry matter)

**Soil concentration (mg/kg)**

322 Environmental Risk Assessment of Soil Contamination

**Metal**

<LD – below limit of detection

more than from the pollution source.

**6.2. Heavy metal bioaccumulation in plants**

*vulgaris* which have only 12 – 36 % dry matter.

**Table 2.** Mean concentration of heavy metals in perennial grasses (mg/kg dry matter)

For phytoremediation process to be effective it is better to use those biosystems species adapted to the climatic and soil conditions of the area to be de-polluted. For this reason, the species used in the studies were chosen from those plants that normally grow in the industrial area of the city of Targoviste, perennial grass which are effective to mowing and rebuild their vegetative mass. In addition, the losses caused by death of leaves are greatly reduced.

The bioaccumulation capacity of plants was estimated as the ratio of metal content in soil and the metal concentration in plant. This ratio is called bioaccumulation factor (BF) [7] and we evaluated as weak accumulators the species which have a BF value between 0.8 - 1.2, as good accumulators the species with a value of BF between 1.5 - 5.0 and hyperacumulators those species with higher BF than 5.0 (Table 3).

Absorption and accumulation of metals in perennial grasses was influenced by both species and the soil underneath, pH, moisture and metal content in soil. The bioaccumulation of the studied metals was differently influenced by pH of soil and metal content.

Even *F. pratensis* and *L. perenne* showed the highest values of Cu, Zn, Sn concentration, they did not show the highest accumulation capacity for those metals. The best accumulator for Cu, Zn and Sn were the plants of *C. dactylon* species which showed BF values of 1.12, 1.37 and 6.06 respectively for Cu, Zn and Sn. Lead was very well accumulated by *L. campestris* which showed a very high level of metal bioaccumulation, 12.3. Tin was the metal with best bioaccumulation in perennial grasses.


**Table 3.** Bioaccumulation factor (BF) of heavy metals in plant species – metal accumulation capacity of plants (not accumulative, weak accumulative, good accumulative or hyper accumulative)

None of the studied species of perennial plants showed accumulative capacities for either Co or Mn. This was probably because of the exclusion mechanism of these plants for the two elements. For Ni, three of studied species showed accumulative capacity: *L. campestris, C. dactylon* and *A. alba*, while for Mn five studied species showed accumulative capacity. For the phytoremediation of soils polluted with Ni and Cr, the best species to use is *A. alba*, because it showed the highest values of BF, 1.63 and 2.68 respectively.

#### **6.3. Heavy metal extraction from plant biomass**

For phytoremediation process to be effective it is better to use those biosystems species adapted to the climatic and soil conditions of the area to be de-polluted. For this reason, the species used in the studies were chosen from those plants that normally grow in the industrial area of the city of Targoviste, perennial grass which are effective to mowing and rebuild their vegetative mass. In addition, the losses caused by death of leaves are greatly reduced.

The bioaccumulation capacity of plants was estimated as the ratio of metal content in soil and the metal concentration in plant. This ratio is called bioaccumulation factor (BF) [7] and we evaluated as weak accumulators the species which have a BF value between 0.8 - 1.2, as good accumulators the species with a value of BF between 1.5 - 5.0 and hyperacumulators those

Absorption and accumulation of metals in perennial grasses was influenced by both species and the soil underneath, pH, moisture and metal content in soil. The bioaccumulation of the

Even *F. pratensis* and *L. perenne* showed the highest values of Cu, Zn, Sn concentration, they did not show the highest accumulation capacity for those metals. The best accumulator for Cu, Zn and Sn were the plants of *C. dactylon* species which showed BF values of 1.12, 1.37 and 6.06 respectively for Cu, Zn and Sn. Lead was very well accumulated by *L. campestris* which showed a very high level of metal bioaccumulation, 12.3. Tin was the metal with best bioaccumulation

**Metal BF Accumulation gradient Metal BF Accumulation gradient**

1.12±0.1 *C. dactylon* – weak 4.54±0.2 *A. tenuis* – good

0.98±0.1 *A. tenuis* – weak **Co** <0.8 Not accumulative 1.00±0.1 *A. alba* – weak **Ni** 1.26±0.05 *L. campestris* – good 1.31±0.3 *L. perenne –* good 1.27±0.42 *C. dactylon* – good 1.37±0.1 *C. dactylon* – good 1.63±0.63 *A. alba* – good

3.00±0.8 *A. alba –* good **Cr** 0.83±0.08 *F. pratensis –* weak 4.10±0.8 *F. pratensis –* good 1.16±0.23 *C. dactylon* – weak 4.11±0.6 *L. perenne –* good 1.51±0.11 *A. tenuis* – good 5.85±0.1 *A. tenuis* – hyper 2.11±0.10 *S. capillata –* good 6.06±0.3 *C. dactylon* – hyper 2.68±0.75 *A. alba* – good

**Table 3.** Bioaccumulation factor (BF) of heavy metals in plant species – metal accumulation capacity of plants (not

accumulative, weak accumulative, good accumulative or hyper accumulative)

**Cu** 0.88±0.2 *A. alba* – weak **Pb** 1.04±0.1 *L. perenne –* weak

**Zn** 0.92±0.1 *L. campestris* – weak 12.3±0.9 *L. campestris* – hyper

**Sn** 2.43±0.1 *S. capillata –* good **Mn** <0.8 Not accumulative

studied metals was differently influenced by pH of soil and metal content.

species with higher BF than 5.0 (Table 3).

324 Environmental Risk Assessment of Soil Contamination

in perennial grasses.

Following the phytoremediation of soil polluted with heavy metals, a crop of 400 kg of plants wasobtainedwhichcontainheavymetals,buttheconcentrationsoftheseelementsinthebiomass is very low for the use of these plants for heavy metal extraction process. In order to concen‐ tratethemetal,plantsweresubjectedtoadryingprocessinwhichthemassofsubstancedecreased by64%,registeringapercentageofmetalconcentrationof243.9%.Thepercentageofashresulted fromthe incinerationofdry biomass was 14.3% andthepercentage ofmetal concentrationinthe plant ash increased by values between 544.5 - 2282.1% (Table 4) The differences of metal concentration by incineration was because of volatilization of some elements or because of the adsorptiononflyingashwhichwaslostduringtheincineration.Becauseofthat,futureresearches will be needed for the construction of an integrated system of plant biomass incineration, designed with filters and cyclone for the recovery of all particles of ash.

In this experiment, the ash was used for metal extraction by leaching. The results showed low effectiveness of this method, because the metals from ash were not fully extracted in the leachate. The weakest extraction in the leachate was for tin, only 16.3% from the total quantity of tin in the ash. A fair extraction was only for Ni and Cr which were extracted 92.6% and 87.8% respectively from the ash (Table 5).


Future research should be conducted to establish methods of metal extraction by leaching of ash with better results for the majority of heavy metals to be recovered.

**Table 4.** Metal concentration in plant biomass by drying and incineration (%)


\* 10 g of ash were used to prepare 400 ml solution

**Table 5.** Heavy metal extraction by leaching of ash

In the process of electrolysis, the cathode layout has changed on the surface. At macroscopic level could be observed the metal deposition after the electrolysis - oxides spots and at the microscopic level could be observed a smoothing of the surface (Figure 6).

There was a difference in mass of the cathode of 0.7 mg, from 4.6242 g before to 4.6249 g after electrolysis.

**Figure 6.** Metal depositions on electrode at 1x, 40x and 80x magnification [2]

The quantitative evaluation of metal deposition on cathode was made by EDXRF and different metal concentrations were observed. The most effective deposition was for Ni, Mn and Cr, which showed concentration of 3.07, 2.2 and 7.3 g/kg respectively.

#### **6.4. Balance of phytoremediation and heavy metal extraction**

The experiment of phytoremediation of heavy metal polluted soil in the vicinity of Targoviste city showed the results for one growth season of perennial grass *Lolium perenne* (Table 6). The metal concentration in soil as evaluated before and after the plant culture and after mowing the results showed a decreasing of metal concentration in soil with 0.3 – 5.9%. The concentra‐ tion of cobalt and nickel had the lowest decreasing. The best accumulated heavy metals by plants were Zn and Pb. The accumulation of Pb was because of the high concentration of this metal in soil and the Zn accumulation was because of the synergic effect of the Pb concentration in soil on the accumulation of Zn [2].


<LD – below limit of detection

**Metal**

electrolysis.

**Ash concentration**

326 Environmental Risk Assessment of Soil Contamination

\* 10 g of ash were used to prepare 400 ml solution

**Table 5.** Heavy metal extraction by leaching of ash

**Cu** 1454.08 11.20 30.8% **Zn** 2295.92 30.45 53.1% **Sn** 3367.35 13.70 16.3% **Pb** 76.53 1.25 65.3% **Co** 306.12 < LD - **Ni** 2142.86 49.60 92.6% **Mn** 3214.29 35.5 44.2% **Cr** 3622.45 79.55 87.8%

**(mg/kg) Solution concentration (mg/l) Percentage of metal extraction into**

In the process of electrolysis, the cathode layout has changed on the surface. At macroscopic level could be observed the metal deposition after the electrolysis - oxides spots and at the

There was a difference in mass of the cathode of 0.7 mg, from 4.6242 g before to 4.6249 g after

The quantitative evaluation of metal deposition on cathode was made by EDXRF and different metal concentrations were observed. The most effective deposition was for Ni, Mn and Cr,

The experiment of phytoremediation of heavy metal polluted soil in the vicinity of Targoviste city showed the results for one growth season of perennial grass *Lolium perenne* (Table 6). The

microscopic level could be observed a smoothing of the surface (Figure 6).

**Figure 6.** Metal depositions on electrode at 1x, 40x and 80x magnification [2]

which showed concentration of 3.07, 2.2 and 7.3 g/kg respectively.

**6.4. Balance of phytoremediation and heavy metal extraction**

**solution\* (%)**

**Table 6.** Results of the phytoremediation of heavy metal polluted soil near Targoviste

The synergic effect of the Pb concentration in soil can be observed also in the value of Zn bioaccumulation factor, 3.177. During the experiment of phytoremediation, *Lolium perenne* showed good accumulative capacity for chromium also.

By reporting the results to the quantity of ash used in the experiment, small quantities of heavy metals were obtained (Table 7).

According with the biomass quantity that can be harvested from one hectare cultivated with *Lolium perenne* species (40 tons) and ash content of raw material (5.1%), in a growing season of this species, can be extracted from soil about 0.13 g of Cu, 0.27 g of Zn, 0.07 g of Sn, 0.04 g of Co, 0.88 g of Ni, 0.63 g of Mn, and 2.09 g of Cr. The efficiency of heavy metals extraction by electrolysis can be improved by increasing the leaching efficiency.

Even if these amounts are very small and the economic value of process is almost nonexistent, the immense advantage of metal recovery is the extraction of these elements from soil and the decreasing of toxicity risk caused by the presence of heavy metals in the environment.


\* 2% of Mn deposit is probably because of anodic dissolution

\*\* The results were probably contaminated because of high content of Cr in the stainless steel (14%) Weight of metal deposition – 0.7 mg; fresh biomass per hectare – 40 t; percentage of ash – 5.1%

**Table 7.** Results of heavy metal extraction from *Lolium perenne,* by electrolysis
