**2. Materials and methods**

## **2.1. Study site**

**1. Introduction**

36 Soil Contamination and Alternatives for Sustainable Development

CO<sup>2</sup>

The soil is the upper layer of the solid surface of the planet, formed by weathering rocks, where plants can or may be rooted in and which is an ecological environment for certain types of living beings [1]. Soil is a living, dynamic and non-renewable resource, and its condition and functioning are vital for food production, and for the maintenance of local, regional and global environmental quality. The processes of decomposition and respiration that take place in it play a key role in maintaining the balance between the production and consumption of

 in the biosphere [2]. The soil is composed of mineral particles of variable sizes, organic matter and numerous species of morphologically and physiologically distinct microorganisms. Currently, there is a growing concern about the sustainable use of land in terms of agriculture, environmental quality and human health, as well as its degradation. Soil health can be considered as "*the continuous ability of the soil to function as a living system, within the limits of the ecosystem and land use, to sustain biological productivity, to promote the quality of air and* 

The biological activity of the soil can be reflected in processes such as respiration and enzymatic activity [4]. Enzymatic activity in the soil is mainly of microbial origin that is derived from the intracellular enzymes associated with the cells or the physiological processes of the organisms. Enzymes are direct mediators for the catabolism of the biological components of the soil (organic and mineral). Therefore, these catalysts provide a meaningful evaluation of reaction rates of important processes that occur in the soil [5]. Breathing and activities of soil enzymes can be used as direct measures of microbial activity, soil productivity, and the effects of inhibition due to the presence of contaminants [6]. Microorganisms can react quickly to changes in the environment with alterations in metabolic activity, biomass and the structure of the community [7]. Due to the above, enzymes and respiration have been proposed as indicators for the monitoring of soil quality and the variation of microbial activity [8]. Respiration and enzymatic activity can be influenced in different ways by heavy metals. Dehydrogenase is present only in living organisms and represents active biomass, and it carries out a wide range of oxidation activities that are responsible for the degradation of soil organic matter [9]. Phosphatase is an extracellular enzyme; it hydrolyzes organic phosphorus compounds in different inorganic forms, which can be assimilated by plants [10]. Urease participates in the hydrolysis of urea into carbon dioxide and ammonia, which originates from microbes and shows extracellular activity [11]. Phosphatases are a group of enzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. In this group, we can find the monoester-phosphate hydrolases, in which acid and alkaline phosphatase are found, which are nonspecific enzymes that catalyze the hydrolysis of glycerol phosphate [3]. Glycosidases (β-glucosidase and β-galactosidase) have the ability to intervene in the biogeochemical cycle of carbon and act on glycosides by catalyzing the release of sugars [12]. Overall, a general pattern of inhibition of enzyme activity and respiration due to heavy metal exposure in the

Mining is considered one of the oldest and most fundamental activities of mankind [14]. Mexico has been characterized as a mining power. At present, it occupies the first places in the production of arsenic, cadmium, lead, mercury, fluorite, barium, among others, worldwide.

*water environments, and maintain the health of plants, animals and humans*" [3].

soil has been observed in some studies [13].

The mining district of Santa María de la Paz is located in the municipalities of Villa de la Paz and Matehuala in the state of San Luis Potosí (**Figure 1**). In this district, there is a skarn deposit of Pb-Zn-Ag (Cu-Au) (metamorphic rocks constituted by silicates of Ca, Fe and Mg derived from a protolith of limestones and dolomites in which large amounts of Si, Al, Fe and Mg have been introduced), which has been exploited continuously for a little more than two centuries. The municipal seat of Villa de la Paz is located on the following coordinates: 100°42′47" west longitude and 23°40'31" north latitude, at an altitude of 1837 m above sea level, with an extension of 131.33 km2 and a population of 5350 inhabitants [22]. The municipality has a temperate dry climate. The annual average temperature fluctuates around 18°C; the hottest month is June with 28°C and the coldest month is January with 3°C. Rainfall is 486 mm per year [23]. In the region, there are no important fluvial currents, only intermittent streams, which are formed in the mountains and transport water only during torrential rain events, without any economic or social use are located, but the water they carry is captured on different boards that are used to provide cattle with a trough service and to a lesser extent for land irrigation [24]. Lithosol, xerosol and rendzina soils with petrocalcic and calcareous phases predominate in a remarkable way [25] (CEFIM, 2012). The predominant vegetation types are thorny desert scrub, microphyll, nopal, izotal, cardonal and grassland scrub. Fauna is characterized by species such as hare, rattlesnake, wild cat, wild birds and field mice. The productive activities of the area are mainly: (1) agricultural (tuna, pastures and meadows groomed, corn and beans), (2) livestock with activities of production of bovine meat (8 tons/year), swine (9 tons/year), sheep (1 ton/year), goats (19 tons/year) and production of bovine milk (18,000 L/year) and goat milk (23,000 L/year) [26] and (3) mining industry, which occupies 38.66% of the total area of the municipal capital, therefore the site is a typical mining area [23].

During the time of operation of the mining activities in the area, contamination by heavy metals and arsenic has been generated in soil, water and sediment [24]. In recent years, several studies have been conducted in the area in which the presence of high concentrations of heavy metals and As in water, air and sediment has been demonstrated [24, 27–29]. In the study by Razo et al. [24], tailings, dams and slag deposits were identified as the main sources of pollution and dispersion of particles due to wind (**Figure 2**). On the other hand, it has been proven that there are health risks to human and ecological populations in the area, due to exposure to heavy metals and As [30–36].

#### **2.2. Soil sampling**

Twenty-five composite samples of surface soil were collected in a distance gradient located from the source of contamination (tailings); the gradient was established on a linear path at distances of 0, 10, 100, 1000 and 10,000 m (**Figure 1**); for the location of the route, the predominant direction of the winds and the intermittent water runoff in the area were considered. For each distance, five subsamples of 10 cm3 of soil collected in an area of 1 m2 were gathered. Sampling was carried out on October 2016. The samples were homogenized, sieved (2 mm), placed in 50 mL sterile conical tubes and placed at 4°C for transport to the laboratory, where they were stored for 7 days at −20°C for their analysis.

#### **2.3. Analysis of arsenic and lead in soil**

The samples were dried in an oven (at 30°C) until they reached a constant weight (from 24 to 36 h), and moisture percentage was determined. Samples (0.5 g of soil) were placed in teflon cups with 10 mL of 25% ultrapure HNO3 . Acid digestion was performed in a closed system with a microwave oven (MDS-2000-CEM) at 100 W of power and 80 psi of pressure for 1 h with a TAP of 30 min. The digests were filtered and calibrated to 10 mL. The quantification was performed by atomic absorption spectrophotometry with a hydride generator in the case of As (PerkinElmer AAnalist 100 Flame AA) and a graphite furnace for Pb (PerkinElmer PinAAcle 900 T). In order to calculate the concentrations, calibration curves were made with standards, and as a quality control, blanks and reference materials were used (Montana soil l-NIST-2710a). The percentages of recovery were from 90 to 110%. The samples were processed in duplicate, and the results expressed in mg/kg.

#### **2.4. Physicochemical parameters**

pH was determined using the water measurement method [37] in a 1:2 ratio. About 6 mL of deionized water were added to a sample of 3 g of soil and then were kept under agitation for three cycles of 5 min at 800 rpm. The electrical conductivity was measured using the method mentioned in [37] at a soil-water ratio of 1:5. About 15 mL of deionized water were added to a sample of 3 g of soil and then were kept under agitation for three cycles of 5 min at 800 rpm. The measurements of pH and electrical conductivity were calculated in the supernatant after the sample had been resting for 15 min in a benchtop multiparameter meter (HANNA-HI2550). Organic matter was determined using the wet digestion method [38]. In

a sample of 0.250 g of soil. The mixture was allowed to stand for 5 min after which 17.5 mL of deionized water were added, and it was stirred at 150 rpm for 30 min. The quantification was performed after 24 h using a UV–visible spectrophotometer (Biomate 3 s Thermo Fisher

Scientific) at 600 nm in 2 mL of supernatant. All samples were made in duplicate.

solution (0.5 M) and 5 mL of concentrated H2

Evaluation of the Biological Activity of Soil in a Gradient Concentration of Arsenic and Lead…

http://dx.doi.org/10.5772/intechopen.80031

39

SO4

were added to

addition, 2.5 ml of Na2

Cr<sup>2</sup> O7

**Figure 2.** Mine tailing deposits and acid drainage.

**Figure 1.** Study area and location of sampling sites.

Evaluation of the Biological Activity of Soil in a Gradient Concentration of Arsenic and Lead… http://dx.doi.org/10.5772/intechopen.80031 39

**Figure 1.** Study area and location of sampling sites.

sheep (1 ton/year), goats (19 tons/year) and production of bovine milk (18,000 L/year) and goat milk (23,000 L/year) [26] and (3) mining industry, which occupies 38.66% of the total area

During the time of operation of the mining activities in the area, contamination by heavy metals and arsenic has been generated in soil, water and sediment [24]. In recent years, several studies have been conducted in the area in which the presence of high concentrations of heavy metals and As in water, air and sediment has been demonstrated [24, 27–29]. In the study by Razo et al. [24], tailings, dams and slag deposits were identified as the main sources of pollution and dispersion of particles due to wind (**Figure 2**). On the other hand, it has been proven that there are health risks to human and ecological populations in the area, due to exposure

Twenty-five composite samples of surface soil were collected in a distance gradient located from the source of contamination (tailings); the gradient was established on a linear path at distances of 0, 10, 100, 1000 and 10,000 m (**Figure 1**); for the location of the route, the predominant direction of the winds and the intermittent water runoff in the area were considered. For

Sampling was carried out on October 2016. The samples were homogenized, sieved (2 mm), placed in 50 mL sterile conical tubes and placed at 4°C for transport to the laboratory, where

The samples were dried in an oven (at 30°C) until they reached a constant weight (from 24 to 36 h), and moisture percentage was determined. Samples (0.5 g of soil) were placed in teflon

with a microwave oven (MDS-2000-CEM) at 100 W of power and 80 psi of pressure for 1 h with a TAP of 30 min. The digests were filtered and calibrated to 10 mL. The quantification was performed by atomic absorption spectrophotometry with a hydride generator in the case of As (PerkinElmer AAnalist 100 Flame AA) and a graphite furnace for Pb (PerkinElmer PinAAcle 900 T). In order to calculate the concentrations, calibration curves were made with standards, and as a quality control, blanks and reference materials were used (Montana soil l-NIST-2710a). The percentages of recovery were from 90 to 110%. The samples were pro-

pH was determined using the water measurement method [37] in a 1:2 ratio. About 6 mL of deionized water were added to a sample of 3 g of soil and then were kept under agitation for three cycles of 5 min at 800 rpm. The electrical conductivity was measured using the method mentioned in [37] at a soil-water ratio of 1:5. About 15 mL of deionized water were added to a sample of 3 g of soil and then were kept under agitation for three cycles of 5 min at 800 rpm. The measurements of pH and electrical conductivity were calculated in the

of soil collected in an area of 1 m2

. Acid digestion was performed in a closed system

were gathered.

of the municipal capital, therefore the site is a typical mining area [23].

38 Soil Contamination and Alternatives for Sustainable Development

to heavy metals and As [30–36].

each distance, five subsamples of 10 cm3

**2.3. Analysis of arsenic and lead in soil**

cups with 10 mL of 25% ultrapure HNO3

**2.4. Physicochemical parameters**

they were stored for 7 days at −20°C for their analysis.

cessed in duplicate, and the results expressed in mg/kg.

**2.2. Soil sampling**

**Figure 2.** Mine tailing deposits and acid drainage.

supernatant after the sample had been resting for 15 min in a benchtop multiparameter meter (HANNA-HI2550). Organic matter was determined using the wet digestion method [38]. In addition, 2.5 ml of Na2 Cr<sup>2</sup> O7 solution (0.5 M) and 5 mL of concentrated H2 SO4 were added to a sample of 0.250 g of soil. The mixture was allowed to stand for 5 min after which 17.5 mL of deionized water were added, and it was stirred at 150 rpm for 30 min. The quantification was performed after 24 h using a UV–visible spectrophotometer (Biomate 3 s Thermo Fisher Scientific) at 600 nm in 2 mL of supernatant. All samples were made in duplicate.

#### **2.5. Respiration and enzyme assays**

Soil respiration was determined using the MicroResp method [39], which consists of filling a 96-well plate microplate (1.2 mL per well) with soil, a carbon source (25 μL of glucose at 25%) and water (375 μL). The plate was subsequently sealed (silicone rubber gasket with interconnection holes) with a CO2 colorimetric detection trap consisting of a microplate (300 μL per well) with 150 μL of agar enriched with KCl (0.15 mol L−1) and cresol red dye (32.7 μmol L−1). Incubation was carried out at 25°C for 6 h in dark conditions. The absorbance in the detection plate was measured at 570 nm. The amount of CO2 released was calculated using a calibration curve. The results were expressed in μg C-CO2 g−1 soil h−1.

**2.6. Statistical analysis**

best r2

determined at 5%.

source.

**3. Results and discussion**

**3.1. Arsenic and lead in soil**

The median and the interquartile range are reported. To compare the concentrations of As, Pb, physicochemical parameters and biological activity (respiration and enzymatic activity) by the distance to the source, the Kruskal-Wallis test was used in logarithmically transformed data. Linear models based on distance (DistLM, models based on Euclidean distances with "step-wise" procedure obtained by 9999 permutations and selected under the criterion of

Evaluation of the Biological Activity of Soil in a Gradient Concentration of Arsenic and Lead…

http://dx.doi.org/10.5772/intechopen.80031

41

) were performed to evaluate the association between biological activity and physicochemical parameters, as well as polluting elements. The DistLM model was calculated with logarithmically transformed and normalized data, excluding electrical conductivity due to its high association with the rest of the co-variables (As, Pb, pH and OM). In order to visualize the ordering patterns of the samples and the relationships between the variables, a redundancy analysis (dbRDA) was performed, representing the axes of greatest variation and the correlation between the covariates. The univariate analysis was performed using the GraphPad Prism Version 6 software, and the multivariate was performed using the software PRIMER 6 Version 6.1.18 & PERMANOVA Version 1.0.8 of PRIMER-E Ltd. Statistical significance was

The average content of As in the Earth's crust is 1.8 mg/kg, and, that of the soils has been found to be at 6.83 mg/kg. The most mobile forms of As are absorbed in a pH range of 7–9. The ions of As are known to be easily fixed in soils by Fe and Al hydroxides, the clay fraction, P and Ca compounds and organic matter [48]. The arsenic concentrations in this study ranged from 4.7 to 463.2 mg/kg. Changes in arsenic concentration were found mainly between the distances of 0–100 m and 1000–10,000 m (kW-H4,25 = 21.71 p = 0.0002, **Figure 3a**); with the highest concentrations being from the samples located in the first 100 m from the

Pb is a toxic metal naturally present in the Earth's crust (15 mg/kg). Mining, metallurgy, manufacturing and recycling activities, and, in some countries, the use of lead paints and gasolines, are among the main sources of environmental pollution [49]. The average level of Pb in soils has been measured at 27 mg/kg [48]. In our study, lead concentrations (mg/kg) ranged from 171.7 to 2319.0; the zones located between distances of 0 and 10,000 m showed the greatest difference (kW-H4,25 = 13.61 p = 0.0086, **Figure 3b**). Arsenic and lead concentrations of the sites located from 0 to 100 m from the source are above the levels established for the remediation of contaminated soils dictated by Mexican regulations (NOM-147-SEMARNAT/SSA1–2004),

In Villa de la Paz, Martínez-Toledo et al. [50] found values (mg/kg) of As from 1461.9 to 28274.0 and of Pb from 466.1 to 3486.4 in a site close to the source of contamination. In another study, Márquez-Reyes [51] determined the average total concentrations (mg/kg) of 13,443.4 of As and 1301.7 of Pb. González-Mille [52] reported average concentrations (mg/kg), for the site, of 222.1 of As and 204.3 of Pb. Chipres [53] found the presence of heavy metals of environmental

which establishes the limits at 22 mg/kg of As and 400 mg/kg of Pb.

Arylsulfatase was quantified according to the method of Tabatabai and Bremner [40]. The method is based on the hydrolytic capacity of the enzyme on an artificial substrate (p-nitrophenyl sulfate) whose product p-nitrophenol is evaluated spectrophotometrically at a wavelength of 420 nm. The activity of arylsulfatase was expressed in μg of p-nitrophenol g−1 h−1.

The β-glucosidase was determined according to the method of [41]. The method is based on the quantification of p-nitrophenol obtained by the action of glycosides after incubation of the soil with the substrate β-D-glucopyranoside in medium at pH 6. The incubation was carried out at 37°C for 1 h; the released p-nitrophenol was removed by filtration after having added CaCl2 and adjusted with THAM buffer at pH 12. The absorbance was recorded at 405 nm. The activity of β-glucosidase was expressed in μg of p-nitrophenol g−1 h−1.

The activity of the enzyme dehydrogenase was determined by the method of [42–44]. The method is based on the measurement of iodonitrotetrazolium formazan (INTF) produced by the reduction of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium (INT) in soil incubated with INT in buffered medium under dark conditions for 1 h at 40°C. The absorbance was measured at 464 nm. The activity of the dehydrogenase was expressed in μg of INTF g−1 h−1.

The enzymatic activity of phosphatase was estimated according to the method of [45, 46]. The method is based on the spectrophotometric determination of p-nitrophenol released when the soil is incubated at 37°C for 1 h with p-nitrophenyl phosphate buffered solution; the alkaline solutions of this compound have a yellow color. Absorbance was measured at 405 nm. The activity of the phosphatase was expressed in μg of p-nitrophenol g−1 h−1.

Urease was quantified by the technique mentioned in [47]. This technique is based on the determination of the ammonium released in the incubation of a soil solution at 37°C for 2 h. The determination of ammonium is carried out through the Berthrlot reaction with certain modifications. The Berthelot reaction consists of the reaction of ammonium in an alkaline medium with a coloring agent giving monochloramine, which is transformed into 2,2-isopropyl.5,5-methyl-indophenol when thymol is added. In this method, the ammonia produced by the urease activity reacts with salicylate and dichloro isocyanate giving a bluish green color. Absorbance was measured at 610 nm. The activity of urease was expressed in μg of NH4 -N g−1 2 h−1.

The respiration and the concentration of the enzymes in the soil were determined 7 days after their collection. The concentrations were adjusted according to the moisture content. All determinations were made in triplicate in a spectrophotometer (BioTek Synergy H1). In our experiments, all the reagents were analytical grade.

#### **2.6. Statistical analysis**

**2.5. Respiration and enzyme assays**

40 Soil Contamination and Alternatives for Sustainable Development

plate was measured at 570 nm. The amount of CO2

curve. The results were expressed in μg C-CO2

nection holes) with a CO2

CaCl2

NH4


Soil respiration was determined using the MicroResp method [39], which consists of filling a 96-well plate microplate (1.2 mL per well) with soil, a carbon source (25 μL of glucose at 25%) and water (375 μL). The plate was subsequently sealed (silicone rubber gasket with intercon-

well) with 150 μL of agar enriched with KCl (0.15 mol L−1) and cresol red dye (32.7 μmol L−1). Incubation was carried out at 25°C for 6 h in dark conditions. The absorbance in the detection

Arylsulfatase was quantified according to the method of Tabatabai and Bremner [40]. The method is based on the hydrolytic capacity of the enzyme on an artificial substrate (p-nitrophenyl sulfate) whose product p-nitrophenol is evaluated spectrophotometrically at a wavelength of 420 nm.

The β-glucosidase was determined according to the method of [41]. The method is based on the quantification of p-nitrophenol obtained by the action of glycosides after incubation of the soil with the substrate β-D-glucopyranoside in medium at pH 6. The incubation was carried out at 37°C for 1 h; the released p-nitrophenol was removed by filtration after having added

The activity of the enzyme dehydrogenase was determined by the method of [42–44]. The method is based on the measurement of iodonitrotetrazolium formazan (INTF) produced by the reduction of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium (INT) in soil incubated with INT in buffered medium under dark conditions for 1 h at 40°C. The absorbance was measured at 464 nm. The activity of the dehydrogenase was expressed in μg of INTF g−1 h−1. The enzymatic activity of phosphatase was estimated according to the method of [45, 46]. The method is based on the spectrophotometric determination of p-nitrophenol released when the soil is incubated at 37°C for 1 h with p-nitrophenyl phosphate buffered solution; the alkaline solutions of this compound have a yellow color. Absorbance was measured at 405 nm. The activ-

Urease was quantified by the technique mentioned in [47]. This technique is based on the determination of the ammonium released in the incubation of a soil solution at 37°C for 2 h. The determination of ammonium is carried out through the Berthrlot reaction with certain modifications. The Berthelot reaction consists of the reaction of ammonium in an alkaline medium with a coloring agent giving monochloramine, which is transformed into 2,2-isopropyl.5,5-methyl-indophenol when thymol is added. In this method, the ammonia produced by the urease activity reacts with salicylate and dichloro isocyanate giving a bluish green color. Absorbance was measured at 610 nm. The activity of urease was expressed in μg of

The respiration and the concentration of the enzymes in the soil were determined 7 days after their collection. The concentrations were adjusted according to the moisture content. All determinations were made in triplicate in a spectrophotometer (BioTek Synergy H1). In our

and adjusted with THAM buffer at pH 12. The absorbance was recorded at 405 nm. The

The activity of arylsulfatase was expressed in μg of p-nitrophenol g−1 h−1.

activity of β-glucosidase was expressed in μg of p-nitrophenol g−1 h−1.

ity of the phosphatase was expressed in μg of p-nitrophenol g−1 h−1.

experiments, all the reagents were analytical grade.

g−1 soil h−1.

colorimetric detection trap consisting of a microplate (300 μL per

released was calculated using a calibration

The median and the interquartile range are reported. To compare the concentrations of As, Pb, physicochemical parameters and biological activity (respiration and enzymatic activity) by the distance to the source, the Kruskal-Wallis test was used in logarithmically transformed data. Linear models based on distance (DistLM, models based on Euclidean distances with "step-wise" procedure obtained by 9999 permutations and selected under the criterion of best r2 ) were performed to evaluate the association between biological activity and physicochemical parameters, as well as polluting elements. The DistLM model was calculated with logarithmically transformed and normalized data, excluding electrical conductivity due to its high association with the rest of the co-variables (As, Pb, pH and OM). In order to visualize the ordering patterns of the samples and the relationships between the variables, a redundancy analysis (dbRDA) was performed, representing the axes of greatest variation and the correlation between the covariates. The univariate analysis was performed using the GraphPad Prism Version 6 software, and the multivariate was performed using the software PRIMER 6 Version 6.1.18 & PERMANOVA Version 1.0.8 of PRIMER-E Ltd. Statistical significance was determined at 5%.
