**2. Materials and methods**

#### **2.1. General**

Ethyl acetate (PA), used to extract the reaction mixtures and the salts used to prepare artificial sea water were purchased from a commercial source (Synth, Vetec, Brazil). Ethyl acetate (HPLC grade) for the analytical curve was purchased from a commercial source (Tedia, Rio de Janeiro, Brazil). The malt extract and agar used in solid and liquid culture media were purchased from commercial sources (Acumedia and Himedia, Brazil).

#### **2.2. Pesticides**

The analytical standards of chlorpyrifos and profenofos were purchased from Sigma-Aldrich, Brazil. Commercial pesticide containing profenofos was purchased from Syngenta® under the name Polytrin 400/40 CE. The commercial profenofos used in the marine fungi biodegradation test was donated by Professor Marcos R. de V. Lanza (IQSC-USP). The 4-bromo-2-chlorophenol was purchased from Sigma-Aldrich, Brazil.

#### **2.3. Marine fungi**

The Brazilian marine-derived fungal strains *Aspergillus sydowii*-CBMAI 934, *Aspergillus sydowii*-CBMAI 935 and *Penicillium raistrickii* CBMAI 931 were isolated from the sponge *Chelonaplysilla erecta; Aspergillus sydowii* CBMAI 1241, *Penicillium decaturense* CBMAI 1234 and *Penicillium raistrickii* CBMAI 1235 were isolated from the sponge *Dragmacidon reticulata; Trichoderma* sp. CBMAI 932 was isolated from the sponge *Geodia corticostylifera*. The sponges were collected in the South Atlantic Ocean at São Sebastião in São Paulo state, Brazil, by Professor Roberto G. S. Berlinck (Chemistry Institute of Sao Carlos, University of São Paulo, IQSC-USP). The marine fungi were isolated and purified in the microbiology laboratory of the Department of Ecology and Evolutionary Biology supervised by Professor Mirna H. R. Seleghim (UFSCar-Brazil). The fungi were identified by both conventional and molecular methods at the Chemical, Biological and Agricultural Multidisciplinary Research Center at UNICAMP, São Paulo, Brazil (http://www.cpqba.unicamp.br/). The isolated and identified marine fungi were deposited in the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI).

### **2.4. Composition of marine fungi growth media**

main objective of this study was the screening of Brazilian marine fungi with the enzymes required for detoxification of organophosphate pesticides (phosphotriesterases-PTEs and /or carboxylesterases-CBEs). The biodegradation of profenofos in the presence of these selected fungi was evaluated, assessing the degradation of the pesticide, as well as the formation of the metabolite, 4-bromo-2-chlorophenol. This results are environmentally important, because the pesticides applied to crops can be leached into rivers, lakes and seas under these different

Ethyl acetate (PA), used to extract the reaction mixtures and the salts used to prepare artificial sea water were purchased from a commercial source (Synth, Vetec, Brazil). Ethyl acetate (HPLC grade) for the analytical curve was purchased from a commercial source (Tedia, Rio de Janeiro, Brazil). The malt extract and agar used in solid and liquid culture media were

The analytical standards of chlorpyrifos and profenofos were purchased from Sigma-Aldrich, Brazil. Commercial pesticide containing profenofos was purchased from Syngenta® under the name Polytrin 400/40 CE. The commercial profenofos used in the marine fungi biodegradation test was donated by Professor Marcos R. de V. Lanza (IQSC-USP). The 4-bromo-2-chlorophenol

The Brazilian marine-derived fungal strains *Aspergillus sydowii*-CBMAI 934, *Aspergillus sydowii*-CBMAI 935 and *Penicillium raistrickii* CBMAI 931 were isolated from the sponge *Chelonaplysilla erecta; Aspergillus sydowii* CBMAI 1241, *Penicillium decaturense* CBMAI 1234 and *Penicillium raistrickii* CBMAI 1235 were isolated from the sponge *Dragmacidon reticulata; Trichoderma* sp. CBMAI 932 was isolated from the sponge *Geodia corticostylifera*. The sponges were collected in the South Atlantic Ocean at São Sebastião in São Paulo state, Brazil, by Professor Roberto G. S. Berlinck (Chemistry Institute of Sao Carlos, University of São Paulo, IQSC-USP). The marine fungi were isolated and purified in the microbiology laboratory of the Department of Ecology and Evolutionary Biology supervised by Professor Mirna H. R. Seleghim (UFSCar-Brazil). The fungi were identified by both conventional and molecular methods at the Chemical, Biological and Agricultural Multidisciplinary Research Center at UNICAMP, São Paulo, Brazil (http://www.cpqba.unicamp.br/). The isolated and identified marine fungi were deposited in the Brazilian Collection of Environmental and Industrial

conditions, where they may suffer different biodegradation processes.

purchased from commercial sources (Acumedia and Himedia, Brazil).

**2. Materials and methods**

154 Applied Bioremediation - Active and Passive Approaches

was purchased from Sigma-Aldrich, Brazil.

**2.1. General**

**2.2. Pesticides**

**2.3. Marine fungi**

Microorganisms (CBMAI).

*Composition of Artificial Sea Water* (ASW) (1L): CaCl2.2H2O (1.36g), MgCl2.6H2O (9.68g), KCl (0.61g), NaCl (30.0g), Na2HPO4 (0.014 mg), Na2SO4 (3.47g), NaHCO3 (0.17g), KBr (0.1g), SrCl2.6H2O (0.040g), H3BO3 (0.030g).

*Solid medium for stock cultures:* agar (20 g. L-1) and malt extract (20 g.L-1) in ASW (1L) and adjusted to pH 8 by addition of 3M KOH.

*Solid medium for fungal screening:* agar (20 g. L-1) and malt extract (20 g.L-1) dissolved in ASW (1L) and adjusted to pH 5 by addition of 3M KOH or 1M HCl.

*Liquid medium:* malt extract (20 g.L-1) in ASW (1L), adjusted to pH 7 by addition of 3M KOH or 1M HCl.

*Liquid mineral medium supplemented with KNO3(12.5 ppm)*: KNO3 (12.5 mg.L-1) dissolved in ASW (1L), adjusted to pH 7 by addition of 3M KOH or 1M HCl.

The culture media were sterilized in autoclave for 20 minutes (at 121 °C, 1.5 kPa). All manip‐ ulations involving marine fungi were carried out under sterile conditions in a Veco laminar flow cabinet. The stock cultures of the marine microorganisms were stored on solid culture medium (25 mL), in Petri dishes, maintained at 4°C in the refrigerator.

### **2.5. Cultivation of marine fungi on solid medium in the presence of profenofos**

Marine fungi were screened by culturing on Petri dishes containing 25 mL of solid culture medium (2.0 g of malt extract, 2.0 g of agar and 100 mL of ASW) with the addition of profenofos and without (control culture). After the medium sterilization in the autoclave, the agar was cooled to 40-45°C and the profenofos was added at three different concentrations: 5.0, 10.0 and 15.0 μL per plate, solubilized in 100.0, 200.0 and 300.0 μL of dimethyl sulfoxide (DMSO), respectively. At room temperature, fungal mycelia from recent cultures were transferred to the surfaces of the agar plates with an inoculating loop. The fungi were incubated for 10 days at 35°C. Tolerance of profenofos was estimated by the size of the colony formed on the surface of the plates, relative to the control culture.

#### **2.6. Analytical curve**

Stock solutions of 500.0 ppm of profenofos, 4-bromo-2-chlorophenol (main metabolite) and chlorpyrifos (used as internal standard) were prepared.

*Profenofos 500.0 ppm:* 3.4 μL (1.3 mmol) of profenofos analytical standard and ethyl acetate (10.0 mL).

*4-bromo-2-chlorophenol 500.0 ppm*: 5.0 mg (2.4 mol) of 4-bromo-2-chlorophenol and ethyl acetate (10.0 mL).

*Chlorpyrifos 500.0 ppm*: 5.0 mg (1.4 mol) of chlorpyrifos and ethyl acetate (10.0 mL).

All standard solutions were prepared in a volumetric flask and made up to the containing 10.0 mL mark with ethyl acetate (HPLC grade). From these stock solutions were prepared the working solutions, at concentrations of 5.0, 10.0, 20.0, 30.0 and 50.0 ppm of profenofos and 4 bromo-2-chlorophenol, in ethyl acetate (HPLC grade). The concentration of internal standard, chlorpyrifos, was maintained at 30.0 ppm in all assays. Next, 1.0 mL aliquots from the stock solutions were transferred into 1.5 mL vials. Triplicates samples, for each concentration of analyte, were analyzed by GC-MS-SIM.

*2.8.1. Biodegradation of 4-bromo-2-chlorophenol by A. sydowii CBMAI 935 and P. raistrickii CBMAI*

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These reactions were prepared in 250-mL Erlenmeyer flasks containing 100.0 mL of liquid medium at pH 7 (Section 2.8) in which the 4-bromo-2-chlorophenol (50.0 ppm, 2.4 mmol) was added. The reaction was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The inoculations, extractions and analyses proceeded as described in Section 2.8. The results are

*2.8.2. Biodegradation of profenofos by A. sydowii CBMAI 935 and P. raistrickii CBMAI 931 in liquid*

Two small slices of solid medium (1.2 cm x 1.2 cm) bearing the marine fungal mycelia were transferred to 250 mL Erlenmeyer flasks containing 100 mL of liquid mineral medium (1.25 g of KNO3 and 100.0 mL of ASW, pH 7), previously sterilized in autoclave for 20 minutes at 121°C. Next, 100.0 ppm (37.2 μL) of commercial profenofos was added to the medium. The reaction was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as described in Section 2.8. The results are summarized in Table 7.

To a 250 mL Erlenmeyer flask containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7), previously sterilized in the autoclave for 20 minutes at 121°C, 50.0 ppm (18.6 μL, Section 2.5) of commercial profenofos was added. The reaction was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as described in

*2.8.4. Growth of marine fungi A. sydowii CBMAI 935 and P. raistrickii CBMAI 931 in the absence of*

In a 250 mL Erlenmeyer flasks containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7), previously sterilized in the autoclave for 20 minutes at 121°C, two small slices of solid medium (1.2 cm x 1.2 cm) bearing the marine fungi was added, without any profenofos. The culture medium was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as described in Section 2.8. The results are summarized in

**2.9. Biodegradation of the pesticide profenofos at various concentrations by** *P. raistrickii*

In four 250 mL Erlenmeyer flasks containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7), previously sterilized in the autoclave for 20 minutes at 121°C, commercial profenofos was added separately at 15.0 ppm (18.6 μL), 30 ppm (11.2 μL), 50.0 ppm (18.6 μL) and 60.0 ppm (22.3 μL). In the flasks were inoculated two small slices of solid medium (1.2 cm x 1.2 cm) bearing mycelium of *P. raistrickii* CBMAI 931. These reactions were incubated in an

*931 in liquid medium*

summarized in Table 6.

*profenofos*

Table 9.

**CBMAI 931**

*minimal medium supplemented with KNO3*

*2.8.3. Degradation of profenofos in the absence of marine fungi*

Section 2.8. The results are summarized in Table 8.

#### **2.7. Determination of profenofos concentration in commercial sample**

The sample was prepared with 20.0 μL of commercial pesticide profenofos and 60.0 μL (2.4 mmol, 30.0 ppm internal standard) of stock solution of chlorpyrifos 500.0 ppm in ethyl acetate (HPLC grade), in a 100.0 mL volumetric flask. Duplicate samples of 1.0 mL were prepared and analyzed by GC-MS-SIM.

#### **2.8. Biodegradation of profenofos by** *A. sydowii* **CBMAI 935 and** *P. raistrickii* **CBMAI 931 in liquid medium**

The fungi *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 were inoculated in solid culture medium (2.0 g of malt extract, 2.0 g of agar) in the presence of profenofos (50.0 ppm) and incubated for 7 days at 35°C. Two small slices of solid medium (1.2 cm x 1.2 cm) bearing the marine fungal mycelia were transferred to 250 mL Erlenmeyer flasks containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7). The liquid medium containing the inocula were incubated in an orbital shaker (Technal TE-421 or Superohm G-25) for 4 days (130 rpm, 32°C). After that, 50.0 ppm (18.6 μL, Section 2.6) of commercial profenofos was added. The reaction was incubated in the orbital shaker for 30 days (130 rpm, 32°C).

Extractions were performed at 10, 20 and 30 days. The reaction culture was filtered on a Buchner funnel to separate the mycelia from liquid medium. The mycelial mass obtained was rinsed and suspended in water and ethyl acetate (1:1). The mixture was stirred magnetically for 30 minutes and filtered again using Buchner funnel.

For the 10-days reaction, the extractions were then analyzed separately on the first mycelial extract and filtered medium. The liquid medium and mycelial extract were acidified to pH 6 and extracted separately, three times with ethyl acetate (3 x 25 mL) (Table 3).

For reactions at 20 and 30 days, the liquid medium and mycelial extract (after the Buchner filtration and extraction of mycelia with water and ethyl acetate, 1:1) were put together in an Erlenmeyer flask, acidified to pH 6 and extracted three times with ethyl acetate (3 x 25 mL). The filtered mycelial cells were dried in an oven (35 °C, 24 h) and then weighed (Tables 4-5).

After extractions, the organic phase was dried over anhydrous Na2SO4, followed by solvent filtration and evaporation, resulting in a final volume of 100.0 mL. The residual (no degraded) profenofos and the 4-bromo-2-chlorophenol released were analyzed by a gas cromatography coupled to a mass spectrometer in single-ion monitoring mode (GC-MS-SIM). Under these conditions, the concentration of pesticide and metabolite were determined by comparing the peak area of the samples with an analytical curve. The biodegradation results are summarized in Tables 6-10. Further degradation and growth experiments were performed, to test some parameters (Sections 2.8.1-2.8.4).

#### *2.8.1. Biodegradation of 4-bromo-2-chlorophenol by A. sydowii CBMAI 935 and P. raistrickii CBMAI 931 in liquid medium*

These reactions were prepared in 250-mL Erlenmeyer flasks containing 100.0 mL of liquid medium at pH 7 (Section 2.8) in which the 4-bromo-2-chlorophenol (50.0 ppm, 2.4 mmol) was added. The reaction was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The inoculations, extractions and analyses proceeded as described in Section 2.8. The results are summarized in Table 6.

#### *2.8.2. Biodegradation of profenofos by A. sydowii CBMAI 935 and P. raistrickii CBMAI 931 in liquid minimal medium supplemented with KNO3*

Two small slices of solid medium (1.2 cm x 1.2 cm) bearing the marine fungal mycelia were transferred to 250 mL Erlenmeyer flasks containing 100 mL of liquid mineral medium (1.25 g of KNO3 and 100.0 mL of ASW, pH 7), previously sterilized in autoclave for 20 minutes at 121°C. Next, 100.0 ppm (37.2 μL) of commercial profenofos was added to the medium. The reaction was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as described in Section 2.8. The results are summarized in Table 7.

#### *2.8.3. Degradation of profenofos in the absence of marine fungi*

working solutions, at concentrations of 5.0, 10.0, 20.0, 30.0 and 50.0 ppm of profenofos and 4 bromo-2-chlorophenol, in ethyl acetate (HPLC grade). The concentration of internal standard, chlorpyrifos, was maintained at 30.0 ppm in all assays. Next, 1.0 mL aliquots from the stock solutions were transferred into 1.5 mL vials. Triplicates samples, for each concentration of

The sample was prepared with 20.0 μL of commercial pesticide profenofos and 60.0 μL (2.4 mmol, 30.0 ppm internal standard) of stock solution of chlorpyrifos 500.0 ppm in ethyl acetate (HPLC grade), in a 100.0 mL volumetric flask. Duplicate samples of 1.0 mL were prepared and

**2.8. Biodegradation of profenofos by** *A. sydowii* **CBMAI 935 and** *P. raistrickii* **CBMAI 931**

The fungi *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 were inoculated in solid culture medium (2.0 g of malt extract, 2.0 g of agar) in the presence of profenofos (50.0 ppm) and incubated for 7 days at 35°C. Two small slices of solid medium (1.2 cm x 1.2 cm) bearing the marine fungal mycelia were transferred to 250 mL Erlenmeyer flasks containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7). The liquid medium containing the inocula were incubated in an orbital shaker (Technal TE-421 or Superohm G-25) for 4 days (130 rpm, 32°C). After that, 50.0 ppm (18.6 μL, Section 2.6) of commercial profenofos was added.

Extractions were performed at 10, 20 and 30 days. The reaction culture was filtered on a Buchner funnel to separate the mycelia from liquid medium. The mycelial mass obtained was rinsed and suspended in water and ethyl acetate (1:1). The mixture was stirred magnetically

For the 10-days reaction, the extractions were then analyzed separately on the first mycelial extract and filtered medium. The liquid medium and mycelial extract were acidified to pH 6

For reactions at 20 and 30 days, the liquid medium and mycelial extract (after the Buchner filtration and extraction of mycelia with water and ethyl acetate, 1:1) were put together in an Erlenmeyer flask, acidified to pH 6 and extracted three times with ethyl acetate (3 x 25 mL). The filtered mycelial cells were dried in an oven (35 °C, 24 h) and then weighed (Tables 4-5).

After extractions, the organic phase was dried over anhydrous Na2SO4, followed by solvent filtration and evaporation, resulting in a final volume of 100.0 mL. The residual (no degraded) profenofos and the 4-bromo-2-chlorophenol released were analyzed by a gas cromatography coupled to a mass spectrometer in single-ion monitoring mode (GC-MS-SIM). Under these conditions, the concentration of pesticide and metabolite were determined by comparing the peak area of the samples with an analytical curve. The biodegradation results are summarized in Tables 6-10. Further degradation and growth experiments were performed, to test some

The reaction was incubated in the orbital shaker for 30 days (130 rpm, 32°C).

and extracted separately, three times with ethyl acetate (3 x 25 mL) (Table 3).

for 30 minutes and filtered again using Buchner funnel.

parameters (Sections 2.8.1-2.8.4).

**2.7. Determination of profenofos concentration in commercial sample**

analyte, were analyzed by GC-MS-SIM.

156 Applied Bioremediation - Active and Passive Approaches

analyzed by GC-MS-SIM.

**in liquid medium**

To a 250 mL Erlenmeyer flask containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7), previously sterilized in the autoclave for 20 minutes at 121°C, 50.0 ppm (18.6 μL, Section 2.5) of commercial profenofos was added. The reaction was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as described in Section 2.8. The results are summarized in Table 8.

#### *2.8.4. Growth of marine fungi A. sydowii CBMAI 935 and P. raistrickii CBMAI 931 in the absence of profenofos*

In a 250 mL Erlenmeyer flasks containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7), previously sterilized in the autoclave for 20 minutes at 121°C, two small slices of solid medium (1.2 cm x 1.2 cm) bearing the marine fungi was added, without any profenofos. The culture medium was incubated in an orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as described in Section 2.8. The results are summarized in Table 9.

#### **2.9. Biodegradation of the pesticide profenofos at various concentrations by** *P. raistrickii* **CBMAI 931**

In four 250 mL Erlenmeyer flasks containing liquid medium (2.0 g of malt extract and 100.0 mL of ASW, pH 7), previously sterilized in the autoclave for 20 minutes at 121°C, commercial profenofos was added separately at 15.0 ppm (18.6 μL), 30 ppm (11.2 μL), 50.0 ppm (18.6 μL) and 60.0 ppm (22.3 μL). In the flasks were inoculated two small slices of solid medium (1.2 cm x 1.2 cm) bearing mycelium of *P. raistrickii* CBMAI 931. These reactions were incubated in an orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as in Section 2.8. The results are summarized in Table 10.

added to the solid cultures were 5.0, 10.0 and 15.0 μL per Petri dish, corresponding to con‐

After 10 days of growth at 35 °C, the colony diameters were measured and the average diameter (cm) of the colonies formed on each Petri dish was recorded. Since most of the colonies showed non-circular radial growth (Figure 5), they were measured between the furthest points. Figure 5 summarizes the qualitative results of the marine fungi growth on solid culture media in the

When several colonies grow in a Petri dish, one colony can compete and/or inhibit the growth of another. In this experiment on solid medium, it was important to assess fungal growth on the plate surface to detect the presence or absence of microbial growth. However, the meas‐ urement of colonies had no quantitative purpose, and the test was done only to estimate the

**Marine fungi Colony diameter (cm)**

\*Estimated measure, because the number of spores did not allow observation of the set of colonies

*Aspergillus sydowii* CBMAI 934 4.0 x 3.0 1.0 x 1.5 1.0 x 1.0 1.0 x 1.0 *Aspergillus sydowii* CBMAI 935 Whole plate\* 3.0 x 2.5 3.0 x 2.5 3.0 x 2.5 *Aspergillus sydowii* CBMAI 1241 3.5 x 2.5 3.0 x 2.5 3.0 x 2.5 2.0 x 2.0 *Penicillium* d*ecaturense* CBMAI 1234 2.5 x 2.5 1.0 x 1.5\* 3.0 x 2.0 1.0 x 1.0\* *Penicillium raistrickii* CBMAI 931 3.5 x 3.0 3.0 x 3.0 3.0 x 2.5 2.5 x 2.5 *Penicillium raistrickii* CBMAI 1235 4.0 x 3.0 2.0 x 2.0 1.0 x 1.0 1.5 x 1.0 *Trichoderma* sp. CBMAI 932 Whole plate\* 3.5 x 2.5 3.0 x 2.0 2.0 x 2.0

**Table 2.** Growth of marine fungi on solid agar medium of malt extract 2% with absence and addition of profenofos

Fungal development and growth requires a variety of inorganic and organic nutrients in the medium. Carbon is one of the most important elements for microbial growth, as carbon compounds provide energy for cell growth and serve as the basic units to build the cell materials. Nitrogen is also essential to the organisms, as well as other elements (hydrogen, oxygen and phosphorus) (Pelczar *et al.,* 1997). Thus, fungal growth in the presence of pesticides may indicate fungal tolerance to the pesticide toxicity; pesticide metabolism as a mechanism of defense of the microorganism to eliminate the xenobiotic compound; or even pesticide use as a source of nutrient for fungal growth, since the organophosphate pesticide profenofos has

In the screening of fungal strains on solid medium, in the presence of profenofos, excepting by the marine fungi *A. sydowii* CBMAI 934 and *P. raistrickii* CBMAI 1235, all other microor‐ ganisms (*P. raistrickii* CBMAI 931, *A. sydowii* CBMAI 935, *A. sydowii* CBMAI 1241 and *Tricho‐*

**Control culture 80.0 ppma 160.0 ppmb 240.0 ppmc**

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centrations of 80.0, 160.0 and 240.0 ppm, respectively (Table 2).

fungal growth.

a

c

5.0 µL profenofos and 100.0 µL DMSO b10.0 µL profenofos and 200.0 µL DMSO

15.0 µL profenofos and 300.0 µL DMSO

carbon, oxygen, sulfur and phosphorus in its structure.

(35 °C, 10 days, pH 5).

absence and presence of profenofos, for the strains which growth-better.

#### **2.10. GC-MS analyses**

The GC-MS system was a Shimadzu GC2010plus gas chromatograph coupled to a massselective detector (ShimadzuMS2010plus) in electron ionization (EI, 70 eV) mode. The GC-MS oven was fitted with a DB5 fused silica column (J&W Scientific 30m x 0.25mm x 0.25 μm). The chromatographic conditions were: initial oven temperature 100 °C (for 5 min), increased to 250 °C (for 10 min) at 5 °C/min; run time 45.0 min; injector temperature 200 °C; detector temper‐ ature 200 °C; injector split ratio 1:1; helium carrier gas at a pressure of 60 kPa. The analytes were first analyzed in SCAN mode in order to select the ion and the retention time for each compound. The selected-ion mode (SIM) analyses were performed to measure the biodegra‐ dation of profenofos. Table 1 shows the retention time and selected ion for each compound, used in the SIM-mode analyses.


**Table 1.** Method data of the SIM-mode analyses.
