**Effects of Water Stress on Germination and Growth of Wheat, Photosynthetic Efficiency and Accumulation of Metabolites**

Rui Guo, Wei Ping Hao, Dao Zhi Gong, Xiu Li Zhong and Feng Xue Gu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51205

## **1. Introduction**

Especially over the last 100 years, our unbridled exploitation of the world's natural resources has severely damaged its vegetation and has also resulted in worrying accumulations of in‐ dustrial wastes and greenhouse gases. Together, these have upset natural ecosystem balances and have created many environment and climatic problems, including rising temperatures, in‐ creasing desertification, serious soil loss, soil salinization and damaging accumulations of soil nitrogen [39, 31, 37]. In many nations, the recent increased incidences of severe drought and associated desertification are coming into especially sharp focus because of their sudden, long term and devastating consequences for the local human population.

Drought imposes one of the commonest and most significant constraints to agricultural pro‐ duction, seriously affecting crop growth, gene expression, distribution, yield and quality [45, 44, 53]. There are numerous reports on photosynthetic and metabolites characteristics under water stress [22, 52, 25, 5]. Generally, photosynthesis is inhibited by water stress, also affects photosynthetic components and chloroplast stress [54, 52]. Plants have evolved a number of mechanisms to adapt to and survive water stress, Some plant species have evolved mecha‐ nisms to cope with the stress, including drought avoidance, dehydration avoidance, or de‐ hydration tolerance. Such adaptive mechanisms are the results of a multitude of morphoanatomical, physiological, biochemical, and molecular changes [1, 2, 6]. But to our knowledge, only a few report about the effects of different level water stress on photosyn‐ thetic and metabolites of wheat seedlings.

© 2013 Guo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Guo et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Wheat is an important crop, with some cultivars tolerant to water stress. The purpose of this study was to investigate the effects of water stress on the growth, chlorophyll fluorescence and accumulations of proline, betaine and carbohydrates of wheat seedlings, by using PEG simulated water stress. It was also desired to elucidate mechanisms of water stress damage and to identify possible adaptive mechanisms to water stress. Understanding how wheat manages water stress is important for the reclamation of drought-prone soils and crop pro‐ duction, and possibly also to discover water-stress resistance genes and hence to develop drought-resistance biotechnology in this crop.

rate (RGR) was defined as (ln DW after treatment – ln DW before treatment) / treatment du‐ ration. The water content (WC) percentage was calculated as: 100×(FW–DW)/FW [52].

The maximal photochemical efficiency of PSII (PSII=Fv/Fm), the photosynthetic efficiency of PSII (Y(II)=Fm'-F/Fm'), non-photochemical quenching (NPQ=Fm-Fm'/Fm'), non-photochemi‐ cal quenching coefficient (qN=Fm-Fm'/Fm-Fo'), photochemical quenching (qP=Fm'-F/Fm'- Fo'), the efficiency of excitation energy capture by open PSII reaction centers (Fv'/Fm') and apparent photosynthetic electron transport rate (ETR) were determined between 09:00 and 11:00 h from fully-expanded leaves using an Imaging-PAM (Walz, Effeltrich, Germany], [12, 48]. The leaves were held in the dark for about 20 min before measurement. The intensities

spectively. The contents of carotenoids (Car) and chlorophyll (Chl) *a* and *b* were extracted using acetone, and spectrophotometeric determination at 440, 645 and 663 nm of each sam‐ ple was done three times. The calculations were Chl a = 12.7×OD663-2.69×OD645; Chl b =

Proline was extracted with 3% sulfosalicylic acid for 30 min at 70°C and measured with nin‐ hydrin [55]. Betaine was extracted with 80% methanol for 20 min at 70°C and measured as described by Grieve and Grattan (1983). Total soluble sugars (SS) were extracted for 30 min

One hundred wheat seeds were germinated on filter paper in germination boxes. The dry seeds were submerged in 100 mL of each of the PEG-6000 solutions described above (with distilled water as the control). The boxes were maintained at 20°C in the dark for 10 d, five replicates of each PEG treatment were prepared. Percentages of ger‐ minated seeds were scored daily, based on the emergence of the radicles. The germina‐ tive Energy(Ge), germinative Percentage(Gp), and germination activity Index(Ai) of wheat seeds were modified using Ge = n/Nx100% (n: the number of germination of seeds in 4 days; N: the total number of seeds); Gp = nl/Nlx100% (n1: the number of germina‐

Statistical analysis included one-way analysis of variance (ANOVA) in SPSS (Version 13.0, SPSS, Chicago, IL, USA) and Duncan's method to detect differences in physiological param‐ eters in plants under water stress (*P*≤0.05). All measurements represent the means and

22.9×OD645-4.86×OD663 and Car = 4.7×OD440-0.27×(20.2×OD645+8.02×OD663).

s and 2500μmol/m2

Effects of Water Stress on Germination and Growth of Wheat

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369

s PAR, re‐

**2.4. Measurement of chlorophyll fluorescence and pigments**

of the actinic and saturating light settings were 185μmol/m2

**2.5. Measurement of metabolites and organic acids**

at 70°C in 70% alcohol, and measured using anthrone.

tion of seeds at 10 days; N1: the total number of seeds).

**2.6. Measurement of germination**

**3. Statistical analysis**

standard errors (SE) of five replicates.

## **2. Materials and methods**

## **2.1. Design of simulated water stress conditions**

Water stress conditions were simulated to polyethylene glycol-6000 (PEG) at one of three concentrations: 0, 5, 15 and 25%. The osmotic potentials of the solutions was measured using a water potential meter (Psypro Wescor Corporation, US) [49]. Table 1 results shows how osmotic potential decreases with increasing PEG-6000 concentration.


**Table 1.** The osmotic potential (OP) of solutions of polyethylene glycol (PEG).

#### **2.2. Plant materials and growing conditions**

Seeds of wheat (*Triticum aestivum*) FengYou-68 were sown 20 seeds in per germination box Seedlings were watered daily with 0.5 Hoagland's nutrient solution [14]. All the boxes were placed in growth chambers [HPG-400, Haerbin, China] with a 16-h photoperiod (Sylvania cool white fluorescent lamps, 200 mmol m-2 s-1, 400–700 nm). The temperature was 25 ± 2°C (day) and 21 ± 1.5°C (night).

After three days, 25 boxes containing uniform seedlings were selected and randomly divid‐ ed into five sets of five replicates. One set was used to determine the seedling growth pa‐ rameters just prior to treatment, a second set was used as the untreated control (0% PEG-6000, watered with Hoagland's nutrient solution), and the three remaining sets were stressed with one or other of the PEG-6000 solutions. Each PEG subtreatment was applied to a set of five boxes, daily for 7 days.

#### **2.3. Measurement of growth**

After the seventh day of treatment, the fresh weights (FW) were recorded after removing surface water by blotting and the dry weights (DW) determined after drying for 15 min in an oven at 80°C and then in a vacuum dryer at 40°C to constant weight. The relative growth rate (RGR) was defined as (ln DW after treatment – ln DW before treatment) / treatment du‐ ration. The water content (WC) percentage was calculated as: 100×(FW–DW)/FW [52].

## **2.4. Measurement of chlorophyll fluorescence and pigments**

The maximal photochemical efficiency of PSII (PSII=Fv/Fm), the photosynthetic efficiency of PSII (Y(II)=Fm'-F/Fm'), non-photochemical quenching (NPQ=Fm-Fm'/Fm'), non-photochemi‐ cal quenching coefficient (qN=Fm-Fm'/Fm-Fo'), photochemical quenching (qP=Fm'-F/Fm'- Fo'), the efficiency of excitation energy capture by open PSII reaction centers (Fv'/Fm') and apparent photosynthetic electron transport rate (ETR) were determined between 09:00 and 11:00 h from fully-expanded leaves using an Imaging-PAM (Walz, Effeltrich, Germany], [12, 48]. The leaves were held in the dark for about 20 min before measurement. The intensities of the actinic and saturating light settings were 185μmol/m2 s and 2500μmol/m2 s PAR, re‐ spectively. The contents of carotenoids (Car) and chlorophyll (Chl) *a* and *b* were extracted using acetone, and spectrophotometeric determination at 440, 645 and 663 nm of each sam‐ ple was done three times. The calculations were Chl a = 12.7×OD663-2.69×OD645; Chl b = 22.9×OD645-4.86×OD663 and Car = 4.7×OD440-0.27×(20.2×OD645+8.02×OD663).

#### **2.5. Measurement of metabolites and organic acids**

Proline was extracted with 3% sulfosalicylic acid for 30 min at 70°C and measured with nin‐ hydrin [55]. Betaine was extracted with 80% methanol for 20 min at 70°C and measured as described by Grieve and Grattan (1983). Total soluble sugars (SS) were extracted for 30 min at 70°C in 70% alcohol, and measured using anthrone.

#### **2.6. Measurement of germination**

Wheat is an important crop, with some cultivars tolerant to water stress. The purpose of this study was to investigate the effects of water stress on the growth, chlorophyll fluorescence and accumulations of proline, betaine and carbohydrates of wheat seedlings, by using PEG simulated water stress. It was also desired to elucidate mechanisms of water stress damage and to identify possible adaptive mechanisms to water stress. Understanding how wheat manages water stress is important for the reclamation of drought-prone soils and crop pro‐ duction, and possibly also to discover water-stress resistance genes and hence to develop

Water stress conditions were simulated to polyethylene glycol-6000 (PEG) at one of three concentrations: 0, 5, 15 and 25%. The osmotic potentials of the solutions was measured using a water potential meter (Psypro Wescor Corporation, US) [49]. Table 1 results shows how

PEG-6000 concentration 0% 5% 15% 25%

Seeds of wheat (*Triticum aestivum*) FengYou-68 were sown 20 seeds in per germination box Seedlings were watered daily with 0.5 Hoagland's nutrient solution [14]. All the boxes were placed in growth chambers [HPG-400, Haerbin, China] with a 16-h photoperiod (Sylvania cool white fluorescent lamps, 200 mmol m-2 s-1, 400–700 nm). The temperature was 25 ± 2°C

After three days, 25 boxes containing uniform seedlings were selected and randomly divid‐ ed into five sets of five replicates. One set was used to determine the seedling growth pa‐ rameters just prior to treatment, a second set was used as the untreated control (0% PEG-6000, watered with Hoagland's nutrient solution), and the three remaining sets were stressed with one or other of the PEG-6000 solutions. Each PEG subtreatment was applied to

After the seventh day of treatment, the fresh weights (FW) were recorded after removing surface water by blotting and the dry weights (DW) determined after drying for 15 min in an oven at 80°C and then in a vacuum dryer at 40°C to constant weight. The relative growth

OP (MPa) -0.05 -0.09 -0.34 -0.95

drought-resistance biotechnology in this crop.

368 Soil Processes and Current Trends in Quality Assessment

**2.1. Design of simulated water stress conditions**

osmotic potential decreases with increasing PEG-6000 concentration.

**Table 1.** The osmotic potential (OP) of solutions of polyethylene glycol (PEG).

**2.2. Plant materials and growing conditions**

(day) and 21 ± 1.5°C (night).

a set of five boxes, daily for 7 days.

**2.3. Measurement of growth**

**2. Materials and methods**

One hundred wheat seeds were germinated on filter paper in germination boxes. The dry seeds were submerged in 100 mL of each of the PEG-6000 solutions described above (with distilled water as the control). The boxes were maintained at 20°C in the dark for 10 d, five replicates of each PEG treatment were prepared. Percentages of ger‐ minated seeds were scored daily, based on the emergence of the radicles. The germina‐ tive Energy(Ge), germinative Percentage(Gp), and germination activity Index(Ai) of wheat seeds were modified using Ge = n/Nx100% (n: the number of germination of seeds in 4 days; N: the total number of seeds); Gp = nl/Nlx100% (n1: the number of germina‐ tion of seeds at 10 days; N1: the total number of seeds).

## **3. Statistical analysis**

Statistical analysis included one-way analysis of variance (ANOVA) in SPSS (Version 13.0, SPSS, Chicago, IL, USA) and Duncan's method to detect differences in physiological param‐ eters in plants under water stress (*P*≤0.05). All measurements represent the means and standard errors (SE) of five replicates.

### **4. Results**

#### **4.1. Growth**

The RGR and WC of shoots and roots all decreased with increasing PEG concentration, with the greatest reductions occurring under the highest water stresses (Fig. 1 A - D, *P*≤0.05). From the slopes of equations (1) and (2) (Table 2), it was calculated that the RGR for root and shoot increased by 0.229 and 0.231, respectively, per 1% increase in PEG-6000 concentra‐ tion. Meanwhile, the WC of root and shoot decreased by 24.03 and 21.00, respectively, for each 1% increase in PEG concentration (see equations (3) and ((4)) in Table 2).

**4.2. Chlorophyll fluorescence and pigments**

0.80±0.00a 0.79±0.00a 0.76±0.00a 0.59±0.00b

different at *P*≤0.05 according to Duncan's method.

PEG-6000 concentration

> 0% 5% 15% 25%

PEG-6000 concentration

**4.3. Metabolites**

The Fv/Fm, Y(II), qP and ETR decreased with increasing PEG concentration, while NPQ and qN contents increased significantly, the effects were much more pronounced under high PEG concentration (Table 3; *P*≤0.05). The contents of Chl *a* and Chl *b* under PEG induced wa‐ ter stress were less than in the control, each parameter decreased gradually with increasing PEG concentration. The Chl *a/b* ratio was higher with PEG than in the control (Table 4,

Fv/Fm Y(II) NPQ qN qP ETR

Chl*a* Chl*b* Chl*a*+ Chl*b* Chl*a*/Chl*b* Car

0% 1.14±0.06a 0.29±0.00a 1.43±0.08a 3.93±0.04 0.31±0.00a 5% 1.13±0.08a 0.28±0.00a 1.41±0.06a 4.04±0.08 0.34±0.00a 15% 1.02±0.01a 0.22±0.00b 1.24±0.02ab 4.64±0.08 0.32±0.00a 25% 0.86±0.00b 0.17±0.00c 1.03±0.01b 5.06±0.09 0.28±0.00b

**Table 4.** Effects of PEG induced water stress on contents of photosynthetic pigments (g kg-1FM) in seedlings of wheat.

The contents of proline increased with increasing PEG concentration, with that in the shoot being significantly higher than that in the root (Fig. 2, A and B; *P*≤0.05). In the roots, increas‐ ing water stress had a positive effect on betaine content, causing a significant rise at 25% PEG concentration, however, in the shoots a negative effect (Fig. 2, C and D; *P*≤0.05). The impacts of water stress on soluble sugar were similar as proline and betaine, it contents sig‐

0.64±0.00b 0.63±0.00b 0.78±0.00a 0.58±0.00c 0.76±0.00a 0.75±0.00a 0.51±0.00b 0.00±0.00

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38.30±0.00a 38.00±0.00a 21.20±0.00b 0.00±0.00

0.29±0.00b 0.27±0.00b 0.48±0.00a 0.17±0.00c

**Table 3.** Effects of PEG induced water stress on contents of photosynthetic pigments (g kg-1FM) in seedlings of wheat. The values are the means of five replicates. Means followed by different letters in the same stress type are significantly

*P*≤0.05). The content of Car was scarcely changed by water stress (Table 4, *P*≤0.05).

0.49±0.00a 0.49±0.00a 0.27±0.00b 0.00±0.00

The values are the means of five replicates. Means followed by different letters.

nificantly increased under high water stress (Fig. 2 E and F; P≤0.05).

**Figure 1.** Effects of water stress on shoot (A) and root (B) relative growth rate (RGR) and water content (WC). The values are the means of five replicates. Means followed by different letters in the same stress type are significantly different at *P*≤0.05 according to Duncan's method.


**Table 2.** A regression analysis between RGR, WC and PEG concentration was performed, where *Y*R represented the root RGR and WC, *Y* S the shoot, and *x* was PEG concentration.

### **4.2. Chlorophyll fluorescence and pigments**

**4. Results**

370 Soil Processes and Current Trends in Quality Assessment

**4.1. Growth**

The RGR and WC of shoots and roots all decreased with increasing PEG concentration, with the greatest reductions occurring under the highest water stresses (Fig. 1 A - D, *P*≤0.05). From the slopes of equations (1) and (2) (Table 2), it was calculated that the RGR for root and shoot increased by 0.229 and 0.231, respectively, per 1% increase in PEG-6000 concentra‐ tion. Meanwhile, the WC of root and shoot decreased by 24.03 and 21.00, respectively, for

**Figure 1.** Effects of water stress on shoot (A) and root (B) relative growth rate (RGR) and water content (WC). The values are the means of five replicates. Means followed by different letters in the same stress type are significantly

> *Y*R=-0.229x+1.254 (1) 0.79 0.229 *Y*S=-0.231x+1.389 (2) 0.80 0.231

> *Y*R=-24.03x+132.52 (3) 0.64 24.03 *Y*S=-21.00x+125.28(4) 0.68 21.00

**Table 2.** A regression analysis between RGR, WC and PEG concentration was performed, where *Y*R represented the

Regression equation *<sup>R</sup>*<sup>2</sup> Decrease in RGR and WC per 1% increment in PEG-6000

concentration

different at *P*≤0.05 according to Duncan's method.

root RGR and WC, *Y* S the shoot, and *x* was PEG concentration.

RGR

WC

each 1% increase in PEG concentration (see equations (3) and ((4)) in Table 2).

The Fv/Fm, Y(II), qP and ETR decreased with increasing PEG concentration, while NPQ and qN contents increased significantly, the effects were much more pronounced under high PEG concentration (Table 3; *P*≤0.05). The contents of Chl *a* and Chl *b* under PEG induced wa‐ ter stress were less than in the control, each parameter decreased gradually with increasing PEG concentration. The Chl *a/b* ratio was higher with PEG than in the control (Table 4, *P*≤0.05). The content of Car was scarcely changed by water stress (Table 4, *P*≤0.05).


**Table 3.** Effects of PEG induced water stress on contents of photosynthetic pigments (g kg-1FM) in seedlings of wheat. The values are the means of five replicates. Means followed by different letters in the same stress type are significantly different at *P*≤0.05 according to Duncan's method.


**Table 4.** Effects of PEG induced water stress on contents of photosynthetic pigments (g kg-1FM) in seedlings of wheat. The values are the means of five replicates. Means followed by different letters.

#### **4.3. Metabolites**

The contents of proline increased with increasing PEG concentration, with that in the shoot being significantly higher than that in the root (Fig. 2, A and B; *P*≤0.05). In the roots, increas‐ ing water stress had a positive effect on betaine content, causing a significant rise at 25% PEG concentration, however, in the shoots a negative effect (Fig. 2, C and D; *P*≤0.05). The impacts of water stress on soluble sugar were similar as proline and betaine, it contents sig‐ nificantly increased under high water stress (Fig. 2 E and F; P≤0.05).

shoot, but in root it level show completely opposite change (Fig. 3 D1 and D2, *P*≤0.05). Water stress had a significant negative impact on CA and LA levels in shoots, but there had no reg‐

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**Figure 3.** Effects of water stress on the levels of Oxalic Acid (A1), Citric Acid (B1), Malic Acid (C1), Formic Acid (D1), Lactic Acid (E1) and Succinic Acid (F1) in the root, and Oxalic Acid (A2), Citric Acid (B2), Malic Acid (C2), Formic Acid (D2), Lactic Acid (E2) and Succinic Acid (F2) shoot of wheat seedlings. The values are the means of five replicates. Means followed by different letters in the same stress type are significantly different at *P*≤0.05 according to Duncan's

The table 5 shows that the trend in changes in Gp and Ge of wheat seeds under water stress conditions was similar; there was a decreased trend with increased PEG-6000 concentration

(*P*≤0.05), the reductions were greater when concentration above 15% (*P*≤0.05).

method.

**5.1. Germination**

ular impact on the levels of CA and LA in roots (Fig. 3 B and E, *P*≤0.05).

**Figure 2.** Effects of PEG induced water stress on the contents of proline, betaine and soluble sugar in roots and shoots of wheat seedling. The values are the means of five replicates. Means followed by different letters in the same stress type are significantly different at *P*≤0.05 according to Duncan's method.

#### **5. Organic acids**

*OA*, *CA*, *MA*, *FA*, *LA* and *SA* were all detected in both the shoots and roots of the wheat seedlings under water stress (Fig. 3). In response to water stress, the *OA*, *MA* and *SA* content of the roots and shoots decreased with PEG-6000 concentration increased, declined signifi‐ cant reduction above 15% PEG-6000 concentration (Fig. 3 A1, A2, C1, C2, F1 and F2, *P*≤0.05). The level of *FA* increased under low water stress whereas it decreased under high stress in shoot, but in root it level show completely opposite change (Fig. 3 D1 and D2, *P*≤0.05). Water stress had a significant negative impact on CA and LA levels in shoots, but there had no reg‐ ular impact on the levels of CA and LA in roots (Fig. 3 B and E, *P*≤0.05).

**Figure 3.** Effects of water stress on the levels of Oxalic Acid (A1), Citric Acid (B1), Malic Acid (C1), Formic Acid (D1), Lactic Acid (E1) and Succinic Acid (F1) in the root, and Oxalic Acid (A2), Citric Acid (B2), Malic Acid (C2), Formic Acid (D2), Lactic Acid (E2) and Succinic Acid (F2) shoot of wheat seedlings. The values are the means of five replicates. Means followed by different letters in the same stress type are significantly different at *P*≤0.05 according to Duncan's method.

#### **5.1. Germination**

**Figure 2.** Effects of PEG induced water stress on the contents of proline, betaine and soluble sugar in roots and shoots of wheat seedling. The values are the means of five replicates. Means followed by different letters in the same stress

*OA*, *CA*, *MA*, *FA*, *LA* and *SA* were all detected in both the shoots and roots of the wheat seedlings under water stress (Fig. 3). In response to water stress, the *OA*, *MA* and *SA* content of the roots and shoots decreased with PEG-6000 concentration increased, declined signifi‐ cant reduction above 15% PEG-6000 concentration (Fig. 3 A1, A2, C1, C2, F1 and F2, *P*≤0.05). The level of *FA* increased under low water stress whereas it decreased under high stress in

type are significantly different at *P*≤0.05 according to Duncan's method.

372 Soil Processes and Current Trends in Quality Assessment

**5. Organic acids**

The table 5 shows that the trend in changes in Gp and Ge of wheat seeds under water stress conditions was similar; there was a decreased trend with increased PEG-6000 concentration (*P*≤0.05), the reductions were greater when concentration above 15% (*P*≤0.05).


creased, while those of NPQ and qN increased with increasing PEG concentrations. These results indicate that electron transport activity and the photosynthetic apparatus of wheat

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Chl and Car are the main photosynthetic pigments of plants, so these are good indicators of the photosynthesis capability of a plant. Under water stress, with the exception of Car which barely changed, the contents of Chl *a* and *b* decreased slightly at first but then decreased more sharply at the 15% PEG concentration (Table 4; *P*≤0.05). This may be linked with the observation that under -0.34MPa water potential conditions Chl synthesis was severely in‐ hibited with the result that the functioning of the photosynthetic apparatus became serious‐ ly impaired [25, 5]. Compared with the control, the water stress effects on Chl *a/b* were high and this appears to be closely related to the metabolic regulation of Chl; this possibility is

Proline and betaine are also known to play important roles in osmotic adjustment with their accumulation under water stress being observed in many species [46, 38]. Here, the results show that, along with a decrease in osmotic potential, the accumulation of free proline and betaine increased significantly both in the roots and the shoots. This increase would lower the osmotic potential [i.e. make it more strongly negative] in the cells which would help to maintain turgor and thus sustain the normal physiological and biochemical processes in the

Soluble sugars are the main osmotic adjustment substances and so are important indicators of drought tolerance. The results show that the soluble sugars contents of wheat seedlings increases under high PEG concentration. This indicates that they may help to regulate and maintain the activity of physiological processes within the plant in a high water-stress envi‐

The accumulation of organic acids is a physiological response of plants to stress, when plants are suffered by water stress, they can through cells apperceive and transmit drought signal [42]. There nearly no impact on the content of organic acids under blew 15% water stress, it decreased significantly under high stress, but in shoot FA completely opposite change (Fig. 3, *P*≤0.05). The results confirmed that the organic acids metabolic regulation was closely related to the plant water stress resistance. The change of organic acid may be adaptive mechanism by which wheat seedlings maintain their intracellular osmotic balance

Germination is one of the most critical periods in the life cycle of plants. Under water stress, low water potential is a determining factor inhibiting seed germination [51, 43]. The inhibit‐

seedlings with certain drought-resistance are damaged.

**7.2. Impact of water stress treatments on metabolites**

ronment by raising the osmotic potential of the cells [14].

**7.3. Impact of water stress treatments on organic acids**

**7.4. Impact of water stress treatments on germination**

worth further investigation.

face of drought (Fig. 2, *P*≤0.05).

under water stress [47, 55].

**Table 5.** Effect of water stress on two indices (Gp and Ge) of germination for wheat. Germinative percentage - Gp and germinative Energy - Gp.

## **6. Discussion**

PEG is an osmotic agent, which play an important role in the regulation of mineral elements, hormone, protein metabolism and effects of signal transduction [50, 41]. The main function of PEG is to slow down the moisture rate of import and export seeds, which benefit to re‐ duce membrane system injury in process of seed imbibition and repair impaired membrane system [27, 16]. PEG has been widely used in seed priming and simulated water stress test, the wheat seedlings were treated by three different PEG concentrations.

## **7. Impact of water stress treatments on growth**

In plants in general, an appropriate growth strategy is key to fitness in a competitive situation, so too in wheat seedlings, their growth strategy is critical to survival [10]. The RGR value of a plant reflects its vigour and is considered a good index of its exposure to stresses of all sorts [26, 52]. The RGR response of wheat seedlings exposed to increasing PEG concentrations (Fig. 1 A, P ≤ 0.05), revealed a decrease for roots and shoots (Table 2, *P* ≤ 0.05). This may reflect the im‐ pact of water stress on root cell development, which would likely impair nutrient uptake as well as having detrimental effects on photosynthesis, essential for biomass accumulation and therefore on shoot and root elongation. The change trend for WC was similar to that for RGR but the extension of WC in the root was about 1.14-times that of the shoot (Fig 1. B; Table 2;*P*≤0.05). Water stress therefore appears to reduce the absorption and utilization of water to such an extent that the tolerance mechanisms employed by these plants in a drought are insuf‐ ficient to maintain normal growth.

#### **7.1. Impact of water stress treatments on chlorophyll fluorescence and pigments**

The chlorophyll fluorescence kinetics react to the "intrinsic" characteristic of photosynthesis and can rapidly and sensitively reflect a plant's physiological status and its relationship with the environment [Huang et al., 2009]. In this study, PSII values decreased with increas‐ ing PEG concentration but these began to decline significantly in 15% PEG concentration. The results indicate that photoinhibition occurs under water stress as a result of damage to the reaction center of photosystem II (Table 3, *P*≤0.05). The values of Y(II), qP and ETR de‐ creased, while those of NPQ and qN increased with increasing PEG concentrations. These results indicate that electron transport activity and the photosynthetic apparatus of wheat seedlings with certain drought-resistance are damaged.

Chl and Car are the main photosynthetic pigments of plants, so these are good indicators of the photosynthesis capability of a plant. Under water stress, with the exception of Car which barely changed, the contents of Chl *a* and *b* decreased slightly at first but then decreased more sharply at the 15% PEG concentration (Table 4; *P*≤0.05). This may be linked with the observation that under -0.34MPa water potential conditions Chl synthesis was severely in‐ hibited with the result that the functioning of the photosynthetic apparatus became serious‐ ly impaired [25, 5]. Compared with the control, the water stress effects on Chl *a/b* were high and this appears to be closely related to the metabolic regulation of Chl; this possibility is worth further investigation.

## **7.2. Impact of water stress treatments on metabolites**

Treatment Gp (%) Ge (%) CK 96.66 96.00 5% PEG 95.33 91.67 15% PEG 83.67 80.33 25% PEG 64.00 63.67

**Table 5.** Effect of water stress on two indices (Gp and Ge) of germination for wheat. Germinative percentage - Gp and

PEG is an osmotic agent, which play an important role in the regulation of mineral elements, hormone, protein metabolism and effects of signal transduction [50, 41]. The main function of PEG is to slow down the moisture rate of import and export seeds, which benefit to re‐ duce membrane system injury in process of seed imbibition and repair impaired membrane system [27, 16]. PEG has been widely used in seed priming and simulated water stress test,

In plants in general, an appropriate growth strategy is key to fitness in a competitive situation, so too in wheat seedlings, their growth strategy is critical to survival [10]. The RGR value of a plant reflects its vigour and is considered a good index of its exposure to stresses of all sorts [26, 52]. The RGR response of wheat seedlings exposed to increasing PEG concentrations (Fig. 1 A, P ≤ 0.05), revealed a decrease for roots and shoots (Table 2, *P* ≤ 0.05). This may reflect the im‐ pact of water stress on root cell development, which would likely impair nutrient uptake as well as having detrimental effects on photosynthesis, essential for biomass accumulation and therefore on shoot and root elongation. The change trend for WC was similar to that for RGR but the extension of WC in the root was about 1.14-times that of the shoot (Fig 1. B; Table 2;*P*≤0.05). Water stress therefore appears to reduce the absorption and utilization of water to such an extent that the tolerance mechanisms employed by these plants in a drought are insuf‐

**7.1. Impact of water stress treatments on chlorophyll fluorescence and pigments**

The chlorophyll fluorescence kinetics react to the "intrinsic" characteristic of photosynthesis and can rapidly and sensitively reflect a plant's physiological status and its relationship with the environment [Huang et al., 2009]. In this study, PSII values decreased with increas‐ ing PEG concentration but these began to decline significantly in 15% PEG concentration. The results indicate that photoinhibition occurs under water stress as a result of damage to the reaction center of photosystem II (Table 3, *P*≤0.05). The values of Y(II), qP and ETR de‐

the wheat seedlings were treated by three different PEG concentrations.

**7. Impact of water stress treatments on growth**

ficient to maintain normal growth.

germinative Energy - Gp.

374 Soil Processes and Current Trends in Quality Assessment

**6. Discussion**

Proline and betaine are also known to play important roles in osmotic adjustment with their accumulation under water stress being observed in many species [46, 38]. Here, the results show that, along with a decrease in osmotic potential, the accumulation of free proline and betaine increased significantly both in the roots and the shoots. This increase would lower the osmotic potential [i.e. make it more strongly negative] in the cells which would help to maintain turgor and thus sustain the normal physiological and biochemical processes in the face of drought (Fig. 2, *P*≤0.05).

Soluble sugars are the main osmotic adjustment substances and so are important indicators of drought tolerance. The results show that the soluble sugars contents of wheat seedlings increases under high PEG concentration. This indicates that they may help to regulate and maintain the activity of physiological processes within the plant in a high water-stress envi‐ ronment by raising the osmotic potential of the cells [14].

### **7.3. Impact of water stress treatments on organic acids**

The accumulation of organic acids is a physiological response of plants to stress, when plants are suffered by water stress, they can through cells apperceive and transmit drought signal [42]. There nearly no impact on the content of organic acids under blew 15% water stress, it decreased significantly under high stress, but in shoot FA completely opposite change (Fig. 3, *P*≤0.05). The results confirmed that the organic acids metabolic regulation was closely related to the plant water stress resistance. The change of organic acid may be adaptive mechanism by which wheat seedlings maintain their intracellular osmotic balance under water stress [47, 55].

### **7.4. Impact of water stress treatments on germination**

Germination is one of the most critical periods in the life cycle of plants. Under water stress, low water potential is a determining factor inhibiting seed germination [51, 43]. The inhibit‐ ing action of water stress on the wheat germination was increased with PEG-6000 concentra‐ tion increasing (Table 5).

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## **8. Conclusion**

In summary, the growth of wheat seedlings was inhibited by water stress, especially in roots. The function of water regulation occurs outside root, or in apoplast of root, or both outside root and in apoplast of root. Therefore, we propose that the water-potential adjust‐ ment of the roots may be a key physiological mechanism for wheat resisting water stress. Proline, betaine and soluble sugar content increase to a greater extent in response to water stress, these data suggest that wheat seedlings may initially sense high drought environ‐ ments, the harmful effects of water stress on the distribution and accumulation of carbohy‐ drates, it was reflecting the specific detrimental effects of a drought environment. With the extension of PEG-6000 concentrations, wheat seedlings photosynthetic electron transport and photosynthetic primary reaction inhibited, heat disseminate which possess photoprotec‐ tive effect increased. It implies that there was a closed relationship between the effects of water stress on chlorophyll fluorescence parameters of wheat seedlings. These results pro‐ vide useful data that will facilitate the development of strategies for the creation of engi‐ neered wheat varieties that are more tolerant towards water stress.

## **Acknowledgements**

This work supported by grants from the Project of the National Natural Science Foundation of China (No. 31170303, 30870238, 30871447, 50709040, 31070398). The basic research special fund operations (1610122012001), the international scientific and technological cooperation projects (No. 2010DFB30550).

## **Author details**

Rui Guo\* , Wei Ping Hao, Dao Zhi Gong, Xiu Li Zhong and Feng Xue Gu

\*Address all correspondence to: guor219@yahoo.com

Institute of Environment and Sustainable Development in Agriculture(IEDA), Chinese Academy of Agricultural Sciences (CAAS)/Key Laboratory of Dry land Agriculture, MOA, Beijing 100081, China

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ing action of water stress on the wheat germination was increased with PEG-6000 concentra‐

In summary, the growth of wheat seedlings was inhibited by water stress, especially in roots. The function of water regulation occurs outside root, or in apoplast of root, or both outside root and in apoplast of root. Therefore, we propose that the water-potential adjust‐ ment of the roots may be a key physiological mechanism for wheat resisting water stress. Proline, betaine and soluble sugar content increase to a greater extent in response to water stress, these data suggest that wheat seedlings may initially sense high drought environ‐ ments, the harmful effects of water stress on the distribution and accumulation of carbohy‐ drates, it was reflecting the specific detrimental effects of a drought environment. With the extension of PEG-6000 concentrations, wheat seedlings photosynthetic electron transport and photosynthetic primary reaction inhibited, heat disseminate which possess photoprotec‐ tive effect increased. It implies that there was a closed relationship between the effects of water stress on chlorophyll fluorescence parameters of wheat seedlings. These results pro‐ vide useful data that will facilitate the development of strategies for the creation of engi‐

This work supported by grants from the Project of the National Natural Science Foundation of China (No. 31170303, 30870238, 30871447, 50709040, 31070398). The basic research special fund operations (1610122012001), the international scientific and technological cooperation

Institute of Environment and Sustainable Development in Agriculture(IEDA), Chinese Academy of Agricultural Sciences (CAAS)/Key Laboratory of Dry land Agriculture, MOA,

, Wei Ping Hao, Dao Zhi Gong, Xiu Li Zhong and Feng Xue Gu

\*Address all correspondence to: guor219@yahoo.com

neered wheat varieties that are more tolerant towards water stress.

tion increasing (Table 5).

376 Soil Processes and Current Trends in Quality Assessment

**Acknowledgements**

projects (No. 2010DFB30550).

**Author details**

Beijing 100081, China

Rui Guo\*

**8. Conclusion**


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**Chapter 14**

**The Role of Soil Properties in Plant Endemism – A**

The soil as the support of all terrestrial ecosystems is distributed as a continuous landscape and varies according to drainage, geomorphology and litho-climatic conditions [1]. There are five major factors that control the formation of soil: parent materials, climate, biota, top‐ ography and time [2]. These factors present interdependence; for example contrasting cli‐ matic regimes are likely to be associated with contrasting types of vegetation. Nonetheless in certain situation one of the factors had the dominant influence in determining differences among of set of soil [3]. In addition processes of soil genesis are operating under the influ‐ ence of environmental factors; therefore we can understand the relationship between partic‐ ular soils and the landscape and ecosystem in which they function [4]. Climate is perhaps the most influential of the four factor acting on large geographical areas (large scale), contri‐ buting to the development of specific types of soils and vegetation patterns [5], such as Geli‐ sols (tundra vegetation: lichens, grasses and low shrub), Histosols (water loving plants: pond weeds, cattails, sedges, reed, mosses), Spodosols (mainly coniferous species), Alfisols (deciduous forest), Mollisols (grasses), Aridisols (xerophytic plants), and Oxisols (tropical rain forest). However, at regional level (medium scale) soil variability is often related to small changes in topography and thickness of parent materials or to the effect of organism. In Trans-Mexican Volcanic Belt, is common finding soil sequences or catenas related with specific vegetation species such as Andosols (Pines-volcanic ashes), Cambisols (deciduous trees-colluvial material), fluvisols (crop lands-alluvial material) y solonchack (halophyte grasses-lacutrine material) [6]. Finally at local scales, variation in edaphic characters often provides the best statistical explanation for variation in floristic composition. Frequently,

> © 2013 Bárcenas-Argüello et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Bárcenas-Argüello et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Revision of Conservation Strategies**

María Luisa Bárcenas-Argüello,

http://dx.doi.org/10.5772/53056

Teresa Terrazas

**1. Introduction**

Ma. del Carmen Gutiérrez-Castorena and

Additional information is available at the end of the chapter


## **The Role of Soil Properties in Plant Endemism – A Revision of Conservation Strategies**

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in plant stress physiology. *Photosynthesis Research*, 25, 147-150.

mi tescommunis ). *ActaBot. Boreal. Occident. Sin.*, 22(3), 561-565.

tivars seedlings. *Acta Bot. Boreal. Occident. Sin.*, 24, 812-816.

sues of flat pea (Lathyrus sylvestris L.). *Enviro Expt Bot.*, 30, 497-504.

seeds. *Journal of Forestry Sci and Tech.*, 4, 142-145.

49, 775-788.

*Planta*, 120, 279-289.

380 Soil Processes and Current Trends in Quality Assessment

Plant Physio1. : , 116, 1403-1412.

*and wheat.*, 27(2), 45-50.

*Chinese Materia Medica*, 34, 127-131.

and Populus euphratica. *Forest Research*, 21(4), 566-570.

María Luisa Bárcenas-Argüello, Ma. del Carmen Gutiérrez-Castorena and Teresa Terrazas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53056

## **1. Introduction**

The soil as the support of all terrestrial ecosystems is distributed as a continuous landscape and varies according to drainage, geomorphology and litho-climatic conditions [1]. There are five major factors that control the formation of soil: parent materials, climate, biota, top‐ ography and time [2]. These factors present interdependence; for example contrasting cli‐ matic regimes are likely to be associated with contrasting types of vegetation. Nonetheless in certain situation one of the factors had the dominant influence in determining differences among of set of soil [3]. In addition processes of soil genesis are operating under the influ‐ ence of environmental factors; therefore we can understand the relationship between partic‐ ular soils and the landscape and ecosystem in which they function [4]. Climate is perhaps the most influential of the four factor acting on large geographical areas (large scale), contri‐ buting to the development of specific types of soils and vegetation patterns [5], such as Geli‐ sols (tundra vegetation: lichens, grasses and low shrub), Histosols (water loving plants: pond weeds, cattails, sedges, reed, mosses), Spodosols (mainly coniferous species), Alfisols (deciduous forest), Mollisols (grasses), Aridisols (xerophytic plants), and Oxisols (tropical rain forest). However, at regional level (medium scale) soil variability is often related to small changes in topography and thickness of parent materials or to the effect of organism. In Trans-Mexican Volcanic Belt, is common finding soil sequences or catenas related with specific vegetation species such as Andosols (Pines-volcanic ashes), Cambisols (deciduous trees-colluvial material), fluvisols (crop lands-alluvial material) y solonchack (halophyte grasses-lacutrine material) [6]. Finally at local scales, variation in edaphic characters often provides the best statistical explanation for variation in floristic composition. Frequently,

© 2013 Bárcenas-Argüello et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Bárcenas-Argüello et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

systematic variations in the parent material are closely related to endemism [7]. Endemic species have relatively narrow tolerance to changes in their environment, and can be de‐ pendant on certain geologic and edaphic features. There are studies that provide detailed in‐ formation on species and plant communities, some include relief features, and others report physical and chemical analyses which allow us to infer about soil fertility [8,9] while some other studies have performed statistical analysis to correlate such variables [10-12]. This chapter presents a revision of the current knowledge on the role of soil properties as for pH, H2O2 reaction, and reactions to HCl in the distribution of endemic plant species and synthe‐ tize in table 1 studies that give details examples of this relationship (plant-soil endemicity).

**Species/References**

(All monocotyledons)

*Lupinus* subcarnosus (Leguminosae) [72]

*Lasthenia californica* (Asteraceae) [73,74]

*Coccoloba cereifera* (Polygonaceae), [75]

*Erigonum nervulosum (*Polygonaceae) *Streptanthus brachatus* and *S. morrisonii* (Brassicaceae) [76]

**2. Endemism**

[71]

**Soi/Substrate**

Sandy soils, gravelly soils and quartzitic outcrops

Serpentine barren

**Table 1.** Studies in which species distribution and soil features are reported.

looking for consistency with geological models [21-23].

soils

Sandy soil Allozymic variation in

Serpentine soils Genetic, physiological

proteins

studies

enzymes and other

and phylogenetic

Each 25 m2 quadrant was classified according to soil types and were sampled for chemical and granulometric analyses

Low concentrations of

Endemic, in botany, means that a plant species is considered native to the country –regionwhere it can be found [13] and the term is applied to the distribution of organisms [14]. Al‐ though climatic factors are the most studied [15,16]; endemism is a non-ecological [17], geological event. Climate limits the flora [18,19], while geological characteristics largely de‐ fine habitat diversity [4]. Moreover, edaphically severe habitats commonly support edaphic endemics, which are plant species that do not occur elsewhere [20]. Although that might be not enough to recognize the endemic species, edaphological characters (macro and micro) are essential to establish phylogenetic hypothesis of endemic taxa and areas of endemism,

Ca, Mg, P, N

**type Technique Results/Conclusions Localities**

The Role of Soil Properties in Plant Endemism – A Revision of Conservation Strategies

Edaphically restricted species is less genetically variable

Races A and C of *L. californica* coexist on serpentine soil, but inhabit soil of differing physical and chemical properties

The spatial distribution of *Coccoloba* was largely related to the arrangement

Differences in Ca and Mg between serpentine soils allow distinct species

of sandfields

distribution

East-central Texas USA

http://dx.doi.org/10.5772/53056

383

Palo Alto, California in the Santa Cruz Mountains USA

Serra Do Cipó, southeastern Brazil

Lake County, California, USA


*Yucca filifera*


**Table 1.** Studies in which species distribution and soil features are reported.

## **2. Endemism**

systematic variations in the parent material are closely related to endemism [7]. Endemic species have relatively narrow tolerance to changes in their environment, and can be de‐ pendant on certain geologic and edaphic features. There are studies that provide detailed in‐ formation on species and plant communities, some include relief features, and others report physical and chemical analyses which allow us to infer about soil fertility [8,9] while some other studies have performed statistical analysis to correlate such variables [10-12]. This chapter presents a revision of the current knowledge on the role of soil properties as for pH, H2O2 reaction, and reactions to HCl in the distribution of endemic plant species and synthe‐ tize in table 1 studies that give details examples of this relationship (plant-soil endemicity).

**type Technique Results/Conclusions Localities**

specialist

than 6.1

absent

Soil pH *Satureja arkansana* was

Three species are endemics Coastal of

This plant is an edaphic

absent on soil with a pH less

In areas with soil pH greater than 6.1 *C. lanceolata* was

This plant grows better on non-alkali soil when grown without competition

The current extraction of gravel its habitat which could pose a direct threat

It was observed that soil characteristics drastically alter the conformation of the vegetation and therefore species are not present

California USA

Chihuahuan Desert, Mexico

Arkansas USA

Yolo, California,

Baja California Sur, Mexico

Northern Gulf Coastal Plain in northeastern Mexico

USA

**Species/References**

*Calochotusobispoensis*, *C*. *tiburonensis*, *C*. *pulchellus* (Liliaceae)

[40]

*Ariocarpus kotschoubeyanus* (Cactaceae) [64]

*Satureja arkansana* (Lamiaceae) and

*Coreopsis lanceolata* (Asteraceae) [25]

*Hemizonia pungens* ssp.

*Guaiacum unijugum* (Zygophyllaceae) [67]

*Agave bracteosa*, *A*. *victoria-reginae*, *A*. *albopilosa Brahea berlandieri Dasylirion berlandieri Hesperaloe funifera* var.

*pungens* (Asteraceae) [20]

*funifera Yucca filifera* **Soi/Substrate**

382 Soil Processes and Current Trends in Quality Assessment

Silty, dry lake beds

Sandstone glade with alkaline soil

Coastal dune and

arroyo environments

Litosols stoniness soils, summit, slopes, Stoniness soils depth soils

Serpentine Concentrations of Ni

Alkali pools Reciprocal transplant

greenhouse experiment

Genetic analysis using microsatellite

Sampling of 39 plots (1 Ha each one) all species were recorded and particle size, pH, depth and organic

matter

and Cu in plant tissue

Cartographic method by conglomerates

> Endemic, in botany, means that a plant species is considered native to the country –regionwhere it can be found [13] and the term is applied to the distribution of organisms [14]. Al‐ though climatic factors are the most studied [15,16]; endemism is a non-ecological [17], geological event. Climate limits the flora [18,19], while geological characteristics largely de‐ fine habitat diversity [4]. Moreover, edaphically severe habitats commonly support edaphic endemics, which are plant species that do not occur elsewhere [20]. Although that might be not enough to recognize the endemic species, edaphological characters (macro and micro) are essential to establish phylogenetic hypothesis of endemic taxa and areas of endemism, looking for consistency with geological models [21-23].

## **3. Soil**

Soil, in soil taxonomy [24] is a natural body comprised of solids (minerals and organic mat‐ ter), liquid, and gases that occurs on the land surface. Soil occupies space, and is character‐ ized By one of the following: horizons or layers, that are distinguishable from the initial materials a result of additions, losses, transfers, and transformations of energy and matter or the ability to support rooted plants in a natural environment.

of soil mosaics formed by slope processes; whereby mountainous regions are characterized

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The soil formation is regulated by the origin of parent material and by the age of the ex‐ posed surface. The nature of the parent material profoundly influences soil characteristics such as the chemical weathering and the quantity and type of clay minerals. While the total composition of the parent rocks is only one of the factors involved in soil formation, it is of considerable interest to analyze some of these rocks, and of soils of similar origin. The time of soils formation refers to the age of the exposed surface. The soils change with time and

Physical, chemical and mineralogical analyses are used in soil taxonomic criteria. For stand‐ ard laboratory methods descriptions see Appendix of Keys to Taxonomy of Soils [24]. The diversity of soil properties resulted in the diversity of soil use and soil ecological functions [38,39]. In edaphic islands such as serpentine and limestone outcrops upon which plant grow are necessary specific analyses; per example, in ultramafic soil trace elements as Mn, Cu, Zn, Cr and the heavy metals Ni and Co are extracted, as well as in calcareous soils are measured soluble and exchangeable P [40,41]. Overall, previous authors agree that vegeta‐

The existing literature comprises a wide range of sampling techniques to obtain vegetation samples. The plant populations may vary in size and in number of individuals per species. According to our field observations, it is necessary to select populations that adequately rep‐ resent the endemic area (Figure 1). Soil sampling must be performed in the same locations where vegetation has been previously sampled. The overlap of both sampling activities al‐ lows correlating changes in vegetation and soil [43]. Thus, the resulting plots are clearly rep‐ resentative of the surrounding area. Accordingly, geology and topography are essential characteristics that need to be thoroughly examined. For instance, spatial variability studies have indicated that even when the relief of the site is gently rolling, erosion processes can affect soil properties [44, 45], and therefore alter the results of sample analysis. We do not recommend to combined soil samples from which the different edaphic variables are meas‐ ured [11], this method can give erroneous conclusions because information at the micro-

The geology and the topography are factors that influence the formation of particular soil type and the establishment of specific biological forms, [38]. The geological origin of rocks can be identified visually and mineralogical composition by X-ray diffraction or other meth‐ ods [31]. Undisturbed rock samples must be collected, without showing any chemical or physical weathering. The slope and rock outcrops are some landscape features easy to dis‐ tinguish. The slope is important by the sediments mobilized by slope processes as for land‐

tion differences are strongly associated with differences in the bedrock [29,42].

by high soil diversity.

**3.2. Soil properties**

**4. Sampling approach**

scale level is lost.

undergo a process of evolution [2].

We intend to clarify that for some plant species, soil has played an essential role in their evo‐ lution and current distribution. Therefore, it is necessary to indicate that according to soil taxonomy is not possible to classify the earthy materials used in pots in greenhouses. In the same sense, plants even grow on trees, but trees are regarded as non soil. Soil covers the earth's surface as a continuum, except on bare rock. Some endemics are restricted to a par‐ ticular geological formation or to one type of rock or rock outcrops. Endemicity found on rocky outcrops, either calcareous or otherwise, has been reported by several researchers [25-31].

In order to understand the relevance of soil for plant endemism, it is necessary to highlight that the geological processes as genesis of unique soil types may provide the necessary isola‐ tion for the genesis of unique biota, and the edaphic factors are used to draw the link be‐ tween environments and taxa [32]. If the environmental scenario is potentially multidimensional [33], some soil properties should be also considered as predictors. Previous research [2] considers that the distinction between soil and environment is arbitrary; and might constitute a theoretical artifact which does not represent natural processes.

#### **3.1. Soil genesis**

The transformation of rock into soil is designated as soil formation. Climate, organisms, re‐ lief, rocks, and the time are soil forming factors. Therefore, soil can be considered as a partic‐ ular combination of its forming factors. For a given combination of factors there is only one soil type [2]. Soil properties such as pH, clay content, porosity, etc, are determined by the combination of these factors. The smallest change in any one of the properties, gives rise to a new soil.

Climate is usually considered the dominant soil forming factor, and cannot be described by a single index [4]; for example, the high proportion of smectite in soil, indicate a highly sea‐ sonal semiarid subtropical climate [34].

Living organism as bacterial species are able to fix N2, dissolved P, weathered extrusive igneous rock, marble, and limestone, and significantly mobilized useful minerals, such as P, K, Mg, Mn, Fe, Cu, and Zn in rock minerals [35]. Additionally, plant root systems al‐ ter the structure of the surrounding soil [36], and the roots of some plants have the abil‐ ity to exude low-molecular-weight organic acids that produce changes in the availability of nutrients [37].

Relief modifies the water relationships in soils, affected by slope processes such as erosion, landslides and other mass movement [38-39]. However, little is known about the dynamics of soil mosaics formed by slope processes; whereby mountainous regions are characterized by high soil diversity.

The soil formation is regulated by the origin of parent material and by the age of the ex‐ posed surface. The nature of the parent material profoundly influences soil characteristics such as the chemical weathering and the quantity and type of clay minerals. While the total composition of the parent rocks is only one of the factors involved in soil formation, it is of considerable interest to analyze some of these rocks, and of soils of similar origin. The time of soils formation refers to the age of the exposed surface. The soils change with time and undergo a process of evolution [2].

## **3.2. Soil properties**

**3. Soil**

[25-31].

**3.1. Soil genesis**

new soil.

of nutrients [37].

sonal semiarid subtropical climate [34].

Soil, in soil taxonomy [24] is a natural body comprised of solids (minerals and organic mat‐ ter), liquid, and gases that occurs on the land surface. Soil occupies space, and is character‐ ized By one of the following: horizons or layers, that are distinguishable from the initial materials a result of additions, losses, transfers, and transformations of energy and matter or

We intend to clarify that for some plant species, soil has played an essential role in their evo‐ lution and current distribution. Therefore, it is necessary to indicate that according to soil taxonomy is not possible to classify the earthy materials used in pots in greenhouses. In the same sense, plants even grow on trees, but trees are regarded as non soil. Soil covers the earth's surface as a continuum, except on bare rock. Some endemics are restricted to a par‐ ticular geological formation or to one type of rock or rock outcrops. Endemicity found on rocky outcrops, either calcareous or otherwise, has been reported by several researchers

In order to understand the relevance of soil for plant endemism, it is necessary to highlight that the geological processes as genesis of unique soil types may provide the necessary isola‐ tion for the genesis of unique biota, and the edaphic factors are used to draw the link be‐ tween environments and taxa [32]. If the environmental scenario is potentially multidimensional [33], some soil properties should be also considered as predictors. Previous research [2] considers that the distinction between soil and environment is arbitrary; and

The transformation of rock into soil is designated as soil formation. Climate, organisms, re‐ lief, rocks, and the time are soil forming factors. Therefore, soil can be considered as a partic‐ ular combination of its forming factors. For a given combination of factors there is only one soil type [2]. Soil properties such as pH, clay content, porosity, etc, are determined by the combination of these factors. The smallest change in any one of the properties, gives rise to a

Climate is usually considered the dominant soil forming factor, and cannot be described by a single index [4]; for example, the high proportion of smectite in soil, indicate a highly sea‐

Living organism as bacterial species are able to fix N2, dissolved P, weathered extrusive igneous rock, marble, and limestone, and significantly mobilized useful minerals, such as P, K, Mg, Mn, Fe, Cu, and Zn in rock minerals [35]. Additionally, plant root systems al‐ ter the structure of the surrounding soil [36], and the roots of some plants have the abil‐ ity to exude low-molecular-weight organic acids that produce changes in the availability

Relief modifies the water relationships in soils, affected by slope processes such as erosion, landslides and other mass movement [38-39]. However, little is known about the dynamics

might constitute a theoretical artifact which does not represent natural processes.

the ability to support rooted plants in a natural environment.

384 Soil Processes and Current Trends in Quality Assessment

Physical, chemical and mineralogical analyses are used in soil taxonomic criteria. For stand‐ ard laboratory methods descriptions see Appendix of Keys to Taxonomy of Soils [24]. The diversity of soil properties resulted in the diversity of soil use and soil ecological functions [38,39]. In edaphic islands such as serpentine and limestone outcrops upon which plant grow are necessary specific analyses; per example, in ultramafic soil trace elements as Mn, Cu, Zn, Cr and the heavy metals Ni and Co are extracted, as well as in calcareous soils are measured soluble and exchangeable P [40,41]. Overall, previous authors agree that vegeta‐ tion differences are strongly associated with differences in the bedrock [29,42].

## **4. Sampling approach**

The existing literature comprises a wide range of sampling techniques to obtain vegetation samples. The plant populations may vary in size and in number of individuals per species. According to our field observations, it is necessary to select populations that adequately rep‐ resent the endemic area (Figure 1). Soil sampling must be performed in the same locations where vegetation has been previously sampled. The overlap of both sampling activities al‐ lows correlating changes in vegetation and soil [43]. Thus, the resulting plots are clearly rep‐ resentative of the surrounding area. Accordingly, geology and topography are essential characteristics that need to be thoroughly examined. For instance, spatial variability studies have indicated that even when the relief of the site is gently rolling, erosion processes can affect soil properties [44, 45], and therefore alter the results of sample analysis. We do not recommend to combined soil samples from which the different edaphic variables are meas‐ ured [11], this method can give erroneous conclusions because information at the microscale level is lost.

The geology and the topography are factors that influence the formation of particular soil type and the establishment of specific biological forms, [38]. The geological origin of rocks can be identified visually and mineralogical composition by X-ray diffraction or other meth‐ ods [31]. Undisturbed rock samples must be collected, without showing any chemical or physical weathering. The slope and rock outcrops are some landscape features easy to dis‐ tinguish. The slope is important by the sediments mobilized by slope processes as for land‐ slides, colluviation, and accumulation of the material that eroded from upper landscape positions [39], this can be recorder in degrees or percent. The geomorphic position can regis‐ ter as summit, shoulder, back slope, toe slope, and floodplain if any (Figure 2).

oxalates (Figure 4), and their abundance is associated with calcareous soils [78]. We recom‐ mend isolate crystals from the plant tissue for better analyses and carried out X-ray diffraction (XDR), chemical composition and morphology with scanning electron microscopy (SEM). We also suggest use the petrographic microscope to know the optical properties of the crystals for be able to identify in the sand fraction of soil [57]. Additionally to relate the soil elements that plants take up soil and incorporate in their tissues, use energy dispersive X-ray (EDX) on crys‐ tals analyses. In order to determine the importance of the biominerals in the soil properties, is necessary that at least three hundred grains from the sandy fraction will count on a grain

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387

mount by line counting method using a petrographic microscope [54, 57].

**Figure 3.** Samples. (A) rock and organic debris for *Cephalocereus apicicephalium*; (B) soil for *C*. *totolapensis*.

**Figure 4.** Calcium oxalate crystals isolated from *Cephalocereus* species. (A) petrographic microscope; (B) scanning

electron microscopy.

**Figure 1.** Delimiting study area in slope or plane environments. (A, B) Nuevo León, México. Habitats for several succu‐ lent species endemic to the Chihuahuan Desert.

**Figure 2.** Geomorphic position of *Cephalocereus* species. (A) *C*. *nizandensis* on summit; (B) *C*. *senilis* on summit and shoulder.

The slope aspect would influence insolation, temperature and moisture. Isolation, tempera‐ ture and moisture also must be into accounted because they have effect on the bedrock or with the vegetation [29, 77]. In the surface it is necessary to test soil and bedrock reactions to HCl as a measure of their calcareousness. Moreover, the soil properties of depth, stoniness, root distribution, H2O2 reaction, structure, and Munsell color should be recorded [31,54]. If the characteristics of the site permit, three or more bulk soil or rhizosphere soil and rock samples along the geomorphic position per site should be collected (Figure 3). In the cases where there was no soil, organic debris must be collected [54].

Finally, the abundance of certain minerals in the soil can influence the physiological response and metabolism in plant species, such as heavy metals accumulation and the synthesis and ac‐ cumulation of biominerals [40, 53]. The most common biominerals in plant tissues are calcium oxalates (Figure 4), and their abundance is associated with calcareous soils [78]. We recom‐ mend isolate crystals from the plant tissue for better analyses and carried out X-ray diffraction (XDR), chemical composition and morphology with scanning electron microscopy (SEM). We also suggest use the petrographic microscope to know the optical properties of the crystals for be able to identify in the sand fraction of soil [57]. Additionally to relate the soil elements that plants take up soil and incorporate in their tissues, use energy dispersive X-ray (EDX) on crys‐ tals analyses. In order to determine the importance of the biominerals in the soil properties, is necessary that at least three hundred grains from the sandy fraction will count on a grain mount by line counting method using a petrographic microscope [54, 57].

slides, colluviation, and accumulation of the material that eroded from upper landscape positions [39], this can be recorder in degrees or percent. The geomorphic position can regis‐

**Figure 1.** Delimiting study area in slope or plane environments. (A, B) Nuevo León, México. Habitats for several succu‐

**Figure 2.** Geomorphic position of *Cephalocereus* species. (A) *C*. *nizandensis* on summit; (B) *C*. *senilis* on summit and

The slope aspect would influence insolation, temperature and moisture. Isolation, tempera‐ ture and moisture also must be into accounted because they have effect on the bedrock or with the vegetation [29, 77]. In the surface it is necessary to test soil and bedrock reactions to HCl as a measure of their calcareousness. Moreover, the soil properties of depth, stoniness, root distribution, H2O2 reaction, structure, and Munsell color should be recorded [31,54]. If the characteristics of the site permit, three or more bulk soil or rhizosphere soil and rock samples along the geomorphic position per site should be collected (Figure 3). In the cases

Finally, the abundance of certain minerals in the soil can influence the physiological response and metabolism in plant species, such as heavy metals accumulation and the synthesis and ac‐ cumulation of biominerals [40, 53]. The most common biominerals in plant tissues are calcium

where there was no soil, organic debris must be collected [54].

lent species endemic to the Chihuahuan Desert.

386 Soil Processes and Current Trends in Quality Assessment

shoulder.

ter as summit, shoulder, back slope, toe slope, and floodplain if any (Figure 2).

**Figure 3.** Samples. (A) rock and organic debris for *Cephalocereus apicicephalium*; (B) soil for *C*. *totolapensis*.

**Figure 4.** Calcium oxalate crystals isolated from *Cephalocereus* species. (A) petrographic microscope; (B) scanning electron microscopy.

## **5. Edaphic endemism**

The interest for environmental conservation has been growing over the last decades. How‐ ever, there has been little or no consensus on priority species or conservation strategies Ta‐ ble 2. The conservation of soil diversity greatly overlaps with such for plants, and both become endangered as a result of land use [46]. The fact that soil taxa are geographically re‐ stricted is important for planning and conservation efforts. Soil characteristics often play an essential role in determining plant community distributions [47]. The endemism of native plant species in edaphically specialized habitats suggest that these native endemic species are uniquely specialized to survive and grow better under the conditions prevalent in these harsh areas. For instance, several authors [48] reported there are almost five edaphically-re‐ stricted or -endemic butterflies, mostly associated with serpentine soils. Some species are ab‐ solutely limited by the edaphic restriction of their host plants. These are a better argument for biodiversity preservation [49].

to reduce water potentials to levels lower than found on nonserpentine soils, as well as keep

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389

Soil constitutes the main source of nutrients for plants. However, for plants growing on bare rock mycorriza have been described as an important factor in promoting edaphic specializa‐ tion [35, 37]. This high degree of host specificity of symbiotic microbes could enhance nu‐ trient uptake in the infertile soils or rocks. In rhizoplane of cacti, several bacterial species were isolated. This bacterium fixed N2, dissolved P, weathered extrusive igneous rock, mar‐ ble and limestone, and significantly mobilized useful minerals, such as P, K, Mg, Mn, Fe, Cu and Zn in rock minerals [35]. Other rock-colonizing cacti usually grow in cracks or fissures that are deeply penetrated by the root system: *Mammillaria fraileana* [31] and *Cephalocereus apicicephalium* and *Cephalocereus nizandensis* (Figure 5) [54]. Little is known about weathering mechanisms, except that the roots of these species can exude low-molecular organic acids (LOAs). The LOAs in root exudates may play an important role in the solubilization and plant availability of mineral nutrients in the rock [55]. Additionally, [20] mentioned that

rock outcrop represent refuges from competition with other (often exotic) species.

**Figure 5.** Rocky habitats. (A) *Cephalocereus apicicephalium* grows in fissures; (B) *C*. *nizandensis* grows in cracks.

The edaphic endemics are now restricted to unusual and sometimes contaminated soils, but may have been able to withstand large concentrations of metals in their tissues or large quantities of calcium oxalate crystals [40]. The ability to tolerate excessively high levels of nickel and other heavy metals may be a physiological adaptation of the genus *Calochortus* and not necessarily an evolutionary response by several species to life on an ultramafic sub‐ strate. The amount of crystalline Ca oxalate in the oldest leaves of *Eucalyptus diversicolor* may be related in part to the high levels of exchange-able soil calcium [56]. *Cephalocereus* species could accumulate great quantities of calcium oxalate crystals even if there is low calcium soluble in the soil [54]. *Cephalocereus nizandensis* and *C. apicicephalium* grow on limestone out‐

stomata closed or nearly closed [53].


**Table 2.** Studies in which species endemism are related to soil features.

Some explanations for the close relationship soil-plant have been looking at morpho-ana‐ tomical changes and physiological response to variations in soil parameters. Soil nutrient status determines leaves with glands or without glands: sclerophyllous (leaves without glands) plants exist almost exclusively on oligotrophic soil; whereas orthophyllous (leaves with glands) growth on more or less equally on both oligotrophic and eutrophic soil [50]. In this sense, exceptionally high levels of species turnover were found along all three soil fertil‐ ity gradients which reflect the high degree of edaphic specialization of the flora [51]. Soil fer‐ tility is difficult to quantify, because it dependent not only of the nitrogen (N) and phosphorus (P) status of the soils, but also on their availability. Differences between species in ability to solubilize mineral nutrients could affect the ability or inability of plants to grow in particular soils. In calcareous soils, species suffer lime-chlorosis by Fe deficiency, and their growth is affected by inability to solubilize the native phosphate [52]. In ultramaphic soils, the vegetation accumulates large quantities of heavy metals in their tissues. So, endem‐ ic plants have developed strategies to grow successfully in these unusual conditions. For ex‐ ample, to minimize water requirements and excessive water loss, serpentine plants are able to reduce water potentials to levels lower than found on nonserpentine soils, as well as keep stomata closed or nearly closed [53].

**5. Edaphic endemism**

388 Soil Processes and Current Trends in Quality Assessment

for biodiversity preservation [49].

The interest for environmental conservation has been growing over the last decades. How‐ ever, there has been little or no consensus on priority species or conservation strategies Ta‐ ble 2. The conservation of soil diversity greatly overlaps with such for plants, and both become endangered as a result of land use [46]. The fact that soil taxa are geographically re‐ stricted is important for planning and conservation efforts. Soil characteristics often play an essential role in determining plant community distributions [47]. The endemism of native plant species in edaphically specialized habitats suggest that these native endemic species are uniquely specialized to survive and grow better under the conditions prevalent in these harsh areas. For instance, several authors [48] reported there are almost five edaphically-re‐ stricted or -endemic butterflies, mostly associated with serpentine soils. Some species are ab‐ solutely limited by the edaphic restriction of their host plants. These are a better argument

**Species Evidence Reference**

*Calochotus* (3 spp.) Soil studies 40

*Calulanthus amplexicaulis* var *barbarae* Soil studies 59

*Mammillaria fraileana* Soil-rock studies 31

Some explanations for the close relationship soil-plant have been looking at morpho-ana‐ tomical changes and physiological response to variations in soil parameters. Soil nutrient status determines leaves with glands or without glands: sclerophyllous (leaves without glands) plants exist almost exclusively on oligotrophic soil; whereas orthophyllous (leaves with glands) growth on more or less equally on both oligotrophic and eutrophic soil [50]. In this sense, exceptionally high levels of species turnover were found along all three soil fertil‐ ity gradients which reflect the high degree of edaphic specialization of the flora [51]. Soil fer‐ tility is difficult to quantify, because it dependent not only of the nitrogen (N) and phosphorus (P) status of the soils, but also on their availability. Differences between species in ability to solubilize mineral nutrients could affect the ability or inability of plants to grow in particular soils. In calcareous soils, species suffer lime-chlorosis by Fe deficiency, and their growth is affected by inability to solubilize the native phosphate [52]. In ultramaphic soils, the vegetation accumulates large quantities of heavy metals in their tissues. So, endem‐ ic plants have developed strategies to grow successfully in these unusual conditions. For ex‐ ample, to minimize water requirements and excessive water loss, serpentine plants are able

**Table 2.** Studies in which species endemism are related to soil features.

*Cephalocereus* (5 spp) Soil-rock studies 54, 57

Soil constitutes the main source of nutrients for plants. However, for plants growing on bare rock mycorriza have been described as an important factor in promoting edaphic specializa‐ tion [35, 37]. This high degree of host specificity of symbiotic microbes could enhance nu‐ trient uptake in the infertile soils or rocks. In rhizoplane of cacti, several bacterial species were isolated. This bacterium fixed N2, dissolved P, weathered extrusive igneous rock, mar‐ ble and limestone, and significantly mobilized useful minerals, such as P, K, Mg, Mn, Fe, Cu and Zn in rock minerals [35]. Other rock-colonizing cacti usually grow in cracks or fissures that are deeply penetrated by the root system: *Mammillaria fraileana* [31] and *Cephalocereus apicicephalium* and *Cephalocereus nizandensis* (Figure 5) [54]. Little is known about weathering mechanisms, except that the roots of these species can exude low-molecular organic acids (LOAs). The LOAs in root exudates may play an important role in the solubilization and plant availability of mineral nutrients in the rock [55]. Additionally, [20] mentioned that rock outcrop represent refuges from competition with other (often exotic) species.

**Figure 5.** Rocky habitats. (A) *Cephalocereus apicicephalium* grows in fissures; (B) *C*. *nizandensis* grows in cracks.

The edaphic endemics are now restricted to unusual and sometimes contaminated soils, but may have been able to withstand large concentrations of metals in their tissues or large quantities of calcium oxalate crystals [40]. The ability to tolerate excessively high levels of nickel and other heavy metals may be a physiological adaptation of the genus *Calochortus* and not necessarily an evolutionary response by several species to life on an ultramafic sub‐ strate. The amount of crystalline Ca oxalate in the oldest leaves of *Eucalyptus diversicolor* may be related in part to the high levels of exchange-able soil calcium [56]. *Cephalocereus* species could accumulate great quantities of calcium oxalate crystals even if there is low calcium soluble in the soil [54]. *Cephalocereus nizandensis* and *C. apicicephalium* grow on limestone out‐ crops, where the Ca is precipitated and *C. totolapensis* preferred acid soils from andesites, siltstones or mica schist (soluble Ca is 19-72 parts per million). The amount of soluble Ca is also very low in where grow *C. columna-trajani* (63-229 parts per million) and *C. senilis* (82-100 parts per million) [57]. The last two species have the larger epidermal crystals of the genus (Figure 6) [58].

**6. Conservation strategies**

private organizations to preserve such areas.

grows in calcarious rocks.

In the landscape, the vegetational differences often serve to delineate the geologic disconti‐ nuities of an area even to the casual observer. The remarkable differences often observed in plant cover for different soil types in adjacent areas, have naturally led to attempts to ex‐ plain these phenomena in terms of the physical or chemical properties of the soil, or of the physiological characteristics of the plants [61]. These areas should be priority sites for con‐ servation to preserve the unique interaction between soil and plant species as well as the mi‐ crobiota and fauna. For example, the halophytic and gypsophytic vegetation of the Ebro-Basin at Los Monegros [62] or flora of the Coastal Calcareous Hills of the Biosphere Reserve Baconao in Cuba [63] are excellent to demonstrate the varied adaptations of plant types and life-forms as strategies to survive on edapho-climatic harsh conditions of various kinds. In the Chihuahuan Desert region it was found that several Cactaceae species, particularly many members of the Cacteae tribe often inhabit extremely specialized habitats, such as gypsum and other unusual soil formations (Figure 7) [64]. The patches of edaphic endemism also frequently exist as refuges for native species in highly invaded ecosystems [20], ultra‐ mafic substrates act as sites in which *Pinus balfouriana* escapes of the competition [65]. More‐ over, the work with *Helianthus exilis* showed the need to protect specialized microhabitat found only within the large serpentine outcrops, the species cannot survived outside the narrow conditions proper of its habitat [66]. However, the scarcity of conclusive studies on role of soil to determine the prevalence of endemic plants hampers the efforts of public and

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**Figure 7.** A) *Aztekium ritterii* grows in outcrops of steep slopes of crystalline grypsum; (B) *Turbinicarpus valdezianus*

The population size can greatly vary among populations of the same specie generating mi‐ cro-endemic nature. The small populations of some species consist of adult individuals that may be 3 as in *Cephalocereus totolapensis* [54] or 4 as in *Guaiacum unijugum* [67]. These popula‐ tion sizes are not reported sometimes considerer no significant, but many regions show a unique assemblage of species or a higher level of species richness or other associated species which could serve to protect this ecosystem. In Australian alpine vegetation the analyses of

**Figure 6.** Prismatic calcium oxalate crystals in epidermal cells. (A) *Cephalocereus columna-trajani*; (B) *C*. *senilis*. Scale A = 40 μm, B = 50 μm.

For studies of evolution of a soil-type adapted endemic species is necessary to perform phylogenetic analysis. The existence of genetically compatible taxa with such distinct edaphic requirements presents a unique opportunity for intensive study of the genetic basis of tolerance to soil-type. A group plant may have evolved in a very dynamic selec‐ tive context with strong edaphic selective pressures. In [59] they examined phylogenetic relationships of the rare serpentine endemic taxon *Caulanthus amplexicaulus* var. *barbarae*. They found that the serpentine taxa were nonmonophyletic evolving independently at least three times, suggesting that tolerance to serpentine may be gained or lost through relatively few genetic changes. In other case, [50] construct phylogeny for the *Pentaschis‐ tis* clade with 82 species in three genera. They investigated the association between leaf anatomy type and soil nutrient type on which species grow. Despite there is little phylo‐ genetic constrain in soil nutrient type. However, only orthophyllous-leaved species diver‐ sify on eutrophic soils. Nevertheless, modern phylogenies on a number of phanerogam genera occurring on New Caledonia (*Acianthus*, *Cupaniopsis*, *Guioa*, *Morinda*, and *Oxera*) have shown a shift in soil preference (from non serpentine to serpentine soils and vice versa). Thus [60] concluded that the ability to grown on serpentine soil is either a plesio‐ morphic or a very homoplasious character and therefore the hypothesis that serpentine soils preserve the indigenous flora in New Caledonia against competition with immi‐ grant species cannot be supported for these groups. Rarely are made specific soil stud‐ ies, the data are taken often of general charts. We suggest making detailed studies as in *Cephalocereus*, according to plant species and soil type [57]. Therefore, it might be possi‐ ble to infer the role of soil in the evolution of endemic plant species using the phyloge‐ netic analysis.

## **6. Conservation strategies**

crops, where the Ca is precipitated and *C. totolapensis* preferred acid soils from andesites, siltstones or mica schist (soluble Ca is 19-72 parts per million). The amount of soluble Ca is also very low in where grow *C. columna-trajani* (63-229 parts per million) and *C. senilis* (82-100 parts per million) [57]. The last two species have the larger epidermal crystals of the

**Figure 6.** Prismatic calcium oxalate crystals in epidermal cells. (A) *Cephalocereus columna-trajani*; (B) *C*. *senilis*. Scale A

For studies of evolution of a soil-type adapted endemic species is necessary to perform phylogenetic analysis. The existence of genetically compatible taxa with such distinct edaphic requirements presents a unique opportunity for intensive study of the genetic basis of tolerance to soil-type. A group plant may have evolved in a very dynamic selec‐ tive context with strong edaphic selective pressures. In [59] they examined phylogenetic relationships of the rare serpentine endemic taxon *Caulanthus amplexicaulus* var. *barbarae*. They found that the serpentine taxa were nonmonophyletic evolving independently at least three times, suggesting that tolerance to serpentine may be gained or lost through relatively few genetic changes. In other case, [50] construct phylogeny for the *Pentaschis‐ tis* clade with 82 species in three genera. They investigated the association between leaf anatomy type and soil nutrient type on which species grow. Despite there is little phylo‐ genetic constrain in soil nutrient type. However, only orthophyllous-leaved species diver‐ sify on eutrophic soils. Nevertheless, modern phylogenies on a number of phanerogam genera occurring on New Caledonia (*Acianthus*, *Cupaniopsis*, *Guioa*, *Morinda*, and *Oxera*) have shown a shift in soil preference (from non serpentine to serpentine soils and vice versa). Thus [60] concluded that the ability to grown on serpentine soil is either a plesio‐ morphic or a very homoplasious character and therefore the hypothesis that serpentine soils preserve the indigenous flora in New Caledonia against competition with immi‐ grant species cannot be supported for these groups. Rarely are made specific soil stud‐ ies, the data are taken often of general charts. We suggest making detailed studies as in *Cephalocereus*, according to plant species and soil type [57]. Therefore, it might be possi‐ ble to infer the role of soil in the evolution of endemic plant species using the phyloge‐

genus (Figure 6) [58].

390 Soil Processes and Current Trends in Quality Assessment

= 40 μm, B = 50 μm.

netic analysis.

In the landscape, the vegetational differences often serve to delineate the geologic disconti‐ nuities of an area even to the casual observer. The remarkable differences often observed in plant cover for different soil types in adjacent areas, have naturally led to attempts to ex‐ plain these phenomena in terms of the physical or chemical properties of the soil, or of the physiological characteristics of the plants [61]. These areas should be priority sites for con‐ servation to preserve the unique interaction between soil and plant species as well as the mi‐ crobiota and fauna. For example, the halophytic and gypsophytic vegetation of the Ebro-Basin at Los Monegros [62] or flora of the Coastal Calcareous Hills of the Biosphere Reserve Baconao in Cuba [63] are excellent to demonstrate the varied adaptations of plant types and life-forms as strategies to survive on edapho-climatic harsh conditions of various kinds. In the Chihuahuan Desert region it was found that several Cactaceae species, particularly many members of the Cacteae tribe often inhabit extremely specialized habitats, such as gypsum and other unusual soil formations (Figure 7) [64]. The patches of edaphic endemism also frequently exist as refuges for native species in highly invaded ecosystems [20], ultra‐ mafic substrates act as sites in which *Pinus balfouriana* escapes of the competition [65]. More‐ over, the work with *Helianthus exilis* showed the need to protect specialized microhabitat found only within the large serpentine outcrops, the species cannot survived outside the narrow conditions proper of its habitat [66]. However, the scarcity of conclusive studies on role of soil to determine the prevalence of endemic plants hampers the efforts of public and private organizations to preserve such areas.

**Figure 7.** A) *Aztekium ritterii* grows in outcrops of steep slopes of crystalline grypsum; (B) *Turbinicarpus valdezianus* grows in calcarious rocks.

The population size can greatly vary among populations of the same specie generating mi‐ cro-endemic nature. The small populations of some species consist of adult individuals that may be 3 as in *Cephalocereus totolapensis* [54] or 4 as in *Guaiacum unijugum* [67]. These popula‐ tion sizes are not reported sometimes considerer no significant, but many regions show a unique assemblage of species or a higher level of species richness or other associated species which could serve to protect this ecosystem. In Australian alpine vegetation the analyses of the relationships between physiognomic variation and environment indicate that edaphic factors are more important than climatic factors in differentiating formations [18]. Thus, eda‐ phic discontinuities should be determining the size and population distribution and should be considered when proposing conservation areas.

and many endemic plants are found in patches of certain soil within a different soil matrix. Additionally as with the soil, vegetation usually changed abruptly at the contact zone.

The Role of Soil Properties in Plant Endemism – A Revision of Conservation Strategies

Edaphic endemic plants are highly vulnerable to extinction due to stochastic events, habitat degradation, climatic change, and invasion by weedy species. Finally, the protected areas may be unable to maintain regional species diversity and representativeness, especially if additional fragments are lost and fragmented landscapes are left unmanaged and they are

, Ma. del Carmen Gutiérrez-Castorena2

3 Botany Department-Biology Institute, National Autonomous University of Mexico, México

[1] Cotler AH. El Uso de la Información Edáfica en los Estudios Ambientales. Gaceta

[2] Jenny H. Factors of Soil Formation. A of System Quantitative Pedology, New York:

[3] Boul SW, Hole FD, McCraken RJ: Génesis y Clasificación de Suelos. México: Trillas;

[4] Brady NC, Weil RR. The Nature and Properties of Soils. New Jersey: Prentice-Hall;

[5] USDA-NRCS.Global Soil Regions 1:1 million scale. Soil Survey Division, World Soil

[6] Cajuste BL, Gutiérrez-Castorena MdelC. El Factor Relieve en la Distribución de Sue‐ los en México. In: Krasilnikov P, Jiménez NFJ, Reyna TT, García CNE. (eds.) Geogra‐

[7] Billings WD. The Environmental Complex in Relation to Plant Growth and Distribu‐

fía de Suelos de México. México: Las Prensas de Ciencias; 2011. p73-86.

tion. The Quarterly Review of Biology 1952;27(3) 251-265.

and Teresa Terrazas3

http://dx.doi.org/10.5772/53056

393

fragile to soil erosion or degradation by chemical contamination.

\*Address all correspondence to: marialuisabarcenas@gmail.com

1 Botany Program, Postgraduate College, Texcoco, , Mexico

2 Soils Program, Postgraduate College, Texcoco, Mexico

**Author details**

City, Mexico

**References**

María Luisa Bárcenas-Argüello1

Ecológica 2003;(68) 33-42.

Dover Press;1994.

Resources. 1998.

1998.

1999.

Frequently, endemic species are less widely distributed and are less well represented in pro‐ tected areas than other threatened species [68]. Baconao Biosphere Reserve presents high flo‐ ristic composition and which endemic species represent 21%; however they are restricted at limestone hills, parent material that cover only 6.6% of total area [63]; and the same applies when registering Asteraceae endemic species to the Mexican state of Oaxaca with 53.4% and many of them not found in any Biosphere Reserve [69]; in the same sense, 12 endemic plant species are restricted to serpentine soil in Puerto Rico, all rare, uncommon and very local‐ ized within their limited distribution, and only two have been placed on the United States Federal list as threatened or endangered [70], and most species endemic to the state of Nue‐ vo Leon (Northeast of Mexico) present in the submontane scrub have restricted distribution and specially cacti are not located within any protected area [71]. Therefore it is important to reconsider the extension of the sites identified as irreplaceable for various members of the flora endemic worldwide.

The effects of growing human populations on natural communities, on ecosystems, and on some endemic plant populations results in degraded state of sites due to human activity as roadway, tourist development, extraction of mineral as gravel, sand, and others. However, an understanding of the interrelations between soil or bedrock and occurrence of endemics, becomes even more important in the context of restoration ecology and the reversal of land degradation. In addition, the role of soil in the determination of endemic plants has not been sufficiently studied; thus, public and private organizations have not intensified their efforts to preserve such areas. Table 1 show some examples of the different soils supporting en‐ demic species and how through different techniques of study has been able to establish a close relationship between soil and plant. As can be seen, the soil-plant endemism is not ex‐ clusive to one type of soil or a plant family.

## **7. Conclusions**

Diverse studies have demonstrated that soil characteristics are correlated with differences in bedrock. Although incipient and timid studies intend to respond to which extent do soil characteristics correlate with vegetation patterns, this is a question that should not be forgot‐ ten in plant endemic studies.

We cannot do random sampling hoping to find a relationship between habitat and popula‐ tion size or presence / absence of some species. Efforts should be directed to characterize the habitat or habitats where the species grows in order to determine whether the type of soil, rock, and bedrock are the most important factors for endemic species development. When the limiting factor is the substrate, and abrupt limit in abundance is expected, as well as in population parameters. Narrow endemism in plants is frequently related to soil specificity, and many endemic plants are found in patches of certain soil within a different soil matrix. Additionally as with the soil, vegetation usually changed abruptly at the contact zone.

Edaphic endemic plants are highly vulnerable to extinction due to stochastic events, habitat degradation, climatic change, and invasion by weedy species. Finally, the protected areas may be unable to maintain regional species diversity and representativeness, especially if additional fragments are lost and fragmented landscapes are left unmanaged and they are fragile to soil erosion or degradation by chemical contamination.

## **Author details**

the relationships between physiognomic variation and environment indicate that edaphic factors are more important than climatic factors in differentiating formations [18]. Thus, eda‐ phic discontinuities should be determining the size and population distribution and should

Frequently, endemic species are less widely distributed and are less well represented in pro‐ tected areas than other threatened species [68]. Baconao Biosphere Reserve presents high flo‐ ristic composition and which endemic species represent 21%; however they are restricted at limestone hills, parent material that cover only 6.6% of total area [63]; and the same applies when registering Asteraceae endemic species to the Mexican state of Oaxaca with 53.4% and many of them not found in any Biosphere Reserve [69]; in the same sense, 12 endemic plant species are restricted to serpentine soil in Puerto Rico, all rare, uncommon and very local‐ ized within their limited distribution, and only two have been placed on the United States Federal list as threatened or endangered [70], and most species endemic to the state of Nue‐ vo Leon (Northeast of Mexico) present in the submontane scrub have restricted distribution and specially cacti are not located within any protected area [71]. Therefore it is important to reconsider the extension of the sites identified as irreplaceable for various members of the

The effects of growing human populations on natural communities, on ecosystems, and on some endemic plant populations results in degraded state of sites due to human activity as roadway, tourist development, extraction of mineral as gravel, sand, and others. However, an understanding of the interrelations between soil or bedrock and occurrence of endemics, becomes even more important in the context of restoration ecology and the reversal of land degradation. In addition, the role of soil in the determination of endemic plants has not been sufficiently studied; thus, public and private organizations have not intensified their efforts to preserve such areas. Table 1 show some examples of the different soils supporting en‐ demic species and how through different techniques of study has been able to establish a close relationship between soil and plant. As can be seen, the soil-plant endemism is not ex‐

Diverse studies have demonstrated that soil characteristics are correlated with differences in bedrock. Although incipient and timid studies intend to respond to which extent do soil characteristics correlate with vegetation patterns, this is a question that should not be forgot‐

We cannot do random sampling hoping to find a relationship between habitat and popula‐ tion size or presence / absence of some species. Efforts should be directed to characterize the habitat or habitats where the species grows in order to determine whether the type of soil, rock, and bedrock are the most important factors for endemic species development. When the limiting factor is the substrate, and abrupt limit in abundance is expected, as well as in population parameters. Narrow endemism in plants is frequently related to soil specificity,

be considered when proposing conservation areas.

392 Soil Processes and Current Trends in Quality Assessment

flora endemic worldwide.

**7. Conclusions**

ten in plant endemic studies.

clusive to one type of soil or a plant family.

María Luisa Bárcenas-Argüello1 , Ma. del Carmen Gutiérrez-Castorena2 and Teresa Terrazas3

\*Address all correspondence to: marialuisabarcenas@gmail.com


3 Botany Department-Biology Institute, National Autonomous University of Mexico, México City, Mexico

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**Chapter 15**

in 2007 (Ministry of the Environ‐

**Leachability and Vegetable Absorption of**

Mieko Yachigo and Shinjiro Sato

http://dx.doi.org/10.5772/55123

**1. Introduction**

Additional information is available at the end of the chapter

by approximately 20% since 1994, reaching to 172 million m3

**Heavy Metals from Sewage Sludge Biochar**

Management of industrial wastes has been one of the most challenging problems in most urban municipalities due to increasing waste volume with limited disposal areas, and energy-consum‐ ing and high-cost treatment processes for disposal. In Japan, although the total volume of the industrial waste generated has been relatively constant since 1990 being approximately 400 milliontonsperyear,remainingcapacityofthefinaldisposalfortheindustrialwastehasdecreased

ment Government of Japan, 2011). This disposal capacity is predicted to be filled in the average of 8.5yearsforJapan,and3.6yearsforTokyoMetropolitanareas.Duemainlytohighcostoftreatment for proper disposal, illegal dumping of the industrial waste has been a new problem despite of severe regulation and monitoring of affected areas. The number and total volume of unsolved cases of illegal dumping and improper disposal of the waste were 2,610 and 1.78 million tons,

Sewage sludge is one of the most produced industrial wastes and comprised approximately 20% (77.2 million tons) of the total volume of industrial wastes generated across Japan in 2010 (Ministry of the Environment Government of Japan, 2011). However, the volume of its final disposal was insignificant (0.37 million tons) since the majority of sewage sludge were reclaimed and/or treated by intermediate processing such as thickening, dewatering, anaero‐ bic digestion, composting, incineration, carbonization, and melting. On the dry solid basis, 78% of the total volume of sewage sludge (1.72 million tons) was recycled in beneficial applications mainly for construction materials (1.39 million tons), energy production (0.02 million tons), and application in agriculture and horticulture as alternative fertilizers and/or soil conditioner (0.31 million tons). Due to new technology and diversification of sewage sludge recycling techniques, further recycling avenues are expected particularly for produc‐

> © 2013 Yachigo and Sato; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 Yachigo and Sato; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

respectively, in 2010 (Ministry of the Environment Government of Japan, 2011).

tion of energy, agriculture, forestry, livestock, and fisheries in the future.


## **Chapter 15**

## **Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar**

Mieko Yachigo and Shinjiro Sato

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55123

## **1. Introduction**

[65] Eckert AJ. Influence of Substrate Type and Microsite Availability on the Persistence of Foxtail Pine (*Pinus balfouriana*, Pinaceae) in the Klamath Mountains, California.

[66] Wolf A. Conservation of Endemic Plants in Serpentine Landscapes. Biological Con‐

[67] McCauley RA, Cortés-Palomec AC, Oyama K. Distribution, Genetic Structure, and Conservation Status of the Rare Microendemic Species, *Guaiacum unijugum* (Zygo‐ phyllaceae) in the Cape Region of Baja California, Mexico. Revista Mexicana de Bio‐

[68] Aguirre GJ, Duivenvoorden JF. Can we Expect to Protect Threatened Species in Pro‐ tected Areas? A Case Study of the Genus *Pinus* in Mexico. Revista Mexicana de Bio‐

[69] Suárez-Mota ME, Villaseñor JL. Las Compuestas Endémicas de Oaxaca, México: Di‐ versidad y Distribución. Boletín de la Sociedad Botánica de México. 2011;88(1) 55-66.

[70] Cedeño-Maldonado JA, Breckon GJ. Serpentine Endemism in the Flora of Puerto

[71] Estrada-Castillón E, Villareal-Quintanilla JA, Jurado-Ybarra E, Cantú-Ayala C, Gar‐ cía-Aranda MA, Sánchez-Salas J, Jiménez-Pérez J, Pando-Moreno M. Clasificación, Estructura y Diversidad del Matorral Sub-Montano Adyacente a la Planicie Costera

del Golfo Norte en el Noreste de México. Botanical Sciences 2012;90(1) 37-52.

[72] Babbel GR, Selander RK. Genetic Variability in Edaphically Restricted and Wide‐

[73] Rajakaruna N, Bohm BA. The Edaphic Factor and Patterns of Variation in *Lasthenia californica* (Asteraceae). American Journal of Botany. 1999;86(11) 1576-1596.

[74] Rajakaruna N, Baldwin BG, Chan R, Desrochers AM, Bohm BA, Whitton J. Edaphic Races and Phylogenetic Taxa in the *Lasthenia californica* Complex (Asteraceae: Helian‐ theae): an Hypothesis of Parallel Evolution. Molecular Ecology 2003;12(6) 1675-1679

[75] Ribeiro KT, Fernandes GW. Patterns of Abundance of a Narrow Endemic Species in a Tropical and Infertile Montane Habitat. Plant Ecology 2000;147(2) 205-218.

[76] McCarten NF. Rare and Endemic Plants of Lake County Serpentine Soils Habitats.

[77] Nyssen J, Vermeersch D. Slope Aspects Affects Geomorphic Dynamics of Coal Min‐

[78] Garvie LAJ. Decay of Cacti and Carbon Cycling. Naturwissenschaften 2006;93(3)

American Journal of Botany 2006;93(11) 1615-1624.

Rico. Caribbean Journal of Science 1996;32(4) 348-356.

spread Plant Species. Evolution 1974;28(4) 619-630.

Sacramento:Report for the Endangered Plant Project; 1988.

114-118.

ing Soil Heaps in Belgium. Geomorphology 2010;123(1-2) 109-121.

servation. 2001;100(1) 35-44

398 Soil Processes and Current Trends in Quality Assessment

diversidad 2010;81(3) 745-758.

diversidad. 2010;81(3) 875-882.

Management of industrial wastes has been one of the most challenging problems in most urban municipalities due to increasing waste volume with limited disposal areas, and energy-consum‐ ing and high-cost treatment processes for disposal. In Japan, although the total volume of the industrial waste generated has been relatively constant since 1990 being approximately 400 milliontonsperyear,remainingcapacityofthefinaldisposalfortheindustrialwastehasdecreased by approximately 20% since 1994, reaching to 172 million m3 in 2007 (Ministry of the Environ‐ ment Government of Japan, 2011). This disposal capacity is predicted to be filled in the average of 8.5yearsforJapan,and3.6yearsforTokyoMetropolitanareas.Duemainlytohighcostoftreatment for proper disposal, illegal dumping of the industrial waste has been a new problem despite of severe regulation and monitoring of affected areas. The number and total volume of unsolved cases of illegal dumping and improper disposal of the waste were 2,610 and 1.78 million tons, respectively, in 2010 (Ministry of the Environment Government of Japan, 2011).

Sewage sludge is one of the most produced industrial wastes and comprised approximately 20% (77.2 million tons) of the total volume of industrial wastes generated across Japan in 2010 (Ministry of the Environment Government of Japan, 2011). However, the volume of its final disposal was insignificant (0.37 million tons) since the majority of sewage sludge were reclaimed and/or treated by intermediate processing such as thickening, dewatering, anaero‐ bic digestion, composting, incineration, carbonization, and melting. On the dry solid basis, 78% of the total volume of sewage sludge (1.72 million tons) was recycled in beneficial applications mainly for construction materials (1.39 million tons), energy production (0.02 million tons), and application in agriculture and horticulture as alternative fertilizers and/or soil conditioner (0.31 million tons). Due to new technology and diversification of sewage sludge recycling techniques, further recycling avenues are expected particularly for produc‐ tion of energy, agriculture, forestry, livestock, and fisheries in the future.

© 2013 Yachigo and Sato; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Yachigo and Sato; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pyrolysis process of sewage sludge is one of the useful approaches for sewage sludge recycling, and its product called sewage sludge biochar (SSB) can be used as supplemental or alternative material to coal for thermal power plants. Some municipalities in Japan have initiated to use SSB as a part of thermal sources for energy production. In Tokyo, since 2007, 8,700 tons of SSB have been produced per year from 99,000 tons of sewage sludge and mixed with coal for energy plant to contribute approximately 1% of the total calorific value by the plant. The SSB produced is pyrolysed at 500o C for one hour and generates 2,000 kcal kg–1 of heat calorie, which is equivalent to one third of that of coal. Aichi Prefecture started a similar project as Tokyo in 2012, and uses 2,700 tons of SSB pyrolysed at 500o C from 33,000 tons of sewage sludge every year to generate 4.6 million kWh of energy, which is capable of providing electricity equivalent to annual usages of typical 1,270 households in the area.

**2. Materials and methods**

SSB-H) and low (approximately 300o

A soil used in this study was a forest Andisol collected from a mulberry plantation forest in Hachioji, Tokyo, Japan (35.692, 139.316) on Apr. 29, 2011. Surface 15-cm soils were sampled,

Sewage sludge biochars were produced in commercial plants at high (approximately 800o

through 2-mm sieve for chemical analyses, and homogenized between 2 and 5 mm in size for

A pot study was performed on campus of Soka University, Tokyo, Japan. Japanese mustard spinach (*Brassica rapa*) and common bean (*Phaseolus vulgaris*) were planted in 1.3 L planting pots in which the soil and SSB were mixed at different rates. Experimental design was a completely randomized block design with triplicate, two different plants, and four different application rates for each SSB: 0%, 25%, 50%, and 75% (v/v) for SSB-H, and 0%, 5%, 15%, and 25% (v/v) for SSB-L. Each pot received respective amounts of ammonium nitrate, superphos‐ phate, and potassium chloride before planting based on fertilizer application rates recom‐ mended for the plants by Tokyo Prefecture: 140-52-100 kg N-P-K ha–1 for spinach and 80-39-66 kg N-P-K ha–1 for bean. Each plant was sowed on Sep. 23, 2011, and thinned to 4 spinach individuals and 2 bean individuals per pot 2 weeks later. The plants were grown outside and the pots were covered by plastic sheet only when rainfall events occurred. Water was applied periodically to keep 50% of water holding capacity of the soil-SSB mix. Spinach and bean were harvested 46 and 66 days after sowing on Nov. 8 and Nov. 28, 2011, respectively. Throughout

the experiment, the daily mean temperature fluctuated between 16o

rainfall more than 20 mm occurred 5 times on Oct. 5, 15, 22, Nov, 11, and 19, 2011.

C overnight.

Dry weight (DW) of each part of the plants was determined after drying at 70o

After harvest, the plants were divided into shoot, root, and bean sheath (only for common

The soil, SSBs, and plant tissues after the pot study were ground to pass through 150 μm in size and digested for total elemental analyses for heavy metals. Two grams of the soil sample

a hotplate until it turned like a syrup. Then, 30 mL of a perchloric acid-nitric acid mixture (1:4) was added and heated for further 10 min. After cooling, 25 mL of hydrochloric acid (1:5) was

C overnight. The soil and SSB in the pot were separated using 2-mm

C with 30 mL of concentrated nitric acid in a beaker with a glass watch on

C for 1 hr. After cooling, the content was quantitatively transferred

C overnight, sieved through 2 mm, and used for physicochemical analyses and a

C; SSB-L) temperatures, crashed by hummer, and sieved

Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar

C and 22o

C, and the daily

C overnight.

C;

401

http://dx.doi.org/10.5772/55123

**2.1. Soil and biochar**

dried at 45o

the pot study.

**2.2. Pot study**

bean), and dried at 70o

was heated to 180o

added and heated to 130o

sieve, and separately dried at 45o

**2.3. Soil, biochar, plant analyses**

pot study.

Sewagesludgebiocharcanbealsousedforsupplementaloralternativefertilizermaterialforcrop production.Differentfeedstockpropertiesandpyrolysis temperatures,however,yieldSSBwith differentphysicochemicalproperties,thusexhibitdifferentdegreesofeffectwhenSSBisapplied to soil as soil amendments or fertilizers for improving crop production or soil properties (Chan & Xu,2009).AnSSBproducedat800o CinJapancontainedmoderateamountsofmacronutrientsfor crop production, among which P was relatively high (78.9 g P kg–1), and relatively high amounts of heavy metals (1,100 mg Cu kg–1, 1,630 mg Zn kg–1, 4 mg Cd kg–1, 126 mg Ni kg–1) which were within the limits legislated in Japan (Kawano et al., 2012). Effects of SSB application to soils up to 40% by weight on the growth of Begonia (*Begonia semperflorens*) differed depending on the soil type and were more positively pronounced in soils more infertile such as sand-dune and yellow soils.Awastewatersludgebiocharpyrolysedat550o CinAustraliawasappliedatarateof10tha–1 and improved the production of cherry tomatoes (*Lycopersicon esculentum*) by 64% above the control without SSB application (Hossain et al., 2010). Some heavy metals such as Cd, Cu, and Zn were taken up more by the plant with SSB application (0.04, 6.2, and 22 mg kg–1, respectively) compared with the control, however the plant uptake of all 16 metals and trace elements meas‐ ured in the SSB itself were below the Australian maximum permitted concentrations for food products.AsSSBpyrolysistemperatureincreasedfrom300o Cto700o C,whilebiocharyield,fixed C, volatile matter, total N, and inorganic N contents decreased, pH values, ash, total P, available (Colwell) P, Ca, Mg, Fe, S, Zn, Cd contents increased (Hossain et al., 2011). The total concentra‐ tion of heavy metals such as Cd, Cr, Ni, and Pb tends to be enriched in the SSB, but the bioavaila‐ bility (e.g., DTPA-extractable) of many of these trace elements appears to be reduced during pyrolysis process, compared with those in the raw feedstock (Hossain et al., 2011; Méndez et al., 2012). The variability of the micronutrient concentrations in biochar is due to its volatility and pyrolysis temperature effects on both composition and chemical structure of the biochar (Chan and Xu, 2009).

AgronomiceffectsofSSBapplicationtosoils,therefore,widelyvarydependingonphysicochem‐ ical properties of the sewage sludge, pyrolysis temperature, and soil properties, which need to be furtherclarifiedincludingtheheavymetaldynamicsinorderfortheSSBapplicationtobebroadly accepted in agricultural and horticultural practices. Therefore, objectives of this study were to 1) investigate the leachability of heavy metals, particularly Cu, Zn, and Cd from SSB as applied to soil with varying rates, and 2) evaluate the effect of SSB application to soil on plants' growth and absorption of the heavy metals, using SSB produced at two different pyrolysis temperatures.

## **2. Materials and methods**

## **2.1. Soil and biochar**

Pyrolysis process of sewage sludge is one of the useful approaches for sewage sludge recycling, and its product called sewage sludge biochar (SSB) can be used as supplemental or alternative material to coal for thermal power plants. Some municipalities in Japan have initiated to use SSB as a part of thermal sources for energy production. In Tokyo, since 2007, 8,700 tons of SSB have been produced per year from 99,000 tons of sewage sludge and mixed with coal for energy plant to contribute approximately 1% of the total calorific value by the plant. The SSB produced

equivalent to one third of that of coal. Aichi Prefecture started a similar project as Tokyo in

year to generate 4.6 million kWh of energy, which is capable of providing electricity equivalent

Sewagesludgebiocharcanbealsousedforsupplementaloralternativefertilizermaterialforcrop production.Differentfeedstockpropertiesandpyrolysistemperatures,however,yieldSSBwith differentphysicochemicalproperties,thusexhibitdifferentdegreesofeffectwhenSSBisapplied to soil as soil amendments or fertilizers for improving crop production or soil properties (Chan &

crop production, among which P was relatively high (78.9 g P kg–1), and relatively high amounts of heavy metals (1,100 mg Cu kg–1, 1,630 mg Zn kg–1, 4 mg Cd kg–1, 126 mg Ni kg–1) which were within the limits legislated in Japan (Kawano et al., 2012). Effects of SSB application to soils up to 40% by weight on the growth of Begonia (*Begonia semperflorens*) differed depending on the soil type and were more positively pronounced in soils more infertile such as sand-dune and yellow

and improved the production of cherry tomatoes (*Lycopersicon esculentum*) by 64% above the control without SSB application (Hossain et al., 2010). Some heavy metals such as Cd, Cu, and Zn were taken up more by the plant with SSB application (0.04, 6.2, and 22 mg kg–1, respectively) compared with the control, however the plant uptake of all 16 metals and trace elements meas‐ ured in the SSB itself were below the Australian maximum permitted concentrations for food

C, volatile matter, total N, and inorganic N contents decreased, pH values, ash, total P, available (Colwell) P, Ca, Mg, Fe, S, Zn, Cd contents increased (Hossain et al., 2011). The total concentra‐ tion of heavy metals such as Cd, Cr, Ni, and Pb tends to be enriched in the SSB, but the bioavaila‐ bility (e.g., DTPA-extractable) of many of these trace elements appears to be reduced during pyrolysis process, compared with those in the raw feedstock (Hossain et al., 2011; Méndez et al., 2012). The variability of the micronutrient concentrations in biochar is due to its volatility and pyrolysis temperature effects on both composition and chemical structure of the biochar (Chan

AgronomiceffectsofSSBapplicationtosoils,therefore,widelyvarydependingonphysicochem‐ ical properties of the sewage sludge, pyrolysis temperature, and soil properties, which need to be furtherclarifiedincludingtheheavymetaldynamicsinorderfortheSSBapplicationtobebroadly accepted in agricultural and horticultural practices. Therefore, objectives of this study were to 1) investigate the leachability of heavy metals, particularly Cu, Zn, and Cd from SSB as applied to soil with varying rates, and 2) evaluate the effect of SSB application to soil on plants' growth and absorption of the heavy metals, using SSB produced at two different pyrolysis temperatures.

C for one hour and generates 2,000 kcal kg–1 of heat calorie, which is

CinJapancontainedmoderateamountsofmacronutrientsfor

Cto700o

C from 33,000 tons of sewage sludge every

CinAustraliawasappliedatarateof10tha–1

C,whilebiocharyield,fixed

is pyrolysed at 500o

and Xu, 2009).

Xu,2009).AnSSBproducedat800o

2012, and uses 2,700 tons of SSB pyrolysed at 500o

400 Soil Processes and Current Trends in Quality Assessment

soils.Awastewatersludgebiocharpyrolysedat550o

products.AsSSBpyrolysistemperatureincreasedfrom300o

to annual usages of typical 1,270 households in the area.

A soil used in this study was a forest Andisol collected from a mulberry plantation forest in Hachioji, Tokyo, Japan (35.692, 139.316) on Apr. 29, 2011. Surface 15-cm soils were sampled, dried at 45o C overnight, sieved through 2 mm, and used for physicochemical analyses and a pot study.

Sewage sludge biochars were produced in commercial plants at high (approximately 800o C; SSB-H) and low (approximately 300o C; SSB-L) temperatures, crashed by hummer, and sieved through 2-mm sieve for chemical analyses, and homogenized between 2 and 5 mm in size for the pot study.

## **2.2. Pot study**

A pot study was performed on campus of Soka University, Tokyo, Japan. Japanese mustard spinach (*Brassica rapa*) and common bean (*Phaseolus vulgaris*) were planted in 1.3 L planting pots in which the soil and SSB were mixed at different rates. Experimental design was a completely randomized block design with triplicate, two different plants, and four different application rates for each SSB: 0%, 25%, 50%, and 75% (v/v) for SSB-H, and 0%, 5%, 15%, and 25% (v/v) for SSB-L. Each pot received respective amounts of ammonium nitrate, superphos‐ phate, and potassium chloride before planting based on fertilizer application rates recom‐ mended for the plants by Tokyo Prefecture: 140-52-100 kg N-P-K ha–1 for spinach and 80-39-66 kg N-P-K ha–1 for bean. Each plant was sowed on Sep. 23, 2011, and thinned to 4 spinach individuals and 2 bean individuals per pot 2 weeks later. The plants were grown outside and the pots were covered by plastic sheet only when rainfall events occurred. Water was applied periodically to keep 50% of water holding capacity of the soil-SSB mix. Spinach and bean were harvested 46 and 66 days after sowing on Nov. 8 and Nov. 28, 2011, respectively. Throughout the experiment, the daily mean temperature fluctuated between 16o C and 22o C, and the daily rainfall more than 20 mm occurred 5 times on Oct. 5, 15, 22, Nov, 11, and 19, 2011.

After harvest, the plants were divided into shoot, root, and bean sheath (only for common bean), and dried at 70o C overnight. The soil and SSB in the pot were separated using 2-mm sieve, and separately dried at 45o C overnight.

## **2.3. Soil, biochar, plant analyses**

Dry weight (DW) of each part of the plants was determined after drying at 70o C overnight.

The soil, SSBs, and plant tissues after the pot study were ground to pass through 150 μm in size and digested for total elemental analyses for heavy metals. Two grams of the soil sample was heated to 180o C with 30 mL of concentrated nitric acid in a beaker with a glass watch on a hotplate until it turned like a syrup. Then, 30 mL of a perchloric acid-nitric acid mixture (1:4) was added and heated for further 10 min. After cooling, 25 mL of hydrochloric acid (1:5) was added and heated to 130o C for 1 hr. After cooling, the content was quantitatively transferred to a 100 mL flask and filtered through a Whatman No 1 paper (Committee for Analytical Methods for Soil Environment, 1997). Two grams of the SSB sample in crucible was ashed in an electric furnace at 550o C for 8 hr, and the ash was transferred to a beaker with 5 mL of concentrated nitric acid and heated to 120o C on a hotplate for 3 hr. After cooling, 25 mL of hydrochloric acid (1:5) was added and heated at 120o C for further 60 min. After cooling, the content was quantitatively transferred to a 25 mL flask and filtered through a Whatman No 1 paper (Japan Soil Association, 2010). One gram of each of the shoot, root, and sheath plant samples was heated to 140o C with 10 mL of concentrated nitric acid in a beaker with a glass watch on a hotplate until the content is reduced to approximately 1 mL. After cooling, the content was quantitatively transferred to 25 mL flask using 1% nitric acid, and filtered through a Whatman No 1 paper (Committee for Experimental Methods for Plant Nutrition, 1990).

SSB-H were 5 and 1.7 times greater than those in the SSB-L, respectively. The dilute acid (0.1 *M* HCl)-extractable heavy metals varied among elements and SSB types; Cu concentra‐ tion in SSB-H was 34 times greater than that in SSB-L, while Cd in SSB-H was 5 times less

> **Total Zn**

**g kg–1 mg kg–1**

Andisol 7.1 45.1 2.8 25 43 4.9 0.23 2.06 0.024

SSB-H§ 6.6 532.2 34.1 346 455 1.4 19.2 14.1 0.022

SSB-L§ 5.6 528.4 54.8 69 273 1.5 0.56 27.3 0.12

Seeds of both plants with all treatments germinated 100% except for those of the bean with

The average DW of individual spinach shoot showed a significant decrease with increasing application rates of both SSB-H and SSB-L, respectively (Fig. 1a). The highest DW was 1.1 and 1.4 g with SSB-H 0% and SSB-L 5% treatment, respectively. The average DW of the bean shoot did not show significant differences among the SSB-H treatment, but a significant increase

The average DW of the spinach root showed a significant decrease with increasing application rate of SSB-H, but not among the SSB-L treatment except for SSB-L 25% showing the lowest DW among the treatment (Fig. 1b). The average DW of the bean root showed a significant decrease with the increasing SSB-H application rate, and a significant increase only with SSB-

The average DW of the bean sheath was not significantly affected by the SSB application, ranging from 0.04 and 0.18 g among the SSB-H treatment, and 0.04 g for SSB-L 5% rate (the

**Total Cd**

Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar

**HCl Cu‡** **HCl Zn‡**

http://dx.doi.org/10.5772/55123

**HCl Cd‡** 403

than that in SSB-L.

‡ 0.1 *M* HCl-extractable

**3.2 Dry weight of the plants**

L 15% among the SSB-L treatment.

**pH†**

**Total C**

† 1:2.5 soil:solution with H2O and 1:100 SSB:solution with hot H2O

SSB-L 25% treatment being 66% of the germination rate.

with SSB-L 5% and 15% compared with that of control.

bean did not bear the sheath with SSB-L 15% and 25% rates; Table 2).

§ SSB pyrolysed at high temperature (800oC; SSB-H) and low temperature (300oC; SSB-L)

**Table 1.** Basic properties of the soil (Andisol) and sewage sludge biochar (SSB) used in this study.

**Total N**

**Total Cu**

The heavy metals extractable by 0.1 *M* HCl are used as an indicator for environmental pollution in soils. Various chemical extractants are used for single extraction evaluation of heavy metals and may broadly be divided into 3 main classes: (i) weak replacement of ion salts (MgCl2, CaCl2, NH4CO3), (ii) dilute solutions of either weak acid (acetic acid) or strong acids (HCl, HNO3), and (iii) chelating agents (DTPA, EDPA) (Kashem et al. 2007). The unbuffered ion salt solutions are simple procedure to extract bioavailable metals, while a use of 0.1 *M* HCl solution may reflect bioavailability of metals (CSTPA, 1980). Dried 7.0 g of soil samples (< 2 mm) was weighed to a 50 mL centrifuge tube and 35 mL of 0.1 *M* HCl was added. The tube was shaken horizontally for 1 hr at 160 stroke min–1, centrifuged at 5,000 rpm, and the supernatant was filtered through a Whatman No 1 paper (Committee for Analytical Methods for Soil Environ‐ ment, 1997).

Prior to analytical determination, all filtrates were further filtered through a 0.45 μm mem‐ brane. Total and extractable concentrations of Cu, Zn, and Cd in the soil, SSBs, and plant samples were determined using ICP (ICPS-7000 ver. 2.1, Shimazu).

#### **2.4. Statistical analyses**

Significant differences of the total and extractable concentrations of Cu, Zn, and Cd in the soil, SSBs, and plant samples among different SSB application rates for each SSB type and each plant were tested by ANOVA using STATISTICA 6.1 (StatSoft Inc., Tulsa, Okalahoma, USA). Unless otherwise stated, the differences were significant at *p ≤* 0.05 level.

## **3. Results**

### **3.1. Soil and biochar properties**

The Andisol used in this study had an almost neutral pH (H2O) of 7.1 and relatively average TC and TN concentrations (Table 1). The total heavy metal concentrations in the soil were 25 mg Cu kg–1, 43 mg Zn kg–1, and 4.9 mg Cd kg–1. The SSB used in this study had slightly acidic pH levels of 6.6 (SSB-H) and 5.6 (SSB-L). The total Cd concentrations were similar for both SSB types ranging 1.4–1.5 mg kg–1, while the total Cu and Zn concentrations in the SSB-H were 5 and 1.7 times greater than those in the SSB-L, respectively. The dilute acid (0.1 *M* HCl)-extractable heavy metals varied among elements and SSB types; Cu concentra‐ tion in SSB-H was 34 times greater than that in SSB-L, while Cd in SSB-H was 5 times less than that in SSB-L.


† 1:2.5 soil:solution with H2O and 1:100 SSB:solution with hot H2O

‡ 0.1 *M* HCl-extractable

to a 100 mL flask and filtered through a Whatman No 1 paper (Committee for Analytical Methods for Soil Environment, 1997). Two grams of the SSB sample in crucible was ashed in

content was quantitatively transferred to a 25 mL flask and filtered through a Whatman No 1 paper (Japan Soil Association, 2010). One gram of each of the shoot, root, and sheath plant

watch on a hotplate until the content is reduced to approximately 1 mL. After cooling, the content was quantitatively transferred to 25 mL flask using 1% nitric acid, and filtered through a Whatman No 1 paper (Committee for Experimental Methods for Plant Nutrition, 1990).

The heavy metals extractable by 0.1 *M* HCl are used as an indicator for environmental pollution in soils. Various chemical extractants are used for single extraction evaluation of heavy metals and may broadly be divided into 3 main classes: (i) weak replacement of ion salts (MgCl2, CaCl2, NH4CO3), (ii) dilute solutions of either weak acid (acetic acid) or strong acids (HCl, HNO3), and (iii) chelating agents (DTPA, EDPA) (Kashem et al. 2007). The unbuffered ion salt solutions are simple procedure to extract bioavailable metals, while a use of 0.1 *M* HCl solution may reflect bioavailability of metals (CSTPA, 1980). Dried 7.0 g of soil samples (< 2 mm) was weighed to a 50 mL centrifuge tube and 35 mL of 0.1 *M* HCl was added. The tube was shaken horizontally for 1 hr at 160 stroke min–1, centrifuged at 5,000 rpm, and the supernatant was filtered through a Whatman No 1 paper (Committee for Analytical Methods for Soil Environ‐

Prior to analytical determination, all filtrates were further filtered through a 0.45 μm mem‐ brane. Total and extractable concentrations of Cu, Zn, and Cd in the soil, SSBs, and plant

Significant differences of the total and extractable concentrations of Cu, Zn, and Cd in the soil, SSBs, and plant samples among different SSB application rates for each SSB type and each plant were tested by ANOVA using STATISTICA 6.1 (StatSoft Inc., Tulsa, Okalahoma, USA).

The Andisol used in this study had an almost neutral pH (H2O) of 7.1 and relatively average TC and TN concentrations (Table 1). The total heavy metal concentrations in the soil were 25 mg Cu kg–1, 43 mg Zn kg–1, and 4.9 mg Cd kg–1. The SSB used in this study had slightly acidic pH levels of 6.6 (SSB-H) and 5.6 (SSB-L). The total Cd concentrations were similar for both SSB types ranging 1.4–1.5 mg kg–1, while the total Cu and Zn concentrations in the

samples were determined using ICP (ICPS-7000 ver. 2.1, Shimazu).

Unless otherwise stated, the differences were significant at *p ≤* 0.05 level.

C for 8 hr, and the ash was transferred to a beaker with 5 mL of

C with 10 mL of concentrated nitric acid in a beaker with a glass

C on a hotplate for 3 hr. After cooling, 25 mL of

C for further 60 min. After cooling, the

an electric furnace at 550o

samples was heated to 140o

ment, 1997).

**3. Results**

**2.4. Statistical analyses**

**3.1. Soil and biochar properties**

concentrated nitric acid and heated to 120o

402 Soil Processes and Current Trends in Quality Assessment

hydrochloric acid (1:5) was added and heated at 120o

§ SSB pyrolysed at high temperature (800oC; SSB-H) and low temperature (300oC; SSB-L)

**Table 1.** Basic properties of the soil (Andisol) and sewage sludge biochar (SSB) used in this study.

## **3.2 Dry weight of the plants**

Seeds of both plants with all treatments germinated 100% except for those of the bean with SSB-L 25% treatment being 66% of the germination rate.

The average DW of individual spinach shoot showed a significant decrease with increasing application rates of both SSB-H and SSB-L, respectively (Fig. 1a). The highest DW was 1.1 and 1.4 g with SSB-H 0% and SSB-L 5% treatment, respectively. The average DW of the bean shoot did not show significant differences among the SSB-H treatment, but a significant increase with SSB-L 5% and 15% compared with that of control.

The average DW of the spinach root showed a significant decrease with increasing application rate of SSB-H, but not among the SSB-L treatment except for SSB-L 25% showing the lowest DW among the treatment (Fig. 1b). The average DW of the bean root showed a significant decrease with the increasing SSB-H application rate, and a significant increase only with SSB-L 15% among the SSB-L treatment.

The average DW of the bean sheath was not significantly affected by the SSB application, ranging from 0.04 and 0.18 g among the SSB-H treatment, and 0.04 g for SSB-L 5% rate (the bean did not bear the sheath with SSB-L 15% and 25% rates; Table 2).

in the spinach root was greatest with SSB-H 25% rate and SSB-L 15% rate among SSB-H and SSB-L treatments, respectively, while Cu in the bean root was greatest with SSB-H 50% rate and SSB-L 25%, among each SSB treatment, respectively (Fig. 2d). The Zn concentrations in the spinach root with SSB-H 25% and 50% rates were significantly greater than that of the control, and although not significant, those with the SSB-L treatment were greater than that of the control (Fig. 2e). The bean root absorbed significantly greater Zn with SSB-H 50% and 75% rates, and not significantly but greater Zn with SSB-L 15% and 25% rates, compared with that of the control, respectively. Cadmium in the spinach root was significantly higher with SSB-H 75% rate than those at other rates, but Cd with the SSB-L treatment was lower than that of the control (Fig. 2f). The Cu in the bean root was significantly lower with SSB-H 75% rate and

Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar

ab a ab ab A A

a

a <sup>a</sup> <sup>a</sup>

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

b b ab A A A A

b

A B AB

SSB-L 0

SSB-L 5

SSB-L 15

C

SSB-L 25

Spinach Bean

SSB-H 0

SSB-H 25

SSB-H 50

a b a

c

SSB-H 75

A

SSB-L 0

SSB-L 5

<sup>B</sup> <sup>C</sup> <sup>C</sup>

SSB-L 15

SSB-L 25

(f)

B

A a a a a <sup>A</sup> AB AC C

<sup>a</sup> <sup>a</sup>

b b

A A

http://dx.doi.org/10.5772/55123

405

<sup>A</sup> <sup>A</sup>

with SSB-H 50% rate and SSB-L 25%, among each SSB treatment, respectively (Fig. 2d). The Zn concentrations in the spinach root with SSB-H 25% and 50% rates were significantly greater than that of the control, and although not significant, those with the SSB-L treatment were greater than that of the control (Fig. 2e). The bean root absorbed significantly greater Zn with SSB-H 50% and 75% rates, and not significantly but greater Zn with SSB-L 15% and 25% rates, compared with that of the control, respectively. Cadmium in the spinach root was significantly higher with SSB-H 75% rate than those at other rates, but Cd with the SSB-L treatment was lower than that of the control (Fig. 2f). The Cu in the bean root was significantly lower with SSB-H 75% rate and higher with SSB-L 0% rate compared to other

Any of Cu, Zn, and Cd in the sheath was not significantly affected by either application of SSB-H or SSB-L compared with that of the control, respectively, except for Cd with SSB-H 25% and

Fig. 2. Total concentrations of (a) Cu, (b) Zn, and (c) Cd in the shoot and (d) Cu, (e) Zn, and (f) Cd in the root of Japanese mustard spinach and common bean with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application

**Figure 2.** Total concentrations of (a) Cu, (b) Zn, and (c) Cd in the shoot and (d) Cu, (e) Zn, and (f) Cd in the root of Japanese mustard spinach and common bean with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05)

Any of Cu, Zn, and Cd in the sheath was not significantly affected by either application of SSB-H or SSB-L compared with that of the control, respectively, except for Cd with SSB-H

25% and 50% rates being significantly higher than that of the control (Table 2).

higher with SSB-L 0% rate compared to other rates, respectively.

A <sup>A</sup> <sup>A</sup> <sup>A</sup>

<sup>C</sup> <sup>C</sup> <sup>a</sup> <sup>a</sup> <sup>a</sup> <sup>a</sup>

a a a A <sup>B</sup> AB A

<sup>b</sup> bc c

A

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

<sup>B</sup> <sup>B</sup> <sup>B</sup>

<sup>B</sup> <sup>B</sup> <sup>a</sup>

7

rates, respectively.

a

a <sup>b</sup> <sup>b</sup> b A A

a b b b

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

0.0 0.5 1.0 1.5 2.0

Cd (mg kg-1)

(c)

Cu (mg kg-1)

Zn (mg kg-1)

(b)

(a)

b b

b

A

A

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

Spinach Bean

<sup>B</sup> <sup>B</sup> <sup>B</sup>

a

SSB-H 0

among different application rates for each plant and SSB, respectively.

SSB-H 25

SSB-H 50

SSB-H 75

50% rates being significantly higher than that of the control (Table 2).

B

rates for each plant and SSB, respectively.

bean with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respectively. **Figure 1.** Dry weight of (a) shoot and (b) root parts of Japanese mustard spinach and common bean with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respec‐ tively.

Fig. 1. Dry weight of (a) shoot and (b) root parts of Japanese mustard spinach and common

The average DW of the bean sheath was not significantly affected by the SSB application,


**3.3 Total heavy metal concentrations in the plants**  Copper concentrations in the spinach shoot significantly increased with both SSB treatments over the control, respectively, while, although not significantly different, those in the bean **Table 2.** Dry weight (DW), Cu, Zn, and Cd concentrations in the common bean sheath with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each SSB and plant, respectively. na denotes non-applicable.

#### shoot were lower with both SSB treatments compared with the control, respectively (Fig. 2a). Zinc concentrations in the spinach shoot significantly increased with both SSB **3.3. Total heavy metal concentrations in the plants**

only with SSB-L 15% among the SSB-L treatment.

treatments over the control, respectively, except for SSB-L 5% rate (Fig. 2b). On the other hand, Zn in the bean shoot was not significantly affected by both SSB treatments, except for SSB-L 5% being significantly lower than the control. The shoot part of each of both plants contained significantly greater Cd concentrations with both SSB treatments over the control, respectively (Fig. 2c). The concentrations in the plant root were generally greater than those in the shoot for all heavy metals investigated regardless of the plant, SSB type and application rate. The Cu concentration in the spinach root was greatest with SSB-H 25% rate and SSB-L 15% rate among SSB-H and SSB-L treatments, respectively, while Cu in the bean root was greatest Copper concentrations in the spinach shoot significantly increased with both SSB treatments over the control, respectively, while, although not significantly different, those in the bean shoot were lower with both SSB treatments compared with the control, respectively (Fig. 2a). Zinc concentra‐ tions in the spinach shoot significantly increased with both SSB treatments over the control, respectively, except for SSB-L 5% rate (Fig. 2b). On the other hand, Zn in the bean shoot was not significantly affected by both SSB treatments, except for SSB-L 5% being significantly lower than the control. The shoot part of each of both plants contained significantly greater Cd concentra‐ tions with both SSB treatments over the control, respectively (Fig. 2c).

6 The concentrations in the plant root were generally greater than those in the shoot for all heavy metals investigated regardless of the plant, SSB type and application rate. The Cu concentration in the spinach root was greatest with SSB-H 25% rate and SSB-L 15% rate among SSB-H and SSB-L treatments, respectively, while Cu in the bean root was greatest with SSB-H 50% rate and SSB-L 25%, among each SSB treatment, respectively (Fig. 2d). The Zn concentrations in the spinach root with SSB-H 25% and 50% rates were significantly greater than that of the control, and although not significant, those with the SSB-L treatment were greater than that of the control (Fig. 2e). The bean root absorbed significantly greater Zn with SSB-H 50% and 75% rates, and not significantly but greater Zn with SSB-L 15% and 25% rates, compared with that of the control, respectively. Cadmium in the spinach root was significantly higher with SSB-H 75% rate than those at other rates, but Cd with the SSB-L treatment was lower than that of the control (Fig. 2f). The Cu in the bean root was significantly lower with SSB-H 75% rate and higher with SSB-L 0% rate compared to other rates, respectively.

(f) Cd in the root of Japanese mustard spinach and common bean with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respectively. **Figure 2.** Total concentrations of (a) Cu, (b) Zn, and (c) Cd in the shoot and (d) Cu, (e) Zn, and (f) Cd in the root of Japanese mustard spinach and common bean with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respectively.

with SSB-H 50% rate and SSB-L 25%, among each SSB treatment, respectively (Fig. 2d). The Zn concentrations in the spinach root with SSB-H 25% and 50% rates were significantly greater than that of the control, and although not significant, those with the SSB-L treatment were greater than that of the control (Fig. 2e). The bean root absorbed significantly greater Any of Cu, Zn, and Cd in the sheath was not significantly affected by either application of SSB-H or SSB-L compared with that of the control, respectively, except for Cd with SSB-H 25% and 50% rates being significantly higher than that of the control (Table 2).

Zn with SSB-H 50% and 75% rates, and not significantly but greater Zn with SSB-L 15% and 25% rates, compared with that of the control, respectively. Cadmium in the spinach root was significantly higher with SSB-H 75% rate than those at other rates, but Cd with the SSB-L treatment was lower than that of the control (Fig. 2f). The Cu in the bean root was significantly lower with SSB-H 75% rate and higher with SSB-L 0% rate compared to other

Any of Cu, Zn, and Cd in the sheath was not significantly affected by either application of SSB-H or SSB-L compared with that of the control, respectively, except for Cd with SSB-H

25% and 50% rates being significantly higher than that of the control (Table 2).

7

rates, respectively.

6

non-applicable.

respectively (Fig. 2c).

only with SSB-L 15% among the SSB-L treatment.

a b c d

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

Spinach Bean

SSB-H 0

SSB-H 25

SSB-H 50

0.0 0.5 1.0 1.5 2.0

Dry weight (g)

tively.

(a)

B A

404 Soil Processes and Current Trends in Quality Assessment

C D <sup>a</sup> <sup>a</sup> <sup>a</sup>

a A

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

B B AB

**3.3 Total heavy metal concentrations in the plants** 

tions with both SSB treatments over the control, respectively (Fig. 2c).

**3.3. Total heavy metal concentrations in the plants**

bean did not bear the sheath with SSB-L 15% and 25% rates; Table 2).

among different application rates for each plant and SSB, respectively.

The average DW of the bean sheath was not significantly affected by the SSB application, ranging from 0.04 and 0.18 g among the SSB-H treatment, and 0.04 g for SSB-L 5% rate (the

Fig. 1. Dry weight of (a) shoot and (b) root parts of Japanese mustard spinach and common bean with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05)

**Figure 1.** Dry weight of (a) shoot and (b) root parts of Japanese mustard spinach and common bean with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respec‐

DW Cu Zn Cd DW Cu Zn Cd

SSB-H 0 0.07ab 5.37a 3.04a 0.58a SSB-L 0 0.07A 5.37A 3.04A 0.58A SSB-H 25 0.17ab 1.99a 2.62a 0.28b SSB-L 5 0.04A 10.75A 5.00A 1.46A SSB-H 50 0.18a 1.87a 3.36a 0.26b SSB-L 15 na na na na SSB-H 75 0.04b 5.77a 3.43a 0.73a SSB-L 25 na na na na Table 2. Dry weight (DW), Cu, Zn, and Cd concentrations in the common bean sheath with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each SSB and plant, respectively. na denotes non-applicable.

SSB-H 0 0.07ab 5.37a 3.04a 0.58a SSB-L 0 0.07A 5.37A 3.04A 0.58A

SSB-H 25 0.17ab 1.99a 2.62a 0.28b SSB-L 5 0.04A 10.75A 5.00A 1.46A

SSB-H 50 0.18a 1.87a 3.36a 0.26b SSB-L 15 na na na na

SSB-H 75 0.04b 5.77a 3.43a 0.73a SSB-L 25 na na na na

**Table 2.** Dry weight (DW), Cu, Zn, and Cd concentrations in the common bean sheath with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each SSB and plant, respectively. na denotes

Copper concentrations in the spinach shoot significantly increased with both SSB treatments over the control, respectively, while, although not significantly different, those in the bean shoot were lower with both SSB treatments compared with the control, respectively (Fig. 2a). Zinc concentrations in the spinach shoot significantly increased with both SSB treatments over the control, respectively, except for SSB-L 5% rate (Fig. 2b). On the other hand, Zn in the bean shoot was not significantly affected by both SSB treatments, except for SSB-L 5% being significantly lower than the control. The shoot part of each of both plants contained significantly greater Cd concentrations with both SSB treatments over the control,

The concentrations in the plant root were generally greater than those in the shoot for all heavy metals investigated regardless of the plant, SSB type and application rate. The Cu concentration in the spinach root was greatest with SSB-H 25% rate and SSB-L 15% rate among SSB-H and SSB-L treatments, respectively, while Cu in the bean root was greatest

The concentrations in the plant root were generally greater than those in the shoot for all heavy metals investigated regardless of the plant, SSB type and application rate. The Cu concentration

Copper concentrations in the spinach shoot significantly increased with both SSB treatments over the control, respectively, while, although not significantly different, those in the bean shoot were lower with both SSB treatments compared with the control, respectively (Fig. 2a). Zinc concentra‐ tions in the spinach shoot significantly increased with both SSB treatments over the control, respectively, except for SSB-L 5% rate (Fig. 2b). On the other hand, Zn in the bean shoot was not significantly affected by both SSB treatments, except for SSB-L 5% being significantly lower than the control. The shoot part of each of both plants contained significantly greater Cd concentra‐

g ––––– mg kg-1 ––––– g ––––– mg kg-1 –––––

**g mg kg-1 g mg kg-1**

**DW Cu Zn Cd DW Cu Zn Cd**

a a b c

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

0.0 0.2 0.4 0.6 0.8 1.0

(b)

<sup>A</sup> <sup>A</sup> A

B

SSB-L 25

Spinach Bean

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

a ab b b <sup>A</sup> AB

B AB

**3.4 Total heavy metal concentrations in** 

higher total Zn among the SSB-H treatment (Fig. 3b). The total Cd in the biochar did not significantly change among each of the SSB rates and plants, respectively, except for that with SSB-H 75% rate with the spinach being significantly greater than other SSB-H rates (Fig. 3c).

The total Cu concentration in the soil after the pot study was significantly increased only with SSB-H 75% rate among the SSB-H treatment for both plants, respectively, but not affected among the SSB-L treatment for both plants (Fig. 4a). The total Zn in the soil tended to increase with the SSB-H application rate and was significantly greater with SSB-H 75% rate compared with those with other rates (Fig. 4b). The total Zn in the soil was not significantly different among the SSB-L treatments for both plants. The total Cd in the soil was significantly increased with SSB-H 25% and 50% rates for the spinach, but not affected for the bean (Fig. 4c). The total Cd in the soil with the SSB-L treatment significantly increased compared with that of the

HCl-Zn with SSB-H 75% rate for spinach and SSB-H 50% and 75% rates for bean were significantly greater than those with other rates, respectively (Fig. 4e). The HCl-Zn with SSB-L 15% and 25% were significantly greater than those with other rates for spinach, and that with SSB-L 25% rate was significantly greater than those with other rates for bean. The HCl-Cd with SSB-H 75% rate was significantly higher than those with other rates for spinach, and that with SSB-H 25% rate was significantly lower than those with other rates for bean (Fig. 4f). The HCl-Cd with SSB-L 5% was greater compared with those with other rates for spinach, but there were no significant differences among the SSB-L treatment for

extractable (d) Cu, (e) Zn, and (f) Cd in the soil after the pot study with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates

**Figure 4.** Total concentrations of (a) Cu, (b) Zn, and (c) Cd and dilute acid (0.1*M* HCl)- extractable (d) Cu, (e) Zn, and (f) Cd in the soil after the pot study with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different

Fig. 4. Total concentrations of (a) Cu, (b) Zn, and (c) Cd and dilute acid (0.1*M* HCl)-

It appears that the Andisol used in this study contained less but comparable total Cu and Zn,

**3.5. Total heavy metal concentrations in the soil after the pot study**

control for both plants, respectively.

b

c

<sup>a</sup> <sup>b</sup> <sup>b</sup> <sup>a</sup> <sup>A</sup> <sup>B</sup> BC <sup>C</sup>

<sup>A</sup> <sup>A</sup> <sup>A</sup> <sup>A</sup> <sup>a</sup> <sup>a</sup>

<sup>A</sup> <sup>A</sup> <sup>A</sup> <sup>A</sup> <sup>a</sup> <sup>a</sup> <sup>a</sup>

a

b

b

<sup>a</sup> <sup>a</sup> <sup>a</sup> <sup>a</sup> <sup>A</sup>

A A A A

<sup>a</sup> <sup>a</sup> <sup>a</sup>

<sup>a</sup> ab ab

0.0 0.5 1.0 1.5 2.0 (d)

(f)

0 0.01 0.02 0.03 0.04

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

Spinach Bean

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

b

Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar

c

<sup>a</sup> <sup>a</sup> <sup>a</sup> <sup>b</sup> <sup>B</sup> <sup>C</sup>

<sup>A</sup> <sup>A</sup> <sup>A</sup> B a <sup>a</sup>

<sup>A</sup> <sup>A</sup> <sup>B</sup> <sup>B</sup> <sup>a</sup> <sup>a</sup>

b

b

<sup>A</sup> AB <sup>b</sup> <sup>a</sup> <sup>b</sup> <sup>b</sup> <sup>A</sup> <sup>A</sup> <sup>A</sup> <sup>A</sup>

c

c

http://dx.doi.org/10.5772/55123

407

<sup>A</sup> <sup>A</sup> <sup>B</sup> <sup>A</sup>

<sup>A</sup> AB AB <sup>B</sup>

A A A A

B B B

<sup>a</sup> <sup>a</sup> <sup>a</sup>

<sup>a</sup> <sup>b</sup> <sup>b</sup>

0.0 0.5 1.0 1.5 2.0

Cd (mg kg-1)

(c)

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

Spinach Bean

SSB-H 0

SSB-H 25

SSB-H 50

SSB-H 75

SSB-L 0

SSB-L 5

SSB-L 15

SSB-L 25

Cu (mg kg-1)

Zn (mg kg-1)

(b)

(a)

The total Cu concentration in the SSB-H after the pot study was not significantly affected for the spinach but significantly increased with SSB-H 75% rate for the bean (Fig. 3a). The total Cu concentrations in the SSB-L were not significantly affected by the application rate for both plants. After harvest of the spinach the total Zn in the biochar was not affected by the application rate for both SSBs, but at the bean's harvest only SSB-H 75% rate caused significantly higher total Zn among the SSB-H treatment (Fig. 3b). The total Cd in the biochar did not significantly change among each of the SSB rates and plants, respectively, except for that with SSB-H 75% rate with the spinach being significantly greater than

**the biochar after the pot study** 

other SSB-H rates (Fig. 3c).

**the soil after the pot study** 

**3.5 Total heavy metal concentrations in** 

The total Cu concentration in the soil after the pot study was significantly increased only with SSB-H 75% rate among the

The total Zn in the soil tended to increase with the SSB-H application rate and was

9

bean.

**4. Results and Discussion** 

for each plant and SSB, respectively.

application rates for each plant and SSB, respectively.

**4.1 Heavy metal concentrations in the soil and biochar** 

SSB-H treatment for both plants, respectively, but not affected among the SSB-L treatment for both plants (Fig. 4a). and (c) Cd in the biochar after the pot study with different application rates of sewage **Figure 3.** Total concentrations of (a) Cu, (b) Zn, and (c) Cd in the biochar after the pot study with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote signif‐ icant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respectively.

Fig. 3. Total concentrations of (a) Cu, (b) Zn,

sludge biochar at high (SSB-H) and low

#### (SSB-L) temperatures. Different letters **3.4. Total heavy metal concentrations in the biochar after the pot study**

8

significantly greater with SSB-H 75% rate compared with those with other rates (Fig. 4b). The total Zn in the soil was not significantly different among the SSB-L treatments for both plants. The total Cd in the soil was significantly increased with SSB-H 25% and 50% rates for the spinach, but not affected (*p*<0.05) among different application rates for each plant and SSB, respectively. The total Cu concentration in the SSB-H after the pot study was not significantly affected for the spinach but significantly increased with SSB-H 75% rate for the bean (Fig. 3a). The total Cu concentrations in the SSB-L were not significantly affected by the application rate for both plants. After harvest of the spinach the total Zn in the biochar was not affected by the appli‐ cation rate for both SSBs, but at the bean's harvest only SSB-H 75% rate caused significantly

denote significant differences by Fisher test

for the bean (Fig. 4c). The total Cd in the soil with the SSB-L treatment significantly

The dilute acid (0.1 *M* HCl)-extractable Cu in the soil after the pot study tended to increase with increasing SSB-H application rate for both plants, respectively, and those with 75% for spinach, and 50% and 75% for bean were significantly greater than those with lower rates, respectively (Fig. 4d). The HCl-Cu with SSB-L 25% for spinach and SSB-L 15% for bean was significantly lower and higher, respectively, than those with other rates. Similarly, the

**3.6 Acid-extractable heavy metal concentrations in the soil after the pot study** 

increased compared with that of the control for both plants, respectively.

**3.4 Total heavy metal concentrations in**  higher total Zn among the SSB-H treatment (Fig. 3b). The total Cd in the biochar did not significantly change among each of the SSB rates and plants, respectively, except for that with SSB-H 75% rate with the spinach being significantly greater than other SSB-H rates (Fig. 3c).

#### The total Cu concentration in the SSB-H after the pot study was not significantly **3.5. Total heavy metal concentrations in the soil after the pot study**

**the biochar after the pot study** 

the bean's harvest only SSB-H 75% rate

compared with those with other rates (Fig.

9

bean.

**4. Results and Discussion** 

**4.1 Heavy metal concentrations in the soil and biochar** 

other SSB-H rates (Fig. 3c).

**the soil after the pot study** 

for the bean (Fig. 4c). The total Cd in the soil with the SSB-L treatment significantly

The dilute acid (0.1 *M* HCl)-extractable Cu in the soil after the pot study tended to increase with increasing SSB-H application rate for both plants, respectively, and those with 75% for spinach, and 50% and 75% for bean were significantly greater than those with lower rates, respectively (Fig. 4d). The HCl-Cu with SSB-L 25% for spinach and SSB-L 15% for bean was significantly lower and higher, respectively, than those with other rates. Similarly, the

**3.6 Acid-extractable heavy metal concentrations in the soil after the pot study** 

increased compared with that of the control for both plants, respectively.

Fig. 3. Total concentrations of (a) Cu, (b) Zn, and (c) Cd in the biochar after the pot study with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates

**Figure 3.** Total concentrations of (a) Cu, (b) Zn, and (c) Cd in the biochar after the pot study with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote signif‐ icant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respectively.

The total Cu concentration in the SSB-H after the pot study was not significantly affected for the spinach but significantly increased with SSB-H 75% rate for the bean (Fig. 3a). The total Cu concentrations in the SSB-L were not significantly affected by the application rate for both plants. After harvest of the spinach the total Zn in the biochar was not affected by the appli‐ cation rate for both SSBs, but at the bean's harvest only SSB-H 75% rate caused significantly

SSB-L 15

SSB-L 25

Spinach Bean

SSB-H 25

<sup>a</sup> <sup>a</sup> <sup>a</sup>

<sup>a</sup> <sup>a</sup> <sup>a</sup>

<sup>a</sup> ab <sup>b</sup>

SSB-H 50

SSB-H 75

**3.4. Total heavy metal concentrations in the biochar after the pot study**

SSB-L 5

0

0

0.00

SSB-H 25

0.01

0.02

Cd (mg kg-1)

(c)

0.03

0.04

10

20

Zn (mg kg-1)

(b)

30

40

50

10

20

Cu (mg kg-1)

(a)

406 Soil Processes and Current Trends in Quality Assessment

30

40

50

A A A

<sup>A</sup> <sup>A</sup> <sup>A</sup>

<sup>A</sup> <sup>A</sup> <sup>A</sup>

<sup>a</sup> ab <sup>b</sup>

<sup>a</sup> ab <sup>b</sup>

a a a

SSB-H 50

SSB-H 75

SSB-L 5

SSB-L 15

SSB-L 25

A A A

A A

A A A

A

for each plant and SSB, respectively.

affected for the spinach but significantly increased with SSB-H 75% rate for the bean (Fig. 3a). The total Cu concentrations in the SSB-L were not significantly affected by the application rate for both plants. After harvest of the spinach the total Zn in the biochar was not affected by the application rate for both SSBs, but at The total Cu concentration in the soil after the pot study was significantly increased only with SSB-H 75% rate among the SSB-H treatment for both plants, respectively, but not affected among the SSB-L treatment for both plants (Fig. 4a). The total Zn in the soil tended to increase with the SSB-H application rate and was significantly greater with SSB-H 75% rate compared with those with other rates (Fig. 4b). The total Zn in the soil was not significantly different among the SSB-L treatments for both plants. The total Cd in the soil was significantly increased with SSB-H 25% and 50% rates for the spinach, but not affected for the bean (Fig. 4c). The total Cd in the soil with the SSB-L treatment significantly increased compared with that of the control for both plants, respectively.

4b). The total Zn in the soil was not significantly different among the SSB-L treatments for both plants. The total Cd in the soil was significantly increased with SSB-H 25% and 50% rates for the spinach, but not affected extractable (d) Cu, (e) Zn, and (f) Cd in the soil after the pot study with different application rates of sewage sludge biochar at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respectively. **Figure 4.** Total concentrations of (a) Cu, (b) Zn, and (c) Cd and dilute acid (0.1*M* HCl)- extractable (d) Cu, (e) Zn, and (f) Cd in the soil after the pot study with different application rates of sewage sludge biochar pyrolysed at high (SSB-H) and low (SSB-L) temperatures. Different letters denote significant differences by Fisher test (*p*<0.05) among different application rates for each plant and SSB, respectively.

Fig. 4. Total concentrations of (a) Cu, (b) Zn, and (c) Cd and dilute acid (0.1*M* HCl)-

HCl-Zn with SSB-H 75% rate for spinach and SSB-H 50% and 75% rates for bean were significantly greater than those with other rates, respectively (Fig. 4e). The HCl-Zn with SSB-L 15% and 25% were significantly greater than those with other rates for spinach, and that with SSB-L 25% rate was significantly greater than those with other rates for bean. The HCl-Cd with SSB-H 75% rate was significantly higher than those with other rates for spinach, and that with SSB-H 25% rate was significantly lower than those with other rates for bean (Fig. 4f). The HCl-Cd with SSB-L 5% was greater compared with those with other rates for spinach, but there were no significant differences among the SSB-L treatment for

It appears that the Andisol used in this study contained less but comparable total Cu and Zn,

8

## **3.6. Acid-extractable heavy metal concentrations in the soil after the pot study**

The dilute acid (0.1 *M* HCl)-extractable Cu in the soil after the pot study tended to increase with increasing SSB-H application rate for both plants, respectively, and those with 75% for spinach, and 50% and 75% for bean were significantly greater than those with lower rates, respectively (Fig. 4d). The HCl-Cu with SSB-L 25% for spinach and SSB-L 15% for bean was significantly lower and higher, respectively, than those with other rates. Similarly, the HCl-Zn with SSB-H 75% rate for spinach and SSB-H 50% and 75% rates for bean were significantly greater than those with other rates, respectively (Fig. 4e). The HCl-Zn with SSB-L 15% and 25% were significantly greater than those with other rates for spinach, and that with SSB-L 25% rate was significantly greater than those with other rates for bean. The HCl-Cd with SSB-H 75% rate was significantly higher than those with other rates for spinach, and that with SSB-H 25% rate was significantly lower than those with other rates for bean (Fig. 4f). The HCl-Cd with SSB-L 5% was greater compared with those with other rates for spinach, but there were no significant differences among the SSB-L treatment for bean.

**4.2. Effect of biochar on plant growth**

pyrolysed at 700o

further investigations.

**4.3. Plant absorption of heavy metal**

The DW of spinach shoot and root and bean root significantly decreased with increasing rates of SSB-H due mainly to a lack of soil volume in the pot. In fact, chlorosis due to N deficiency was observed with the SSB-H treatment and more pronounced on leaves of both plants with higher SSB-H rates. Although the plant was fertilized, it appeared that the lack of soil volume in the pot with high SSB-H rates limited plant root growth thus the plant growth. In addition, although the concentration was not measured in soil or plants, both plants with the SSB-L treatment showed a leave curvature due possibly to excess uptake of boron. When SSB-L was applied, however, the shoot of both plants grew more than that of the control and their DW peaked at 5% rate for spinach and 15% for bean. A similar trend was observed when a begonia

Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar

(*Begonia semperflorens*) was grown on an Andisol mixed with SSB pyrolysed at 800o

in the DW peak with 25% rate (v/v) (Kawano et al., 2012). In Kawano's study, the effect of SSB application on the plant growth was most pronounced on a most infertile sandy Entisol (Typic Udipsamments) among tested soils with the highest SSB application. It appears that the positive effect of SSB application to soil on the plant may be more prominent in soils which are less favorable for plant growth (Kawano et al., 2012). On the other hand, when an SSB

30%, 50%, and 100% (v/v) application rates to grow common bean, the DW at harvest with each treatment was in the order of control<100%<30%<50% (2.2, 3.6, 4.0 times of control, respectively), and rhizobia with all SSB treatments developed more than that with control (Teranuma & Mori, 2002). It appears that the optimum application rates of SSB for maximum plant growth widely depend on varying properties of soil, SSB, and plant, on which requires

Some heavy metals such as Zn, Cu, and Ni are important for proper functioning of biological systems and deficiency or excess can lead to a number of disorders (Ward, 1995), while others such as Cd are dangerous pollutant due to their high toxicity to plants and inhibit growth at high absorption (Liu et al., 2006). The range of heavy metal concentrations absorbed in both shoot and root of the plants was in the order of Cd<Cu<Zn, respectively, in this study. The same order of the concentration range of heavy metals was observed in various vegetable crops including similar plants used in this study as spinach (*Amaranthus caudatus*), lettuce (*Lactuca sative*), and peas (*Pisum sativum* L.) (Uwah et al, 2011; Singh et al., 2012). The concentrations of Cu, Zn, and Cd in healthy plants can range 4–15, 25–125, and 0.2–0.8 mg kg–1, respectively (National Institute for Agro-Environmental Sciences, 1977). The total Cu and Zn concentrations in the shoot of both plants in this study may have fallen in safe concentration ranges, respec‐ tively, whereas the total Cd may have been in excess absorption range which may have caused inhibition of the plant growth. In fact, the DW of the shoot of the plants showed inverse relationships with the total concentrations of the heavy metals absorbed in the plants' shoot, except for the bean DW under the SSB-L treatment with the total Cd (Table 3). The inhibitive effect of heavy metal absorption on the plant growth, judged by the slope of the correlation equation, was in the order of Zn<Cu<Cd for both plants and both SSB types, respectively.

C was applied to a volcanic ash soil from Kanto area, Japan with 0% (control),

C resulting

http://dx.doi.org/10.5772/55123

409

## **4. Results and discussion**

### **4.1. Heavy metal concentrations in the soil and biochar**

It appears that the Andisol used in this study contained less but comparable total Cu and Zn, and greater total Cd concentrations compared with the concentration range in similar soils of Japan. The concentrations of Andisols sampled in surrounded subareas of Tokyo ranged from 97 and 140 with the average of 118 mg Cu kg–1, from 121 and 175 with the average of 143 mg Zn kg–1, and from 0.37 and 0.55 with the average of 0.44 mg Cd kg–1 (Terashima et al., 2004). Some heavy metals including Cu, Zn, and Cd have tendency of being retained thus accumu‐ lated in soils given being relatively immobile after weathering in the soil (Terashima et al., 2004). High concentrations of some heavy metals including Cd, Hg, Pb, and Sb were observed because of possible artificial interventions especially in suburb areas of Tokyo (Terashima et al., 2007). In fact, the area where the Andisol used in this study was sampled was a former forest converted to a plantation area for mulberry (*Morus alba* L.) tree. The conversion and maintenance processes of the plantation could have involved some artificial interferences that might involve Cd deposition to the area.

Many properties including heavy metal concentrations in SSB widely vary primarily depend‐ ing on those contained in the feedstock (Chan & Xu, 2009). An SSB pyrolysed at 800o C from sewage sludge in Kyoto, Japan contained 1,100 mg Cu kg–1, 1,630 mg Zn kg–1, 4 mg Cd kg–1 (Kawano et al., 2012), while one pyrolysed at 500o C using sewage sludge in Madrid, Spain contained 222 mg Cu kg–1, 1,250 mg Zn kg–1, 1.79 mg Cd kg–1 (Paz-Ferreiro et al., 2012). It seems that both SSB used in this study fall in the low end of concentration ranges for Cu, Zn, and Cd in the SSB found in the literature. Particularly, Zn concentrations in both SSBs were excep‐ tionally lower than those in the literature. The heavy metal concentration in the SSB appears to accumulate as the pyrolysis temperature increases (Hossain et al., 2011), to which the SSB used in this study followed a similar pattern.

## **4.2. Effect of biochar on plant growth**

**3.6. Acid-extractable heavy metal concentrations in the soil after the pot study**

significant differences among the SSB-L treatment for bean.

**4.1. Heavy metal concentrations in the soil and biochar**

**4. Results and discussion**

408 Soil Processes and Current Trends in Quality Assessment

might involve Cd deposition to the area.

(Kawano et al., 2012), while one pyrolysed at 500o

used in this study followed a similar pattern.

The dilute acid (0.1 *M* HCl)-extractable Cu in the soil after the pot study tended to increase with increasing SSB-H application rate for both plants, respectively, and those with 75% for spinach, and 50% and 75% for bean were significantly greater than those with lower rates, respectively (Fig. 4d). The HCl-Cu with SSB-L 25% for spinach and SSB-L 15% for bean was significantly lower and higher, respectively, than those with other rates. Similarly, the HCl-Zn with SSB-H 75% rate for spinach and SSB-H 50% and 75% rates for bean were significantly greater than those with other rates, respectively (Fig. 4e). The HCl-Zn with SSB-L 15% and 25% were significantly greater than those with other rates for spinach, and that with SSB-L 25% rate was significantly greater than those with other rates for bean. The HCl-Cd with SSB-H 75% rate was significantly higher than those with other rates for spinach, and that with SSB-H 25% rate was significantly lower than those with other rates for bean (Fig. 4f). The HCl-Cd with SSB-L 5% was greater compared with those with other rates for spinach, but there were no

It appears that the Andisol used in this study contained less but comparable total Cu and Zn, and greater total Cd concentrations compared with the concentration range in similar soils of Japan. The concentrations of Andisols sampled in surrounded subareas of Tokyo ranged from 97 and 140 with the average of 118 mg Cu kg–1, from 121 and 175 with the average of 143 mg Zn kg–1, and from 0.37 and 0.55 with the average of 0.44 mg Cd kg–1 (Terashima et al., 2004). Some heavy metals including Cu, Zn, and Cd have tendency of being retained thus accumu‐ lated in soils given being relatively immobile after weathering in the soil (Terashima et al., 2004). High concentrations of some heavy metals including Cd, Hg, Pb, and Sb were observed because of possible artificial interventions especially in suburb areas of Tokyo (Terashima et al., 2007). In fact, the area where the Andisol used in this study was sampled was a former forest converted to a plantation area for mulberry (*Morus alba* L.) tree. The conversion and maintenance processes of the plantation could have involved some artificial interferences that

Many properties including heavy metal concentrations in SSB widely vary primarily depend‐ ing on those contained in the feedstock (Chan & Xu, 2009). An SSB pyrolysed at 800o

sewage sludge in Kyoto, Japan contained 1,100 mg Cu kg–1, 1,630 mg Zn kg–1, 4 mg Cd kg–1

contained 222 mg Cu kg–1, 1,250 mg Zn kg–1, 1.79 mg Cd kg–1 (Paz-Ferreiro et al., 2012). It seems that both SSB used in this study fall in the low end of concentration ranges for Cu, Zn, and Cd in the SSB found in the literature. Particularly, Zn concentrations in both SSBs were excep‐ tionally lower than those in the literature. The heavy metal concentration in the SSB appears to accumulate as the pyrolysis temperature increases (Hossain et al., 2011), to which the SSB

C from

C using sewage sludge in Madrid, Spain

The DW of spinach shoot and root and bean root significantly decreased with increasing rates of SSB-H due mainly to a lack of soil volume in the pot. In fact, chlorosis due to N deficiency was observed with the SSB-H treatment and more pronounced on leaves of both plants with higher SSB-H rates. Although the plant was fertilized, it appeared that the lack of soil volume in the pot with high SSB-H rates limited plant root growth thus the plant growth. In addition, although the concentration was not measured in soil or plants, both plants with the SSB-L treatment showed a leave curvature due possibly to excess uptake of boron. When SSB-L was applied, however, the shoot of both plants grew more than that of the control and their DW peaked at 5% rate for spinach and 15% for bean. A similar trend was observed when a begonia (*Begonia semperflorens*) was grown on an Andisol mixed with SSB pyrolysed at 800o C resulting in the DW peak with 25% rate (v/v) (Kawano et al., 2012). In Kawano's study, the effect of SSB application on the plant growth was most pronounced on a most infertile sandy Entisol (Typic Udipsamments) among tested soils with the highest SSB application. It appears that the positive effect of SSB application to soil on the plant may be more prominent in soils which are less favorable for plant growth (Kawano et al., 2012). On the other hand, when an SSB pyrolysed at 700o C was applied to a volcanic ash soil from Kanto area, Japan with 0% (control), 30%, 50%, and 100% (v/v) application rates to grow common bean, the DW at harvest with each treatment was in the order of control<100%<30%<50% (2.2, 3.6, 4.0 times of control, respectively), and rhizobia with all SSB treatments developed more than that with control (Teranuma & Mori, 2002). It appears that the optimum application rates of SSB for maximum plant growth widely depend on varying properties of soil, SSB, and plant, on which requires further investigations.

#### **4.3. Plant absorption of heavy metal**

Some heavy metals such as Zn, Cu, and Ni are important for proper functioning of biological systems and deficiency or excess can lead to a number of disorders (Ward, 1995), while others such as Cd are dangerous pollutant due to their high toxicity to plants and inhibit growth at high absorption (Liu et al., 2006). The range of heavy metal concentrations absorbed in both shoot and root of the plants was in the order of Cd<Cu<Zn, respectively, in this study. The same order of the concentration range of heavy metals was observed in various vegetable crops including similar plants used in this study as spinach (*Amaranthus caudatus*), lettuce (*Lactuca sative*), and peas (*Pisum sativum* L.) (Uwah et al, 2011; Singh et al., 2012). The concentrations of Cu, Zn, and Cd in healthy plants can range 4–15, 25–125, and 0.2–0.8 mg kg–1, respectively (National Institute for Agro-Environmental Sciences, 1977). The total Cu and Zn concentrations in the shoot of both plants in this study may have fallen in safe concentration ranges, respec‐ tively, whereas the total Cd may have been in excess absorption range which may have caused inhibition of the plant growth. In fact, the DW of the shoot of the plants showed inverse relationships with the total concentrations of the heavy metals absorbed in the plants' shoot, except for the bean DW under the SSB-L treatment with the total Cd (Table 3). The inhibitive effect of heavy metal absorption on the plant growth, judged by the slope of the correlation equation, was in the order of Zn<Cu<Cd for both plants and both SSB types, respectively. Particularly, the correlation coefficient was highest for Cd among the heavy metals with the SSB-H treatment for the spinach (0.719). A similar trend was observed when heavy metals' effectiveness in producing oxidative damage on spinach, assessed by the manifestation of external visual toxicity effects, was in the order of Zn<Cu<Cd (Pandey et al., 2009). Interest‐ ingly, however, the bean shoot showed Cd tolerance to some degree with the SSB-L treatment in this study (its correlation coefficient was positive).

**4.4. Heavy metal leachability from biochar**

**4.5. Heavy metal accumulation and availability in soil**

The total concentrations of Cu, Zn, and Cd in SSB after the pot study did not greatly vary among the application rates within the same SSB type and plant, respectively, and 89–91%, 90–92%, and 98–99% of the original concentrations of Cu, Zn, and Cd were lost from the SSB during the pot study, respectively, regardless of the SSB type, application rate, and plant. While there are numerous studies that deal with sorption capacity of biochars for heavy metals when applied to soil in light of phytoremediation perspective (Cao et al., 2009; Namgay et al., 2010; Uchimiya et al., 2011), studies regarding on leachability (through desorption or dissolution) of heavy metals from biochar and their bioavailability are virtually nonexistent. The heavy metals, however, are known to be desorbed by naturally occurring organic acids such as citric, oxalic, acetic, and lactic acids (Nascimento, 2006; Marchi, 2009) and dissolved organic carbon (Antoniadis & Alloway, 2002) from soils amended with sewage sludge. Regardless of equili‐ brium amounts of Cd adsorbed in an Andisol, more than 80% of Cd was desorbed with one time extraction with citric acid if its concentration was more than 0.1 *M*, and more than 90% was recovered if five soil pore volumes of 0.1 *M* citric acid were continuously run through the Cd-contaminated soil (Abe et al., 2004). An increase of dissolved organic matter in soil by the application of digested dewatered sludge significantly reduced the sorption of Cu on both acidic sandy loam and calcareous clay loam (Zhou & Wong, 2001). The application of SSBs in this study may have increased dissolved organic carbon given the SSBs being acidic, which may have increased Cu desorption (extractability) thus also bioavailability. The heavy metals in the SSB used in this study may have leached due to functions of various interactions between soil and plant roots. Further studies are needed to clarify leachability and bioavailability of heavy metals from biochars whose feedstock contain significant amounts of heavy metals in order to elucidate heavy metal dynamics in soil upon the application of such biochars.

Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar

http://dx.doi.org/10.5772/55123

411

The total concentrations of Cu and Zn in the soil after the pot study, regardless of the treatment, were greater and almost equal compared with those of the original soil (25 and 43 mg kg–1, respectively), while both concentrations significantly increased when SSB-H was applied at 75% rate for both plants. On the other hand, the overall concentrations of the total Cd were smaller after the pot study compared with that at pre-plant condition (4.9 mg kg–1); however those with the SSB-L treatment increased with increasing application rate for both plants. Transfer factors (TF) of heavy metal, as calculated as the ratio of the concentration of heavy metal absorbed in a plant to the concentration of heavy metal in soil, can quantify the relative differences in bioavailability of heavy metals to vegetables or to identify the efficiency of a vegetable species to accumulate a heavy metal (Uwah et al., 2011). The TF values in this study, calculated based on the sum of heavy metal concentrations of the shoot and root as the plant concentration and the sum of heavy metal concentrations in the soil and SSB as the soil concentration, were 0.77, 0.83, and 1.37 for spinach, and 0.69, 0.91, and 1.19 for bean for Cu, Zn, and Cd, respectively, regardless of the SSB application rate. These TF values may explain why Cu appeared to have been accumulated in the soil (TF less than 1 and lowest among the 3 metals for both plants), Zn seemed to have had no changes before and after the pot study


**Table 3.** Correlation equations and coefficients between the dry weight (DW) and total concentrations of Cu, Zn, and Cd absorbed in the shoot of the plants.

Overall concentrations of Cu, Zn, and Cd in the root were greater than those in the shoot of the plants probably because of root's chelating exudates to solubilize heavy metals in the rhizosphere (Inaba & Takenaka, 2000) and rapid absorption by the root and slow translocation to shoot (Nada et al., 2007). Accumulation of heavy metals (Cu, Zn, and Cd) in spinach after exposure to 500 μ*M* supply of the metals was greater in the root than in the leaf and stem at the end of experiment (Pandey et al., 2009). The rate of heavy metal absorption by the plant can be affected by many factors of both soil and plant such as soil pH, plant age, plant species, and nature of soil and climate (Alloway & Ayres, 1997; Uwah, 2009). Although not verified, the application of acidic SSBs used in this study may have decreased the neutral pH of the soil during the pot study, which may have enhanced plant absorption of especially Zn and Cd, as commonly observed irrespective of the vegetable crops and soil types (Kuo et al., 1985; Xue & Harrison, 1991). The effects of SSB application on soil properties, heavy metal bioavailability, and plant growth, therefore, need to be further evaluated for the range of different soils and plants, and over time because heavy metal bioavailability may increase over time in soil (Schauer et al., 1980).

## **4.4. Heavy metal leachability from biochar**

Particularly, the correlation coefficient was highest for Cd among the heavy metals with the SSB-H treatment for the spinach (0.719). A similar trend was observed when heavy metals' effectiveness in producing oxidative damage on spinach, assessed by the manifestation of external visual toxicity effects, was in the order of Zn<Cu<Cd (Pandey et al., 2009). Interest‐ ingly, however, the bean shoot showed Cd tolerance to some degree with the SSB-L treatment

**Plant/SSB Equations Coefficients** Spinach/SSB-H (DW) = –0.00097\*(Cu) + 0.0048 0.651

Spinach/SSB-L (DW) = –0.00076\*(Cu) + 0.0052 0.281

Bean/SSB-H (DW) = –0.00008\*(Cu) + 0.0019 0.007

Bean/SSB-L (DW) = –0.00053\*(Cu) + 0.0041 0.082

**Table 3.** Correlation equations and coefficients between the dry weight (DW) and total concentrations of Cu, Zn, and

Overall concentrations of Cu, Zn, and Cd in the root were greater than those in the shoot of the plants probably because of root's chelating exudates to solubilize heavy metals in the rhizosphere (Inaba & Takenaka, 2000) and rapid absorption by the root and slow translocation to shoot (Nada et al., 2007). Accumulation of heavy metals (Cu, Zn, and Cd) in spinach after exposure to 500 μ*M* supply of the metals was greater in the root than in the leaf and stem at the end of experiment (Pandey et al., 2009). The rate of heavy metal absorption by the plant can be affected by many factors of both soil and plant such as soil pH, plant age, plant species, and nature of soil and climate (Alloway & Ayres, 1997; Uwah, 2009). Although not verified, the application of acidic SSBs used in this study may have decreased the neutral pH of the soil during the pot study, which may have enhanced plant absorption of especially Zn and Cd, as commonly observed irrespective of the vegetable crops and soil types (Kuo et al., 1985; Xue & Harrison, 1991). The effects of SSB application on soil properties, heavy metal bioavailability, and plant growth, therefore, need to be further evaluated for the range of different soils and plants, and over time because heavy metal bioavailability may increase over time in soil

(DW) = –0.00015\*(Zn) + 0.0074 0.345 (DW) = –0.00443\*(Cd) + 0.0066 0.719

(DW) = –0.00008\*(Zn) + 0.0070 0.422 (DW) = –0.00129\*(Cd) + 0.0051 0.121

(DW) = –0.00001\*(Zn) + 0.0021 0.038 (DW) = –0.00022\*(Cd) + 0.0020 0.044

(DW) = –0.00006\*(Zn) + 0.0054 0.219 (DW) = 0.00151\*(Cd) + 0.0010 0.537

in this study (its correlation coefficient was positive).

410 Soil Processes and Current Trends in Quality Assessment

Cd absorbed in the shoot of the plants.

(Schauer et al., 1980).

The total concentrations of Cu, Zn, and Cd in SSB after the pot study did not greatly vary among the application rates within the same SSB type and plant, respectively, and 89–91%, 90–92%, and 98–99% of the original concentrations of Cu, Zn, and Cd were lost from the SSB during the pot study, respectively, regardless of the SSB type, application rate, and plant. While there are numerous studies that deal with sorption capacity of biochars for heavy metals when applied to soil in light of phytoremediation perspective (Cao et al., 2009; Namgay et al., 2010; Uchimiya et al., 2011), studies regarding on leachability (through desorption or dissolution) of heavy metals from biochar and their bioavailability are virtually nonexistent. The heavy metals, however, are known to be desorbed by naturally occurring organic acids such as citric, oxalic, acetic, and lactic acids (Nascimento, 2006; Marchi, 2009) and dissolved organic carbon (Antoniadis & Alloway, 2002) from soils amended with sewage sludge. Regardless of equili‐ brium amounts of Cd adsorbed in an Andisol, more than 80% of Cd was desorbed with one time extraction with citric acid if its concentration was more than 0.1 *M*, and more than 90% was recovered if five soil pore volumes of 0.1 *M* citric acid were continuously run through the Cd-contaminated soil (Abe et al., 2004). An increase of dissolved organic matter in soil by the application of digested dewatered sludge significantly reduced the sorption of Cu on both acidic sandy loam and calcareous clay loam (Zhou & Wong, 2001). The application of SSBs in this study may have increased dissolved organic carbon given the SSBs being acidic, which may have increased Cu desorption (extractability) thus also bioavailability. The heavy metals in the SSB used in this study may have leached due to functions of various interactions between soil and plant roots. Further studies are needed to clarify leachability and bioavailability of heavy metals from biochars whose feedstock contain significant amounts of heavy metals in order to elucidate heavy metal dynamics in soil upon the application of such biochars.

### **4.5. Heavy metal accumulation and availability in soil**

The total concentrations of Cu and Zn in the soil after the pot study, regardless of the treatment, were greater and almost equal compared with those of the original soil (25 and 43 mg kg–1, respectively), while both concentrations significantly increased when SSB-H was applied at 75% rate for both plants. On the other hand, the overall concentrations of the total Cd were smaller after the pot study compared with that at pre-plant condition (4.9 mg kg–1); however those with the SSB-L treatment increased with increasing application rate for both plants. Transfer factors (TF) of heavy metal, as calculated as the ratio of the concentration of heavy metal absorbed in a plant to the concentration of heavy metal in soil, can quantify the relative differences in bioavailability of heavy metals to vegetables or to identify the efficiency of a vegetable species to accumulate a heavy metal (Uwah et al., 2011). The TF values in this study, calculated based on the sum of heavy metal concentrations of the shoot and root as the plant concentration and the sum of heavy metal concentrations in the soil and SSB as the soil concentration, were 0.77, 0.83, and 1.37 for spinach, and 0.69, 0.91, and 1.19 for bean for Cu, Zn, and Cd, respectively, regardless of the SSB application rate. These TF values may explain why Cu appeared to have been accumulated in the soil (TF less than 1 and lowest among the 3 metals for both plants), Zn seemed to have had no changes before and after the pot study (TF close to 1), and Cd appeared to have been accumulated more in the plant than in the soil (TF more than 1). The TF values widely vary depending on properties of soils and plants, however, those calculated in this study may be comparable with those found by Uwah et al. (2011), which were 0.25–0.95, 0.38–0.55, and 0.42–2.75 for Cu, Zn, and Cd, respectively, for spinach and lettuce grown on tropical soils in Nigeria. Further studies are needed to elucidate heavy metal accumulation in soil and selective absorption by plants with the SSB application.

with 5% application rate for spinach and 15% rate for bean. Therefore, it was concluded that the optimum application rates of SSB-H and SSB-L were indeterminate (lower application rates of SSB-H need to be evaluated) and 5–15%, respectively, for the best growth of the plants in this study. The concentration ranges of the heavy metals absorbed in both plants were in the order of Cd<Cu<Zn for both shoot and root. The total Cu and Zn in the shoot may have been in safe concentration ranges, and the total Cd may have been in excess range which may have caused inhibition of the growth. In fact, the plant DW showed inverse relationships with the total concentrations of the heavy metals absorbed in the plants, and the inhibitive effect of heavy metals on the plant growth was in the order of Zn<Cu<Cd. Overall concentrations of the heavy metals in the root were greater than those in the shoot for both plants. The leacha‐ bility of Cu, Zn, and Cd from SSBs was 89–91%, 90–92%, and 98–99% of the original total concentrations, respectively, during the pot study regardless of the SSB type, application rate, and plant. However, the total concentrations of Cu, Zn, and Cd in the soil after the pot study were accumulated, unaffected, and reduced, respectively, compared with those before the pot study, which could be explained by the TF of each heavy metal which were less than 1, close to 1, and more than 1, respectively. Nevertheless, the percentage of the dilute acid-extractable Cu, Zn, and Cd to the total concentrations in the soil after the pot study was 0.3–0.8%, 9.4– 11.8%, and 1.8–2.6%, respectively, which were lower than the environmental threshold concentrations in Japan. However, long-term effects of SSB application on heavy metal dynamics among SSBs, soils, and plants are needed to be evaluated for further acceptance of

Leachability and Vegetable Absorption of Heavy Metals from Sewage Sludge Biochar

http://dx.doi.org/10.5772/55123

413

Department of Environmental Engineering for Symbiosis, Soka University, Tokyo, Japan

[1] Abe, Y, Yamaguchi, N, Mizoguchi, M, Imoto, H, & Miyazaki, T. (2004). Effect of or‐ ganic acid on transport of cadmium in soil, *Proceedings for Annual Meeting of the Japa‐ nese Society of Irrigation Drainage and Reclamation Engineering*, (in Japanese), 116-117.

[2] Alloway, B. J, & Ayers, D. C. (1997). *Chemical Principles for Environmental Pollution*,

[3] Antoniadis, V, & Alloway, B. J. (2002). The role of dissolved organic carbon in the mobility of Cd, Ni and Zn in sewage sludge-amended soils, *Environmental Pollution*,

Blackie Academic and Professional, London, UK., 190-220.

SSB application in agronomic benefits.

Mieko Yachigo and Shinjiro Sato

**Author details**

**References**

117, 515-521.

The heavy metal concentrations extractable by 0.1 *M* HCl acid solution in the soil after the pot study followed similar patterns as the total concentrations in the soil. The Cu and Zn concen‐ trations significantly increased only when SSB-H was applied at 75% rate for both plants, while the Cd concentrations did not show noteworthy differences among the treatment. However, the percentage of the concentration of the acid-extractable Cu, Zn, and Cd to the total concen‐ tration (acid solution extractability) was 0.3–0.8%, 9.4–11.8%, and 1.8–2.6%, respectively. When 4 contaminated and 4 non-contaminated soils from a northern part of Japan were extracted for heavy metals using 0.1 *M* HCl, the extractability ranged 12% and 27–33% for Cu, 9% and 12– 31% for Zn, and 33% and 73–92% for Cd in the contaminated and non-contaminated soils, respectively (Kashem et al., 2007). Although the extractability of heavy metals from soil by the dilute acid may vary depending on soil type, heavy metal, contamination degrees, and so on, 0.1 *M* HCl extractant may be the best choice for assessment of mobility or bioavailability of heavy metals (Kashem et al., 2007).

The environmental threshold concentrations in soils are 125 mg Cu kg–1, 125 mg Zn kg–1, and 0.01 mg Cd L–1 as 0.1 *M* HCl extractable heavy metals, set by the Japanese Environmental Ministry. It seems that neither of SSB types within the application rates and study duration used in this study have caused environmental threads to soils. Based on life cycle assessment analysis on different treatment processes of sewage sludge including anaerobic digestion, pyrolysis, and incineration, it was concluded that the most effective utilization of sewage sludge implied both energy and material reuse, and that the land application of digested sludge was an acceptable and good choice as long as heavy metal contents in the final cake could be minimized (Hospido et al., 2005). Therefore, long-term effects of SSB application on heavy metal dynamics among SSBs, soils, and plants are needed to be evaluated for further accept‐ ance of SSB application in agronomic benefits.

## **5. Conclusions**

Biochars derived from feedstock including heavy metals such as SSB need to be thoroughly evaluated for heavy metal dynamics among SSBs, soils, and plants for environmentally safe and sound application of SSB. Both SSBs used in this study contained a low end of concentra‐ tion ranges of Cu, Zn, and Cd, respectively, found in the literature, and the heavy metal concentrations accumulated in the SSB as the pyrolysis temperature increased as found in the literature. Both spinach and bean plants suffered from N deficiency due mainly to a lack of soil volume with excessively high application of SSB-H, and possibly from B toxicity especially from SSB-L application. However, when SSB-L was applied, the shoot DW of plants peaked with 5% application rate for spinach and 15% rate for bean. Therefore, it was concluded that the optimum application rates of SSB-H and SSB-L were indeterminate (lower application rates of SSB-H need to be evaluated) and 5–15%, respectively, for the best growth of the plants in this study. The concentration ranges of the heavy metals absorbed in both plants were in the order of Cd<Cu<Zn for both shoot and root. The total Cu and Zn in the shoot may have been in safe concentration ranges, and the total Cd may have been in excess range which may have caused inhibition of the growth. In fact, the plant DW showed inverse relationships with the total concentrations of the heavy metals absorbed in the plants, and the inhibitive effect of heavy metals on the plant growth was in the order of Zn<Cu<Cd. Overall concentrations of the heavy metals in the root were greater than those in the shoot for both plants. The leacha‐ bility of Cu, Zn, and Cd from SSBs was 89–91%, 90–92%, and 98–99% of the original total concentrations, respectively, during the pot study regardless of the SSB type, application rate, and plant. However, the total concentrations of Cu, Zn, and Cd in the soil after the pot study were accumulated, unaffected, and reduced, respectively, compared with those before the pot study, which could be explained by the TF of each heavy metal which were less than 1, close to 1, and more than 1, respectively. Nevertheless, the percentage of the dilute acid-extractable Cu, Zn, and Cd to the total concentrations in the soil after the pot study was 0.3–0.8%, 9.4– 11.8%, and 1.8–2.6%, respectively, which were lower than the environmental threshold concentrations in Japan. However, long-term effects of SSB application on heavy metal dynamics among SSBs, soils, and plants are needed to be evaluated for further acceptance of SSB application in agronomic benefits.

## **Author details**

(TF close to 1), and Cd appeared to have been accumulated more in the plant than in the soil (TF more than 1). The TF values widely vary depending on properties of soils and plants, however, those calculated in this study may be comparable with those found by Uwah et al. (2011), which were 0.25–0.95, 0.38–0.55, and 0.42–2.75 for Cu, Zn, and Cd, respectively, for spinach and lettuce grown on tropical soils in Nigeria. Further studies are needed to elucidate heavy metal accumulation in soil and selective absorption by plants with the SSB application.

The heavy metal concentrations extractable by 0.1 *M* HCl acid solution in the soil after the pot study followed similar patterns as the total concentrations in the soil. The Cu and Zn concen‐ trations significantly increased only when SSB-H was applied at 75% rate for both plants, while the Cd concentrations did not show noteworthy differences among the treatment. However, the percentage of the concentration of the acid-extractable Cu, Zn, and Cd to the total concen‐ tration (acid solution extractability) was 0.3–0.8%, 9.4–11.8%, and 1.8–2.6%, respectively. When 4 contaminated and 4 non-contaminated soils from a northern part of Japan were extracted for heavy metals using 0.1 *M* HCl, the extractability ranged 12% and 27–33% for Cu, 9% and 12– 31% for Zn, and 33% and 73–92% for Cd in the contaminated and non-contaminated soils, respectively (Kashem et al., 2007). Although the extractability of heavy metals from soil by the dilute acid may vary depending on soil type, heavy metal, contamination degrees, and so on, 0.1 *M* HCl extractant may be the best choice for assessment of mobility or bioavailability of

The environmental threshold concentrations in soils are 125 mg Cu kg–1, 125 mg Zn kg–1, and 0.01 mg Cd L–1 as 0.1 *M* HCl extractable heavy metals, set by the Japanese Environmental Ministry. It seems that neither of SSB types within the application rates and study duration used in this study have caused environmental threads to soils. Based on life cycle assessment analysis on different treatment processes of sewage sludge including anaerobic digestion, pyrolysis, and incineration, it was concluded that the most effective utilization of sewage sludge implied both energy and material reuse, and that the land application of digested sludge was an acceptable and good choice as long as heavy metal contents in the final cake could be minimized (Hospido et al., 2005). Therefore, long-term effects of SSB application on heavy metal dynamics among SSBs, soils, and plants are needed to be evaluated for further accept‐

Biochars derived from feedstock including heavy metals such as SSB need to be thoroughly evaluated for heavy metal dynamics among SSBs, soils, and plants for environmentally safe and sound application of SSB. Both SSBs used in this study contained a low end of concentra‐ tion ranges of Cu, Zn, and Cd, respectively, found in the literature, and the heavy metal concentrations accumulated in the SSB as the pyrolysis temperature increased as found in the literature. Both spinach and bean plants suffered from N deficiency due mainly to a lack of soil volume with excessively high application of SSB-H, and possibly from B toxicity especially from SSB-L application. However, when SSB-L was applied, the shoot DW of plants peaked

heavy metals (Kashem et al., 2007).

412 Soil Processes and Current Trends in Quality Assessment

ance of SSB application in agronomic benefits.

**5. Conclusions**

Mieko Yachigo and Shinjiro Sato

Department of Environmental Engineering for Symbiosis, Soka University, Tokyo, Japan

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**Section 4**

**Soil Microbial Processes**


## **Soil Microbial Processes**

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**Chapter 16**

**Response of Soil Microbial Biomass and Enzyme**

**Activities Under Three Temperate Tree Species to**

**Elevated CO2 in Changbai Mountain, Northeastern**

Owing to fossil fuel combustion, deforestation, and intense agriculture, the concentrations of atmospheric CO2 [CO2] has risen by 100ppm since the mid 1800s [1], and it has been predict‐ ed to double until the end of this century compared to the pre-industrial value [2]. Numer‐ ous studies have shown a greater biomass gain of plants, higher fine root and leaf litter C/N in some species under elevated CO2 condition [3−7]. Moreover, the rising CO2 also could alter litter chemistry (e.g., total N, lignin and starch content) and fine root turnover. Because microbial growth is limited by the type and amount of organic substrates entering the soil [8, 9], the changes in above- and below-ground plant input under elevated CO2 could potentially alter both the substrate availability and microbial activity. Although the effect of elevated CO2 via plants on soil microorganisms has been few studies investigated [10−12], the detailed plant-

mediated effects still are unclear because of the complexity of microbial processes.

Soil microorganisms play an important role in nutrient cycling, CO2 emission and in forma‐ tion of soil total organic carbon (TOC) pool. Therefore, any effect of the rising [CO2] on soil microorganisms might in turn feedback on the response of terrestrial ecosystem to atmos‐ pheric CO2 and the sequestration of extra carbon [9]. Soil enzymes drive soil organic matter decomposition and nutrient transformations. Soil enzyme also was considered as a sensitive indicator, which could be significantly affected by temporal variability [13]. It is evident that the seasonal patterns of temperature and moisture of north temperate ecosystems can affect the activity of soil enzymes [14]. Although several studies have investigated the effects of increased CO2 on the soil microbial biomass and activity, to our knowledge, only relative

> © 2013 Zheng et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Zheng et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Jun Qiang Zheng, Ying Wang and Shi Jie Han

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52837

**1. Introduction**

**China**
