**2.5. Measurement of CO2-evolved**

The soil was bulked; all the stones, visible roots, and fauna were removed, it was sieved to less

Tannery sludge, produced during leather manufacturing, when processing skin or hide to leather, was sampled from a tannery in Leon (Guanajuato, Mexico). It contained large quantities of hair, fatty fleshings, and soluble proteins, as well as sulphide, lime, chromium-

A pre-incubation process of soil samples was necessary to allow the soil microbial activity to stabilize after the sampling and sieve management. Soils were pre-incubated for 1 week prior to starting the experiment at conditions similar to the experiment, i.e. at 20°C in the dark, in a temperature and humidity controlled room. Three replicates were destructively harvested at days 0–90 or 120 and stored at −20°C for N mineralization and soil microbial activities analysis.

Maintaining soil fertility depends on biomass and activity of soil microorganisms vital in the biological cycles of most major plant nutrients [32]. Microorganisms are also involved in forming soil structure [33]. Several microbiological parameters have been suggested to measure soil environmental quality [34]. For instance, soil respiration and enzyme activities such as dehydrogenase activity and nitrification, can inform about the presence of viable microorganisms, and on the intensity, kind and time length of the effects of pollutants on the

Sub-samples of 40 g of soils were placed in 110-ml glass bottles, which were then put into 1-l jars containing 10 ml H2O and a vessel with 20 ml 1 M NaOH solution. The jars were air-tight sealed with plastic lids and incubated at 25 °C for 7 days. After incubation, vessels with 20 ml 1 M NaOH solution were removed, resealed, and stored for future CO2 analysis. At the

shaking for 30 min with 100 ml 0.5 M K2SO4 solution and filtered through Whatman No. 42 paper. Similarly, control fresh samples were extracted. Extractants were stored at −20°C until

Dehydrogenase activity in soils has been used as measure for overall microbial activity [37]. The method is based on the estimation of triphenyltetrazolium chloride (TTC) reduction rate to triphenyl formazan (TPF) in soils after incubation at 30°C for 24 h. Soil dehydrogenase activity was measured using a modified form of the method used by Casida [37]. Five grams of fresh soil were incubated at 37°C for 24 h in test tubes containing 1 ml 3% 2,3,5- triphenyl‐ tetrazolium chloride (TPF), 67 mg CaCO3, and 2.5 ml distilled water. The accumulation of the end-product triphenyl formazan (TPF)was determined in acetone extracts (50 ml) using a

−

−

–N, NO2−N, and NH4

–N in the extracts was determined by colorimetric

+

−N, done by

than 2 mm and stored at 5°C to use latter.

246 Organic Fertilizers - From Basic Concepts to Applied Outcomes

sulphate, salts, dyes, acids, and leather trimmings.

**2.4. Soil microbial activities and nitrification**

mentioned dates, soil was removed for analysis of NO3

PerkinElmer Lamda 3A Spectrophotometer at 520 nm.

−

–N and NO2

–N by Indophenol blue [36].

**2.2. Tannery sludge**

**2.3. Incubation experiments**

metabolic activity of soils.

analysis. Concentration of NO3

+

method [35] and NH4

Glass bottles of 110 ml containing the amended substrate and unamended control soils were placed in 1-l, wide-mouthed glass jars, with a glass flask of 30 ml containing 20 ml of 1 N NaOH solution to trap the evolved CO2. Jars were tightly closed and incubated at 20°C for up to 270 days at room temperature. The NaOH solution was exchanged every 7 days during the first 2 months, and monthly thereafter. Jars were aired each time for 2 min when they were opened to exchange the NaOH solution, to avoid anaerobic conditions in amended and unamended soils. Every time that the NaOH trapped were collected, a blank with non-soils in the jars were collected, too. The values of CO2 in the blanks were used to correct the CO2 trapped inside the jars. CO2 trapped in 1 M NaOH solution was measured in a 5-ml aliquot by titrimetric methods with a standard 0.1 M HCl solution using the phenolphthalein indicator method [38].

## **2.6. Chemical analysis**

Total organic C in the soil and tannery sludge was measured using dichromate digestion [39], total N was measured using Kjeldahl digestion [40], and total hydrolysable and orthophos‐ phate phosphorus were determined using the stannous chloride method [35]. The particle-size was analyzed using the hydrometer method [41]. To conclude, total Cr in tannery sludge, fleshing waste, and infiltration and runoff solutions was measured using absorption atomic spectrometry with a fitted graphite furnace spectrophotometer (Avanta M System 300, GF 3000 S/N 10288). Tannery sludge and fleshing waste were digested with 4:1 HCl: HNO3 using a digiprep TM digestion system, prior to analysis.

#### **2.7. Fractionation of chromium**

Tessier et al. scheme [42] is widely used, although application of sequential extraction is still subject controversy. The main problems of sequential extraction procedures are the nonselective use of extracts and the trace elements redistribution among phases during the extraction [43]. In spite of these restrictions, sequential extraction procedures have proved to be useful in the environmental analytical chemistry field [44].

Sequential extraction was utilized for partitioning Cr in soil and sludge amended soils into six operationally defined fractions described by Tessier et al. [42] and modified by Xiong et al. [45]. Six operationally defined fractions, exchangeable, bound to carbonates, bound to Mn oxides, bound to Fe oxides, bound to organic matter and residues according to procedure described by Tessier. Summarizing, 2 g of soil were placed in a 50-ml polycarbonate centrifuge tube and subjected to the following extraction program: Exchangeable fraction (I): soil extracted with 25 ml of 1 M ammonium acetate was shaken for 2 h, then centrifuged. Carbonate bound fraction (II): Fraction I residue extracted with 25 ml of 1 M sodium acetate, adjusted to pH 5 with acetic acid then shaken for 5 h and centrifuged. Mn-oxide-bound fraction (III): Fraction II residue extracted with 25 ml 0.1 M hydroxylamine hydrochloride adjusted to pH 2 with nitric acid then shaken for 12 h and centrifuged. Fe-oxide bound fraction (IV): Fraction III residue extracted with 25 ml of 0.2 M ammonium oxalate, adjusted to pH 3 with oxalic acid, and shaken for 24 h, then centrifuged. Organic and sulphide-bound fraction (V): Fraction IV residue extracted with 5 ml of 30% H2O2 adjusted to pH 2 with HNO3 then heated in a water bath at 85°C. After cooling, 20 ml of I M ammonium acetate were added, shaken for 2 h, then centri‐ fuged. Residual fraction (VI): Fraction V residue digested with 3:1 HCl: HNO3 in digestion glass tubes. After digestion was completed, 25 ml water were added and then filtered. The levels of Cr in the six fractions (I to VI), plus a fresh sample, were analyzed with atomic absorption spectrometry as described above, at 1, 3, and 6 months of incubation.

Mobility factor percentage was calculated according to the equation:

$$1\% \text{mobidity factor} = \frac{\left(I + II + III\right)}{\left(I + II + III + IV + V + VI\right)} \times 100\%$$

Hexavalent Cr (CrVI) was quantified employing diphenylcarbazide procedure described by Bartlett [46]. One gram of soil was extracted with 3 ml of 10 mM K2HPO4/KH2PO4, pH 7.2. One milliliterazide reagent was added to 8 ml of extract, mixed and stand for 20 min and read the color at 540 nm. Azide reagent was prepared with 120 ml of 85% phosphoric acid, diluted with 280 ml distilled water, to 0.4 g of S-diphenylcarbizide dissolve in 100 ml of 95% ethanol.

#### **2.8. Rainfall system experiment**

For the experiment of simulated rainstorm, the rainfall system type Morin [47] was used (**Figure 2**).

**Figure 2.** Rainfall system type Morin.

#### **2.9. Soil treatments**

extracted with 5 ml of 30% H2O2 adjusted to pH 2 with HNO3 then heated in a water bath at 85°C. After cooling, 20 ml of I M ammonium acetate were added, shaken for 2 h, then centri‐ fuged. Residual fraction (VI): Fraction V residue digested with 3:1 HCl: HNO3 in digestion glass tubes. After digestion was completed, 25 ml water were added and then filtered. The levels of Cr in the six fractions (I to VI), plus a fresh sample, were analyzed with atomic

( ) % <sup>100</sup>

Hexavalent Cr (CrVI) was quantified employing diphenylcarbazide procedure described by Bartlett [46]. One gram of soil was extracted with 3 ml of 10 mM K2HPO4/KH2PO4, pH 7.2. One milliliterazide reagent was added to 8 ml of extract, mixed and stand for 20 min and read the color at 540 nm. Azide reagent was prepared with 120 ml of 85% phosphoric acid, diluted with 280 ml distilled water, to 0.4 g of S-diphenylcarbizide dissolve in 100 ml of 95% ethanol.

For the experiment of simulated rainstorm, the rainfall system type Morin [47] was used

*mobility factor I II III IV V VI*

( )

*I II III*

+ + = ´ + + + ++

absorption spectrometry as described above, at 1, 3, and 6 months of incubation.

Mobility factor percentage was calculated according to the equation:

248 Organic Fertilizers - From Basic Concepts to Applied Outcomes

**2.8. Rainfall system experiment**

**Figure 2.** Rainfall system type Morin.

(**Figure 2**).

After adjusted to 50% WHC, sub-samples of 10 kg of soil were placed in 15 kg rough use plastic bags. Two different treatments were applied to the soils: tannery sludge (T) to outside (O) and under the canopy (U) of mesquite tree soils, and tannery sludge plus fleshing waste in outside and under the canopy soils (OTF and UTF, respectively). The amount of tannery sludge was chosen to give less than 300 mg Cr kg−1; the critical level of Cr for acceptable use of waste and bio-products in agriculture, as established by the US Environmental Protection Agency [48].

A cotton plug was adjusted to the plastic bags to allow ventilation. After moistening, the treated soils were incubated at 25°C for 1, 3, and 6 months in order to allow mineralization to occur and before they were submitted to a simulated rainfall system. A fresh control was included in order to compare the experimental results with it. The treated soils were air-dried and passed through a 4-mm sieve. They were sampled and then each one was packed to a depth of 4 cm in a 30 × 50-cm box over a 12-cm layer of gravel and 4-cm layer of coarse sand for moisture tension control. Soils were then wetted by capillarity while maintaining the water in a levelled position. Following saturation, the boxes were set at a 3% slope and then allowed to drain for 30 min (**Figure 3**). A total of four runs for each treated group were conducted, each one after an incubation period (0, 1, 3, and 6 months). For each run, the soil treatments were placed on assoil box carrousel and subjected to a simulated rainstorm using the rainfall system type Morin [47] (**Figure 2**). The full description of a simulated rain experiment is in Barajas-Aceves et al. [49].

**Figure 3.** pH after a simulated rainfall in semi-arid soils amended with fleshing waste and or tannery sludge. Bars indi‐ cate standard deviation. Source: Ref. [49].

Simulated rainfall at intensity of 50 mm h−1 and a drop diameter of 3 mm was applied for 60 min. With these specifications, the process simulated the 6 months of a raining period in Dolores Hidalgo, Guanajuato, Mexico. During each run, water percolation through the soil and sediment samples were collected in separated plastic bottles every 5 min for 1 h. Weight of sediment samples and infiltrated water was registered. Double aluminium potassium sulphate (20 ml at 10%) was added to the sediment samples to precipitate suspended solids and separate the water by decantation. The solids in the bottle were dried for 24 h at 105°C and the soil loss was measured by gravimetry, expressed in grams. Once the rainfall was stopped, the soil boxes were left for 24 h to allow the drainage ceased from them, the soil was randomly sampled, allow to dry slowly until it had a moisture equivalent to 40% WHC. Before the rainfall system started, the same soils with the same treatments were sampled and were adjusted to 40% WHC before the soil microbial activities determinations.

The decanted water was transferred to a plastic bottle and weighed. The volume was expressed in cm3 (considering the water density of 1 g cm−3). The runoff was calculated by the equation:

$$Runoff(mm) = \frac{V \times 10}{A}$$

where *V* is the volume of runoff (cm3 ), *A* is the area exposed to rainfall (1500 cm2 ), and10 is the factor to convert cm to mm.

The infiltration was calculated with a similar equation and expressed in mm [50–52].

$$Infibration(mm) = \frac{V \times 10}{A}$$

The results of all these measurements were the sum of the 12 samples per treatment collected in one run.
