**2.4. Ligninolytic enzyme activity and H2O2 quantification**

#### *2.4.1. Laccase determination*

Laccase activity was determined according to More et al. [17] by measuring the oxidation of ABTS [2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)] in a reaction mixture (1 mL) containing 100 μL of ABTS (0.5 mM, Sigma, St. Louis, MO, USA), 800 μL of acetate buffer (100 mM, pH 4.5) and 100 μL of enzyme extract. Absorbance changes in the presence of the enzyme were monitored during 5 min at 420 nm (*ε* = 3.6 × 104 M−1 cm−1). One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute per milligram of protein under the assay conditions.

#### *2.4.2. Manganese peroxidase assay*

MnP activity was determined at 610 nm (*ε* = 4460 M−1 cm−1), following the method described by Kuwahara et al. [18]. The reaction mixture contained the following: 700 μL of enzyme extract, 50 μL of phenol red (0.2%), 50 μL of sodium lactate (0.5 mM), 50 μL of egg albumin (0.1%), 50 μL of manganese sulfate (2 mM) and 50 μL of H2O2 (2 mM). The reaction was carried out in 50 μL of sodium succinate buffer (20 mM) at pH 4.5. After 5 minutes, 50 μL of NaOH (2N) was added to stop the reaction. One enzyme unit was defined as 1 μmol of the product formed per minute per milligram of protein under the assay conditions.

#### *2.4.3. Hydrogen peroxide content*

H2O2 content of the fungal enzyme extracts (FEEs) was determined using the iodide/iodate method, according to Klassen et al. [19]. Three milliliters of the FEEs were mixed with 3 mL of a solution containing KI (33 g), NaOH (1 g) and (NH4)6Mo7O24 × 4H2O (0.1 g) in 500 mL of distilled, deionized water, in addition to 3 mL of a solution containing C8H4KO4 (10 g) in 500 mL of distilled, deionized water. The absorbance of the resulting solution was measured at 351 nm in a 3 cm3 cuvette. The blank absorbance was determined by substituting the FEEs with a sterile Sivakumar culture medium in the reaction mixture. Hydrogen peroxide content was calculated by substituting with H2O2 reagent (30%, J.T. Baker™) according to the standard curve of the absorbance of known concentrations (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mg L−1).

#### **2.5. Biodegradation studies**

Biodegradation of atrazine was evaluated in sterile soil microcosm conditions. First, 20 g of air dried soil was placed in serological flasks (100 cm3 ). Then, the sterile soil was contaminated with atrazine (Sigma-Aldrich Co., USA) at the field application rate of 5mg/kg [8], and 20 mL of methanol were added (analytical grade, Honeywell Burdick & Jackson, Muskegon, MI, USA). Soil-methanol-atrazine was mixed using a sterile spatula until the complete evaporation of methanol under a laminar flow hood.

Three treatments were evaluated: *T. maxima* extract, *P. carneus* extract and their co-culture extract (*T. maxima*-*P. carneus*). Soil microcosms were adjusted to a water holding capacity (WHC) of 40% using 0.215 mL of fungal extract per gram of soil. Four experimental units (serological flasks) were used per treatment. Atrazine degradation was evaluated at 1, 3, 6 and 12 h using high-pressure liquid chromatography (HPLC) analysis. In addition, abiotic (sterile soil) and biotic (nonsterilized soil) controls were used.

#### **2.6. Adsorption-desorption studies**

*carneus* (9 days old) were added. Monocultures of both fungi were established at the same time. Fungal cultures were incubated at 25°C and 120 rpm for 6 days. After this step, the fungal enzyme extracts (FEEs) were centrifuged at 7000 rpm during 10 min. The supernatant was filtered with a 0.2 mm nylon filter; this process allows a cell-free extract to be obtained, which

Laccase activity was determined according to More et al. [17] by measuring the oxidation of ABTS [2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)] in a reaction mixture (1 mL) containing 100 μL of ABTS (0.5 mM, Sigma, St. Louis, MO, USA), 800 μL of acetate buffer (100 mM, pH 4.5) and 100 μL of enzyme extract. Absorbance changes in the presence of the

activity was defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute

MnP activity was determined at 610 nm (*ε* = 4460 M−1 cm−1), following the method described by Kuwahara et al. [18]. The reaction mixture contained the following: 700 μL of enzyme extract, 50 μL of phenol red (0.2%), 50 μL of sodium lactate (0.5 mM), 50 μL of egg albumin (0.1%), 50 μL of manganese sulfate (2 mM) and 50 μL of H2O2 (2 mM). The reaction was carried out in 50 μL of sodium succinate buffer (20 mM) at pH 4.5. After 5 minutes, 50 μL of NaOH (2N) was added to stop the reaction. One enzyme unit was defined as 1 μmol of the product

H2O2 content of the fungal enzyme extracts (FEEs) was determined using the iodide/iodate method, according to Klassen et al. [19]. Three milliliters of the FEEs were mixed with 3 mL of a solution containing KI (33 g), NaOH (1 g) and (NH4)6Mo7O24 × 4H2O (0.1 g) in 500 mL of distilled, deionized water, in addition to 3 mL of a solution containing C8H4KO4 (10 g) in 500 mL of distilled, deionized water. The absorbance of the resulting solution was measured at

with a sterile Sivakumar culture medium in the reaction mixture. Hydrogen peroxide content was calculated by substituting with H2O2 reagent (30%, J.T. Baker™) according to the standard curve of the absorbance of known concentrations (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and

Biodegradation of atrazine was evaluated in sterile soil microcosm conditions. First, 20 g of air

cuvette. The blank absorbance was determined by substituting the FEEs

). Then, the sterile soil was contaminated

M−1 cm−1). One unit of laccase

was used to determine laccase and MnP activity and H2O2 content.

**2.4. Ligninolytic enzyme activity and H2O2 quantification**

196 Soil Contamination - Current Consequences and Further Solutions

enzyme were monitored during 5 min at 420 nm (*ε* = 3.6 × 104

formed per minute per milligram of protein under the assay conditions.

per milligram of protein under the assay conditions.

*2.4.1. Laccase determination*

*2.4.2. Manganese peroxidase assay*

*2.4.3. Hydrogen peroxide content*

351 nm in a 3 cm3

**2.5. Biodegradation studies**

dried soil was placed in serological flasks (100 cm3

3.5 mg L−1).

Experiments were conducted using six sorbate concentrations of atrazine (0.5, 1, 5, 10, 20 and 30 mg/kg). Two grams of all soil samples were added to a polypropylene bottle (20 mL), and immediately 5 mL of a methanol solution with the sufficient amount of atrazine was added to obtain the established concentration. Bottles were shaken vigorously (24 h) and placed on a flat rotator shaker (120 rpm) at room temperature (27 ± 1°C) [20]. Four replicates were used for each initial concentration of atrazine. After an equilibration period (24 h), samples were centrifuged in cold (5°C) at 7000 rpm during 20 min. Then, 0.2 mL of supernatant was filtered through a 0.22 μm nylon syringe. The filtrate was used to analyze the atrazine adsorbed using HPLC.

Desorbed atrazine was determined by examining the solid phase of the centrifuged samples; 5 mL of methanol was added in each bottle and shaken during 24 h at 120 rpm in a flat rotatory shaker. After the agitation period, the bottles were centrifuged and filtered as mentioned above for further atrazine analysis.

#### **2.7. Atrazine analysis**

The analysis of atrazine degradation and its desorption-adsorption was performed using a Thermo-Scientific HPLC system coupled to a diode array detector (SpectraSystem UV8000), a sampling injector (SpectraSystem AS3000) and a pump (SpectraSystem P4000) equipped with a Restek ultra C18 column (5 mm × 150 mm × 4.6 mm). The column was operated at 25°C with a flow rate of 1.0 mL min−1 and an injection volume of 20 μL. An isocratic mobile phase was established using acetonitrile-water at a ratio of 70:30. The HPLC-photodiode array detector was monitored at 215 nm [8]. The HPLC method had a running time of 10 min and a retention time of 3.8 min, which enabled the detection and quantification of atrazine. The atrazine detection limit was 0.05 mg g soil−1. The standard curve for atrazine [atrazine = (peak area – 491818)/804962] was made using a standard analytical solution (Sigma-Aldrich Co., USA) at different concentrations, and the *r*<sup>2</sup> value was >0.99. The extraction efficiency of this method was 105%, and this value was taken into account in the final quantifications.
