**3. Results and discussion**

#### **3.1. Screening marine fungi on solid medium**

The strains studied were the filamentous marine fungi *Aspergillus sydowii* CBMAI 934, *Aspergillus sydowii* CBMAI 935, *Aspergillus sydowii* CBMAI 1241, *Penicillium decaturense* CBMAI 1234, *Penicillium raistrickii* CBMAI 931, *Penicillium raistrickii* CBMAI 1235 and *Trichoderma* sp. CBMAI 932. These are multicellular microorganisms, which grow as mycelia, composed by branching microscopic filament named hyphae. Fungi were grown on solid medium at pH 5, which is a good pH for the cultivation of most fungi, while the optimum may vary from 3.8 to 5.6. These pH values favor fungi growth and inhibit growth of most bacteria, which optimal culture condition is at higher pH (Pelczar *et al.*, 1997).

Initially, the biotransformation of profenofos by marine fungi was conducted on solid culture media. The microorganisms were grown on Petri dishes containing 2% malt extract and artificial seawater (ASW). All the strains investigated were analyzed in the presence and absence of the profenofos pesticide, in duplicate tests. Fungi with biocatalytic potential to degrade profenofos were screened by comparing the growth of fungal colonies on Petri dishes at several concentrations of the pesticide and in its absence (control). Volumes of profenofos added to the solid cultures were 5.0, 10.0 and 15.0 μL per Petri dish, corresponding to con‐ centrations of 80.0, 160.0 and 240.0 ppm, respectively (Table 2).

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

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 fungal growth.


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

a 5.0 µL profenofos and 100.0 µL DMSO

orbital shaker for 30 days (130 rpm, 32°C). The extractions and analyses proceeded as in Section

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,

**Compounds Retention time (min) Selected ion**

The strains studied were the filamentous marine fungi *Aspergillus sydowii* CBMAI 934, *Aspergillus sydowii* CBMAI 935, *Aspergillus sydowii* CBMAI 1241, *Penicillium decaturense* CBMAI 1234, *Penicillium raistrickii* CBMAI 931, *Penicillium raistrickii* CBMAI 1235 and *Trichoderma* sp. CBMAI 932. These are multicellular microorganisms, which grow as mycelia, composed by branching microscopic filament named hyphae. Fungi were grown on solid medium at pH 5, which is a good pH for the cultivation of most fungi, while the optimum may vary from 3.8 to 5.6. These pH values favor fungi growth and inhibit growth of most bacteria, which optimal

Initially, the biotransformation of profenofos by marine fungi was conducted on solid culture media. The microorganisms were grown on Petri dishes containing 2% malt extract and artificial seawater (ASW). All the strains investigated were analyzed in the presence and absence of the profenofos pesticide, in duplicate tests. Fungi with biocatalytic potential to degrade profenofos were screened by comparing the growth of fungal colonies on Petri dishes at several concentrations of the pesticide and in its absence (control). Volumes of profenofos

4-Bromo-2-chlorophenol 10.605 207.85 Chlorpyrifos 27.730 313.90 Profenofos 31.190 138.95

**(***m/z***)**

2.8. The results are summarized in Table 10.

158 Applied Bioremediation - Active and Passive Approaches

**2.10. GC-MS analyses**

used in the SIM-mode analyses.

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

**3.1. Screening marine fungi on solid medium**

culture condition is at higher pH (Pelczar *et al.*, 1997).

**3. Results and discussion**

b10.0 µL profenofos and 200.0 µL DMSO

c 15.0 µL profenofos and 300.0 µL DMSO

**Table 2.** Growth of marine fungi on solid agar medium of malt extract 2% with absence and addition of profenofos (35 °C, 10 days, pH 5).

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 carbon, oxygen, sulfur and phosphorus in its structure.

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‐*

metabolite is a product of the mycelial reaction, enzymes were possibly active in the mycelial mass of the marine fungi *P. raistrickii* CBMAI 931 and *A. sydowii* CBMAI 935. Analytical curves by the internal standard method of GC-MS-SIM analysis were constructed in order to deter‐ mine the concentration of the active ingredient profenofos in the commercial pesticide, the expected degradation product (4-bromo-2-chlorophenol) and the residual profenofos during the reaction. By analyzing, in the SCAN mode, a sample containing 4-bromo-2-chlorophenol (metabolite of profenofos), chlorpyrifos (internal standard) , the ion selected and retention time for each compound was determined (Figure 6, Table 1). For 4-bromo-2-chlorophenol (*m/z* 207.85) and chlorpyrifos (*m/z* 313.90), the molecular ions of each analyte was selected, while the base peak ion (*m/z* 138.95) was selected for profenofos. All samples were analyzed in SIM mode for quantification measurements and SCAN mode in the mass range of 50-550 u.m.a. for

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**Figure 6.** Mass spectra for analyses of the fragmentation patterns to select ions in the SCAN mode. (a) 4-Bromo-2-

The internal standard technique is a useful method for minimizing errors due to variations in the used equipments. A substance used as an internal standard should be similar to the analyte, with a similar retention time, not react with another substance or matrix component, not be a part of the test sample and have a retention time different from those of the other substances in the sample (Ribani *et al.*, 2004). The pesticide chlorpyrifos (analytical grade) was used as the internal standard for the determination of profenofos and its metabolite. A graph was produced, the area ratio (area of the substance / area of the internal standard) versus the

confirmation of the molecules identities.

(a)

(b)

)

(c)

chlorophenol, (b) Chlorpyrifos (c) Profenofos

**Figure 5.** Marine fungi growing on solid culture medium containing various concentrations of profenofos pesticide (10 days at 35°C)

*derma* sp. CBMAI 932) showed excellent growth in the presence of the pesticide at all concentrations after 10 days, as shown in Table 2. Compared to the control culture, there was a slight inhibition of the cultures by the largest amount of pesticide (15.0 μL profenofos) (Figure 5). Since fungal growth was satisfactory in the highest concentration of the pesticide, it was possible to suggest that these strains showed good potential for biocatalytic degradation of profenofos.

There was a difference between the growth on the plate with 10.0 μL of pesticide and the other amounts, for the fungus *P. decaturense* CBMAI 1234. In this Petri dish, there was no sporulation and colonies were well defined, with no significant inhibition of growth in comparison with the control plate. Strains of *A. sydowii* CBMAI 934 and *P. raistrickii* CBMAI 1235 showed only a slight growth, compared to the other fungi. However, these last three fungi were able to grow even at higher concentrations of the pesticide (Table 3).

Finally, after this screening on solid culture medium, the strains of *P. raistrickii* CBMAI 931 and *A. sydowii* CBMAI 935 were selected to investigate and quantify the biodegradation of profenofos in liquid culture medium.

#### **3.2. Analytical curves to determine the concentration of profenofos and 4-bromo-2 chlorophenol by GC-MS-SIM analysis**

The OPs are particularly amenable to biodegradation because they are susceptible to hydrol‐ ysis by enzymes (Chen and Mulchandani, 1998). The best known enzymes that promote hydrolysis of OPs are phosphotriesterases (Ghanem and Raushel, 2005). The expected metabolite from hydrolysis of profenofos is 4-bromo-2-chlorophenol. Therefore, if this metabolite is a product of the mycelial reaction, enzymes were possibly active in the mycelial mass of the marine fungi *P. raistrickii* CBMAI 931 and *A. sydowii* CBMAI 935. Analytical curves by the internal standard method of GC-MS-SIM analysis were constructed in order to deter‐ mine the concentration of the active ingredient profenofos in the commercial pesticide, the expected degradation product (4-bromo-2-chlorophenol) and the residual profenofos during the reaction. By analyzing, in the SCAN mode, a sample containing 4-bromo-2-chlorophenol (metabolite of profenofos), chlorpyrifos (internal standard) , the ion selected and retention time for each compound was determined (Figure 6, Table 1). For 4-bromo-2-chlorophenol (*m/z* 207.85) and chlorpyrifos (*m/z* 313.90), the molecular ions of each analyte was selected, while the base peak ion (*m/z* 138.95) was selected for profenofos. All samples were analyzed in SIM mode for quantification measurements and SCAN mode in the mass range of 50-550 u.m.a. for confirmation of the molecules identities.

*derma* sp. CBMAI 932) showed excellent growth in the presence of the pesticide at all concentrations after 10 days, as shown in Table 2. Compared to the control culture, there was a slight inhibition of the cultures by the largest amount of pesticide (15.0 μL profenofos) (Figure 5). Since fungal growth was satisfactory in the highest concentration of the pesticide, it was possible to suggest that these strains showed good potential for biocatalytic degradation of

**Figure 5.** Marine fungi growing on solid culture medium containing various concentrations of profenofos pesticide

There was a difference between the growth on the plate with 10.0 μL of pesticide and the other amounts, for the fungus *P. decaturense* CBMAI 1234. In this Petri dish, there was no sporulation and colonies were well defined, with no significant inhibition of growth in comparison with the control plate. Strains of *A. sydowii* CBMAI 934 and *P. raistrickii* CBMAI 1235 showed only a slight growth, compared to the other fungi. However, these last three fungi were able to grow

Finally, after this screening on solid culture medium, the strains of *P. raistrickii* CBMAI 931 and *A. sydowii* CBMAI 935 were selected to investigate and quantify the biodegradation of

The OPs are particularly amenable to biodegradation because they are susceptible to hydrol‐ ysis by enzymes (Chen and Mulchandani, 1998). The best known enzymes that promote hydrolysis of OPs are phosphotriesterases (Ghanem and Raushel, 2005). The expected metabolite from hydrolysis of profenofos is 4-bromo-2-chlorophenol. Therefore, if this

**3.2. Analytical curves to determine the concentration of profenofos and 4-bromo-2-**

even at higher concentrations of the pesticide (Table 3).

profenofos in liquid culture medium.

160 Applied Bioremediation - Active and Passive Approaches

**chlorophenol by GC-MS-SIM analysis**

profenofos.

(10 days at 35°C)

**Figure 6.** Mass spectra for analyses of the fragmentation patterns to select ions in the SCAN mode. (a) 4-Bromo-2 chlorophenol, (b) Chlorpyrifos (c) Profenofos

The internal standard technique is a useful method for minimizing errors due to variations in the used equipments. A substance used as an internal standard should be similar to the analyte, with a similar retention time, not react with another substance or matrix component, not be a part of the test sample and have a retention time different from those of the other substances in the sample (Ribani *et al.*, 2004). The pesticide chlorpyrifos (analytical grade) was used as the internal standard for the determination of profenofos and its metabolite. A graph was produced, the area ratio (area of the substance / area of the internal standard) versus the

The results were in good agreement and showed that, to obtain a final concentration of 50.0 ppm in 100.0 mL of liquid culture medium, 18.6 μL of commercial profenofos must be added. According to these data, the total concentration of active ingredient (profenofos) in the working

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The inocula used for the profenofos biodegradation reactions were activated in Petri dishes containing 2% of malt extract solid medium and 50.0 ppm of the pesticide, in order to induce the production of phosphotriesterases or other enzyme classes (e.g., CbE = carboxylesterase) capable of degrading the OP. Enzyme induction occurs at the gene transcription level. Gene transcription is the first step in the flow of genetic information and, for this reason, gene expression is relatively easily affected at this point (Madigan *et al.,* 2010; Tortora *et al.,* 1995). An inducible enzyme is synthesized only when its substrate is present in the sample; hence, the inoculum was grown in the presence of profenofos, so that the enzymes of interest were already being expressed when the fungi were transferred to the liquid medium, to catalyze

The pH of the liquid medium was adjusted to 7, bearing to reports in the literature indicating that phosphotriesterases exhibit enhanced catalytic activity at neutral to basic pH. According to Eivazi and Tabatabal, hydrolysis of the pesticide paraoxon with animal enzymes showed good catalytic activity at pH 7.3. Assays of activity by the release of *p*-nitrophenol showed optimal activity at pH 7-11 (Eivazi and Tabatabai, 1977). The hydrolysis of organophosphates in the environment (in the absence of enzymes) is also affected by pH: the more alkaline the medium, the faster is the hydrolysis. According to Zamy *et al.* the half-life of profenofos in phosphate buffer at pH 8 is fifteen days, the pesticide being hydrolyzed in this buffer. Thus, the period in which the pesticide should be completely biodegraded by hydrolysis is consid‐ erable; however, the presence of enzymes would accelerate the process of degradation (Zamy

The step of mycelium extraction was important because the fungi, as well as bacteria, can absorb compounds with the aid of enzymes secreted into medium, which break or carry the complex organic molecules into the cells (Pelczar *et al.*, 1997). Thus, the extraction with magnetic stirring was used to extract both the pesticide that may be inside the mycelium (since this extraction causes the cell disruption) and adhered to surface of the cell membrane.

*3.4.1. Biodegradation of profenofos by marine fungi P. raistrickii CBMAI 931 and A. sydowii*

At 10 days of reaction, the extracts of the mycelium and liquid medium were subjected to separate analysis by GC-MS-SIM. In Figures 8 and 9 chromatograms of each extraction are shown, with the analyses of the superimposed duplicates. The data concerning biodegradation of profenofos by fungal strains of *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 are

**3.4. Biodegradation of profenofos by marine fungi** *P. raistrickii* **CBMAI 931 and** *A.*

sample was approximately 320.0 g.L-1.

*sydowii* **CBMAI 935**

the biodegradation reactions.

*CBMAI 935 after 10 days of reaction*

summarized in Table 3.

*et al.*, 2004).

**Figure 7.** Analytical curves for (a) profenofos, (b) 4-bromo-2-chlorophenol

concentration ratio (variable concentration of substance / constant concentration of the internal standard) (Ribani *et al.*, 2004). This analytical curve was constructed for profenofos and 4 bromo-2-chlorophenol (metabolite of profenofos) at concentrations of 5.0, 10.0, 15.0, 20.0, 30.0 and 50.0 ppm (Figure 7).

The analytical curve for profenofos fitted by the linear equation y = 1.03965 x + 0.17522, with correlation coefficient r = 0.9955, and the one for the metabolite was fitted by the line y = 3.15088x + 0.2272, with correlation coefficient r = 0.99811.

The Brazil´s regulatory agency ANVISA recommends a correlation coefficient of 0.99; thus, the correlation coefficients obtained for the two analytical curves are within the parameters established in the literature (Ribani *et al.*, 2004).

#### **3.3. Determination of the active ingredients concentration in the profenofos commercial sample**

According to information from Syngenta® (Syngenta, 2012), the composition of the pesticide Polytrin, used in this study, was:

Inert ingredients: 560.00 g. L-1 (56.0% w/v)

Profenofos: 400.00 g. L-1 (40% w/v)

Cypermethrin: 40.00 g. L-1 (4% w/v)

The amount of active ingredient present in the working sample was measured, in order to develop reactions of biodegradation with the commercial sample of profenofos. To determine the volume of pesticide profenofos required to give a concentration of 50.0 ppm in the reaction, analyses were performed in duplicate with an arbitrary amount of pesticide (20.0 μL). The analytical data yielded 54.0 ppm for the concentration of active ingredient in 100 mL of medium.

The results were in good agreement and showed that, to obtain a final concentration of 50.0 ppm in 100.0 mL of liquid culture medium, 18.6 μL of commercial profenofos must be added. According to these data, the total concentration of active ingredient (profenofos) in the working sample was approximately 320.0 g.L-1.

#### **3.4. Biodegradation of profenofos by marine fungi** *P. raistrickii* **CBMAI 931 and** *A. sydowii* **CBMAI 935**

concentration ratio (variable concentration of substance / constant concentration of the internal standard) (Ribani *et al.*, 2004). This analytical curve was constructed for profenofos and 4 bromo-2-chlorophenol (metabolite of profenofos) at concentrations of 5.0, 10.0, 15.0, 20.0, 30.0

)

The analytical curve for profenofos fitted by the linear equation y = 1.03965 x + 0.17522, with correlation coefficient r = 0.9955, and the one for the metabolite was fitted by the line y =

The Brazil´s regulatory agency ANVISA recommends a correlation coefficient of 0.99; thus, the correlation coefficients obtained for the two analytical curves are within the parameters

**3.3. Determination of the active ingredients concentration in the profenofos commercial**

According to information from Syngenta® (Syngenta, 2012), the composition of the pesticide

The amount of active ingredient present in the working sample was measured, in order to develop reactions of biodegradation with the commercial sample of profenofos. To determine the volume of pesticide profenofos required to give a concentration of 50.0 ppm in the reaction, analyses were performed in duplicate with an arbitrary amount of pesticide (20.0 μL). The analytical data yielded 54.0 ppm for the concentration of active ingredient in 100 mL of

and 50.0 ppm (Figure 7).

**sample**

medium.

3.15088x + 0.2272, with correlation coefficient r = 0.99811.

**Figure 7.** Analytical curves for (a) profenofos, (b) 4-bromo-2-chlorophenol

(a) (b)

162 Applied Bioremediation - Active and Passive Approaches

established in the literature (Ribani *et al.*, 2004).

Polytrin, used in this study, was:

Profenofos: 400.00 g. L-1 (40% w/v)

Cypermethrin: 40.00 g. L-1 (4% w/v)

Inert ingredients: 560.00 g. L-1 (56.0% w/v)

The inocula used for the profenofos biodegradation reactions were activated in Petri dishes containing 2% of malt extract solid medium and 50.0 ppm of the pesticide, in order to induce the production of phosphotriesterases or other enzyme classes (e.g., CbE = carboxylesterase) capable of degrading the OP. Enzyme induction occurs at the gene transcription level. Gene transcription is the first step in the flow of genetic information and, for this reason, gene expression is relatively easily affected at this point (Madigan *et al.,* 2010; Tortora *et al.,* 1995). An inducible enzyme is synthesized only when its substrate is present in the sample; hence, the inoculum was grown in the presence of profenofos, so that the enzymes of interest were already being expressed when the fungi were transferred to the liquid medium, to catalyze the biodegradation reactions.

The pH of the liquid medium was adjusted to 7, bearing to reports in the literature indicating that phosphotriesterases exhibit enhanced catalytic activity at neutral to basic pH. According to Eivazi and Tabatabal, hydrolysis of the pesticide paraoxon with animal enzymes showed good catalytic activity at pH 7.3. Assays of activity by the release of *p*-nitrophenol showed optimal activity at pH 7-11 (Eivazi and Tabatabai, 1977). The hydrolysis of organophosphates in the environment (in the absence of enzymes) is also affected by pH: the more alkaline the medium, the faster is the hydrolysis. According to Zamy *et al.* the half-life of profenofos in phosphate buffer at pH 8 is fifteen days, the pesticide being hydrolyzed in this buffer. Thus, the period in which the pesticide should be completely biodegraded by hydrolysis is consid‐ erable; however, the presence of enzymes would accelerate the process of degradation (Zamy *et al.*, 2004).

The step of mycelium extraction was important because the fungi, as well as bacteria, can absorb compounds with the aid of enzymes secreted into medium, which break or carry the complex organic molecules into the cells (Pelczar *et al.*, 1997). Thus, the extraction with magnetic stirring was used to extract both the pesticide that may be inside the mycelium (since this extraction causes the cell disruption) and adhered to surface of the cell membrane.

#### *3.4.1. Biodegradation of profenofos by marine fungi P. raistrickii CBMAI 931 and A. sydowii CBMAI 935 after 10 days of reaction*

At 10 days of reaction, the extracts of the mycelium and liquid medium were subjected to separate analysis by GC-MS-SIM. In Figures 8 and 9 chromatograms of each extraction are shown, with the analyses of the superimposed duplicates. The data concerning biodegradation of profenofos by fungal strains of *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 are summarized in Table 3.

The duplicate reactions in the experiments with the fungus *A. sydowii* CBMAI 935 agreed well with each other: 53.0% and 45.0% of profenofos degraded. However, the duplicate reactions with *P. raistrickii* CBMAI 931, showed a significant discordance between for each reaction (81.4% and 48.0% degraded). As the growth behavior of the fungus varies for each experiment, other factors may have interfered and caused the difference between the results. It should also be noted that although the reactions are performed in duplicate, they occur independently and are, therefore, unique reactions.

*A. sydowii CBMAI 935 (50.0 ppm of profenofos)*

medium) **-** 2.1 2.7c 53.0

medium) **-** 1.4 2.4c 45.0

medium) **-** 8.4 1.1 81.4

medium) **-** 9.3 3.0 48.0

**Table 3.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 10 days,

The concentrations of profenofos in the extracts of the liquid culture medium from the *A. sydowii* CBMAI 935 reaction were estimated by straight line extrapolation, since, owing to the low concentrations in the extracts, the peak areas for profenofos were not sufficient to be

In the GC-MS-SIM analyses, superimposing the mycelium and liquid culture medium extract profiles for each fungus, it was noted a higher concentration of the pesticide in the mycelium extract than in the liquid medium one. In GC-MS-SIM analyses of *P. raistrickii* CBMAI 931 it was also clear that the highest concentration of the metabolite (4-bromo-2-chlorophenol) was in the liquid medium. These data may suggest that the fungal mycelium are absorbing profenofos molecules and, after metabolization, excreting a part of the metabolite into the liquid medium. Rather than being absorbed it is also possible that these molecules are adsorbed to fungal cells membranes. A previous study conducted by us showed that the pesticide DDD was accumulated in the mycelium of the marine fungus *Trichoderma* sp. CBMAI 932 (Ortega *et al., 2011*). The higher concentration of profenofos in the mycelium could be explained by an intracelullar enzyme degrading profenofos. According to Chen and Mulchandani, the

*P. raistrickii CBMAI 931 (50.0 ppm of profenofos)*

Reaction 1 (Extraction of mycelium) 0.96 8.5 20.8

Reaction 2 (Extraction of mycelium) 0.64 7.7 25.1

Reaction 1 (Extraction of mycelium) 0.29 1.7 8.2

Reaction 2 (Extraction of mycelium) 0.36 1.6 23.0

applied to the analytical curve; thus these values are only estimates.

c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

\*total of profenofos degraded (mycelium + liquid medium)

in liquid medium and mycelium (32 °C, 130 rpm, pH 7).

**(g) ca metaboliteb ca profenofos % of profenofos**

Biodegradation of the Organophosphate Pesticide Profenofos by Marine Fungi

**degraded\***

165

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**Duplicate reaction Fungal dry mass**

Reaction 1 (Extraction of liquid

Reaction 2 (Extraction of liquid

Reaction 1 (Extraction of liquid

Reaction 2 (Extraction of liquid

b4-bromo-2-chlorophenol

estimated concentration

a

c

**Figure 8.** GC-MS-SIM analyses: Chromatogram of biodegradation of 50.0 ppm profenofos by *A. sydowii* CBMAI 935, at 10 days. a) Extract of the mycelium. b) Extract of the liquid medium

**Figure 9.** GC-MS-SIM analyses: Chromatogram of biodegradation of 50.0 ppm profenofos by *P.raistrickii* CBMAI 931, at 10 days. a) Extract of the mycelium. b) Extract of the liquid medium


The duplicate reactions in the experiments with the fungus *A. sydowii* CBMAI 935 agreed well with each other: 53.0% and 45.0% of profenofos degraded. However, the duplicate reactions with *P. raistrickii* CBMAI 931, showed a significant discordance between for each reaction (81.4% and 48.0% degraded). As the growth behavior of the fungus varies for each experiment, other factors may have interfered and caused the difference between the results. It should also be noted that although the reactions are performed in duplicate, they occur independently and

**Figure 8.** GC-MS-SIM analyses: Chromatogram of biodegradation of 50.0 ppm profenofos by *A. sydowii* CBMAI 935, at

**Figure 9.** GC-MS-SIM analyses: Chromatogram of biodegradation of 50.0 ppm profenofos by *P.raistrickii* CBMAI 931,

10 days. a) Extract of the mycelium. b) Extract of the liquid medium

at 10 days. a) Extract of the mycelium. b) Extract of the liquid medium

are, therefore, unique reactions.

164 Applied Bioremediation - Active and Passive Approaches

**Table 3.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 10 days, in liquid medium and mycelium (32 °C, 130 rpm, pH 7).

The concentrations of profenofos in the extracts of the liquid culture medium from the *A. sydowii* CBMAI 935 reaction were estimated by straight line extrapolation, since, owing to the low concentrations in the extracts, the peak areas for profenofos were not sufficient to be applied to the analytical curve; thus these values are only estimates.

In the GC-MS-SIM analyses, superimposing the mycelium and liquid culture medium extract profiles for each fungus, it was noted a higher concentration of the pesticide in the mycelium extract than in the liquid medium one. In GC-MS-SIM analyses of *P. raistrickii* CBMAI 931 it was also clear that the highest concentration of the metabolite (4-bromo-2-chlorophenol) was in the liquid medium. These data may suggest that the fungal mycelium are absorbing profenofos molecules and, after metabolization, excreting a part of the metabolite into the liquid medium. Rather than being absorbed it is also possible that these molecules are adsorbed to fungal cells membranes. A previous study conducted by us showed that the pesticide DDD was accumulated in the mycelium of the marine fungus *Trichoderma* sp. CBMAI 932 (Ortega *et al., 2011*). The higher concentration of profenofos in the mycelium could be explained by an intracelullar enzyme degrading profenofos. According to Chen and Mulchandani, the organophosphate hydrolase enzyme is found within cells and, for biodegradation to occur, the pesticide should be transported into the interior of the cell. However, this kind of enzyme may limit biodegradation, because for microorganisms containing a high intracellular activity of degradative enzymes, total detoxification may be limited by the transport mechanism (Chen and Mulchandani, 1998).

*3.4.3. Biodegradation of profenofos by marine fungi P. raistrickii CBMAI 931 and A. sydowii*

Finally, profenofos biodegradation reactions using marine fungi *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 were performed for 30 days (Table 5, Figure 10). In this study, the

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**a.** 30-day reaction containing only 4-bromo-2-chlorophenol (main metabolite) as the substrate, with the objective of assessing the biocatalytic potential of these marine fungi for complete degradation of the pesticide into non-toxic metabolites (Table 6, Figure 11);

**b.** 30-day reaction in mineral medium supplemented with potassium nitrate in order to assess whether the fungi are able to grow on medium with the pesticide as the sole carbon

**c.** 30-day reaction for profenofos, in the absence of fungal mycelium, control experiment in order to determine the spontaneous rate of hydrolysis of the pesticide in the liquid

**d.** 30-day reaction with the fungi, in the absence of pesticide, control experiment in order to determine the growth of the marine fungi by measuring the mycelial mass produced

*A. sydowii CBMAI 935 (50.0 ppm of profenofos)*

mycelium) 0.38 12.0 12.5 75.0

mycelium) 0.39 11.0 16.0 68.0

mycelium) 0.22 17.8 2.3\* 95.4

mycelium) 0.20 21.4 0.6 98.8

**Table 5.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 30 days

*P. raistrickii CBMAI 931 (50.0 ppm of profenofos)*

**mass (g) ca metaboliteb ca profenofos % of profenofos**

**degraded**

*CBMAI 935 after 30 days of reaction*

medium (Table 8);

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

in liquid medium (32 °C, 130 rpm, pH 7).

b4-bromo-2-chlorophenol

\*estimated concentration

a

following experiments were also carried out:

source, and nitrate as the nitrogen source (Table 7);

**Duplicate reaction Fungal dry**

c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

without the pesticide influence (Table 9).

*3.4.2. Biodegradation of profenofos by marine fungi P. raistrickii CBMAI 931 and A. sydowii CBMAI 935 after 20 days of reaction*

The reactions that were performed for 20 days were analyzed by making a single extract from the liquid medium along with the mycelia extract that was subjected to GC-MS-SIM analyses. Table 4 shows data regarding the biodegradation of profenofos for 20 days by strains *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 in liquid medium.

The results for both marine fungi showed a good percentage of biodegradation of the pesticide profenofos at 20 days of reaction. However, *P. raistrickii* CBMAI 931 was more efficient than *A. sydowii* CBMAI 935, reaching an approximately average of 82% profenofos degradation, whereas *A. sydowii* CBMAI 935 showed degradation of approximately 71%.


a c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

b4-bromo-2-chlorophenol

**Table 4.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 20 days in liquid medium (32 °C, 130 rpm, pH 7).

#### *3.4.3. Biodegradation of profenofos by marine fungi P. raistrickii CBMAI 931 and A. sydowii CBMAI 935 after 30 days of reaction*

Finally, profenofos biodegradation reactions using marine fungi *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 were performed for 30 days (Table 5, Figure 10). In this study, the following experiments were also carried out:



a c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

b4-bromo-2-chlorophenol

organophosphate hydrolase enzyme is found within cells and, for biodegradation to occur, the pesticide should be transported into the interior of the cell. However, this kind of enzyme may limit biodegradation, because for microorganisms containing a high intracellular activity of degradative enzymes, total detoxification may be limited by the transport mechanism (Chen

*3.4.2. Biodegradation of profenofos by marine fungi P. raistrickii CBMAI 931 and A. sydowii CBMAI*

The reactions that were performed for 20 days were analyzed by making a single extract from the liquid medium along with the mycelia extract that was subjected to GC-MS-SIM analyses. Table 4 shows data regarding the biodegradation of profenofos for 20 days by strains *A.*

The results for both marine fungi showed a good percentage of biodegradation of the pesticide profenofos at 20 days of reaction. However, *P. raistrickii* CBMAI 931 was more efficient than *A. sydowii* CBMAI 935, reaching an approximately average of 82% profenofos degradation,

*A. sydowii CBMAI 935 (50.0 ppm of profenofos)*

*P. raistrickii CBMAI 931 (50.0 ppm of profenofos)*

**Table 4.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 20 days

**mass (g) ca metaboliteb ca profenofos % of profenofos**

0.36 5.5 13.0 74.0

0.33 4.9 16.3 67.4

0.19 13.8 11.0 78.0

0.21 11.0 7.4 85.2

**degraded**

*sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931 in liquid medium.

**Duplicate reaction Fungal dry**

whereas *A. sydowii* CBMAI 935 showed degradation of approximately 71%.

and Mulchandani, 1998).

166 Applied Bioremediation - Active and Passive Approaches

*935 after 20 days of reaction*

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

in liquid medium (32 °C, 130 rpm, pH 7).

c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

mycelium)

mycelium)

mycelium)

mycelium)

b4-bromo-2-chlorophenol

a

\*estimated concentration

**Table 5.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 30 days in liquid medium (32 °C, 130 rpm, pH 7).

**Figure 11.** GC-MS-SIM analyses: Chromatogram of biodegradation of 4-bromo-2-chlorophenol (50.0 ppm) at 30 days.

Profenofos concentrations for reaction in liquid medium with 2% malt extract and reaction in mineral medium, in the presence of *P. raistrickii* CBMAI 931, were estimated by straight line extrapolation of the analytical curve for profenofos. The same was done for all concentrations of the metabolite, in the reaction with the fungi *P. raistrickii* CBMAI 931 and *A. sydowii* CBMAI

*A. sydowii CBMAI 935 (100.0 ppm of profenofos)*

mycelium) \* 39.5 3.7 92.6

mycelium) \* 38.0 5.7 88.6

mycelium) \* 46.3 2.2c 95.6

mycelium) \* 44.8 1.8c 96.4

**Table 7.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 30 days,

*P. raistrickii CBMAI 931 (100.0 ppm of profenofos)*

**mass (g) ca metaboliteb ca profenofos**

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**% of profenofos degraded**

**Duplicate reaction Fungal dry**

a) *A. sydowii* CBMAI 935. b) *P. raistrickii* CBMAI 931

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

b4-bromo-2-chlorophenol

estimated concentration \*mycelial mass not obtained

c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

in liquid mineral medium supplemented with KNO3 (32 °C, 130 rpm, pH 7).

935.

a

c

**Figure 10.** GC-MS-SIM analyses: Chromatogram of biodegradation of profenofos (50.0 ppm) at 30 days. a) *A. sydowii* CBMAI 935. b) *P. raistrickii* CBMAI 931.


a c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

b4-bromo-2-chlorophenol

**Table 6.** Quantitative biodegradation of 4-bromo-2-chlorophenol (main metabolite) by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 30 days in liquid medium (32 °C, 130 rpm, pH 7).

**Figure 11.** GC-MS-SIM analyses: Chromatogram of biodegradation of 4-bromo-2-chlorophenol (50.0 ppm) at 30 days. a) *A. sydowii* CBMAI 935. b) *P. raistrickii* CBMAI 931

Profenofos concentrations for reaction in liquid medium with 2% malt extract and reaction in mineral medium, in the presence of *P. raistrickii* CBMAI 931, were estimated by straight line extrapolation of the analytical curve for profenofos. The same was done for all concentrations of the metabolite, in the reaction with the fungi *P. raistrickii* CBMAI 931 and *A. sydowii* CBMAI 935.


c estimated concentration

**Figure 10.** GC-MS-SIM analyses: Chromatogram of biodegradation of profenofos (50.0 ppm) at 30 days. a) *A. sydowii*

*A. sydowii CBMAI 935 (50.0 ppm of 4-bromo-2-chlorophenol)*

*P. raistrickii CBMAI 931 (50.0 ppm of 4-bromo-2-chlorophenol)*

**Table 6.** Quantitative biodegradation of 4-bromo-2-chlorophenol (main metabolite) by *A. sydowii* CBMAI 935 and *P.*

**mass (g) ca metaboliteb % of metabolite**

0.39 0.8 98.4

0.37 0.9 98.2

0.30 1.8 96.4

0.35 1.2 97.6

**degraded**

**Duplicate reaction Fungal dry**

CBMAI 935. b) *P. raistrickii* CBMAI 931.

168 Applied Bioremediation - Active and Passive Approaches

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

Reaction 1 (Extraction of liquid medium and

Reaction 2 (Extraction of liquid medium and

c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

*raistrickii* CBMAI 931, at 30 days in liquid medium (32 °C, 130 rpm, pH 7).

mycelium)

mycelium)

mycelium)

mycelium)

b4-bromo-2-chlorophenol

a

\*mycelial mass not obtained

**Table 7.** Quantitative biodegradation of profenofos by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, at 30 days, in liquid mineral medium supplemented with KNO3 (32 °C, 130 rpm, pH 7).


So, a possible explanation for the incomplete conversion of reactants to products may be the 4-bromo-2-chlorophenol further degradation or the conversion to other metabolites by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931. However, at 30 days of reaction, the biode‐ gradation of the pesticide was stagnant (compared to 20 days), so that stagnation in the degradation or conversion of the metabolite may also have occurred, causing a slight accu‐ mulation at 30 days of reaction. This stagnation may be caused either by fungal death or reaching its stationary growth phase, after one of the nutrients in the liquid medium became

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The final concentrations of profenofos in 30 days biodegradation reactions with *P. raistrickii* CBMAI 931 were estimated because of the very low concentration obtained, as previously mentioned. The biodegradation of the pesticide in the presence of this fungus was almost complete, suggesting that this has a greater biocatalytic potential than *A. sydowii* CBMAI 935

The results for the biodegradation of the metabolite were satisfactory, since there was an almost complete degradation (or conversion) of 4-bromo-2-chlorophenol by both fungi (Table 6). However, it was not possible to identify the other metabolites formed in this degradation.

Complementing the biodegradation studies, an experiment was conducted in liquid mineral medium supplemented with potassium nitrate that demonstrated, through the high percent‐ age of degradation of profenofos, that the fungi could be using the pesticide as a source of carbon, since this was the sole carbon source present in the reaction medium. The concentration of pesticide in the mineral medium was 100.0 ppm, higher than in the earlier tests, since the pesticide was the sole source of carbon, it was needed a high concentration for the fungi growth. There was a greater final concentration of the metabolite, which could be partially degraded / converted in other molecules, since only 50% of the pesticide was converted to this

Through the control reaction of profenofos, in the absence of the fungus (and hence without enzymes), the spontaneous hydrolysis of the pesticide in the medium was assessed. This experiment revealed degradation of about 40%, indicating that this pesticide is not persistent in the environment, since a half-life of about a month is relatively low compared to other pesticides, such as organochlorines. However, approximately 60% of the pesticide was not degraded, showing that the enzymatic process is highly effective for promoting the biodegra‐ dation of profenofos. It should be noted, also, that the spontaneous hydrolysis does not

Aly and Badway discussed the hydrolysis of profenofos at 20°C with buffered solutions at pH 5, 7 and 9. A loss of 50%occurred in 106 days at pH5, 43 days at pH7 and 0.7 days at pH 9. Rate constants and half-lives (t1/2) revealed that this insecticide was relatively stable in acid medium and its stability decreased in higher pHs. The studies showed that the mode of decomposition of profenofos in acidic and neutral media is dealkylation, but in an alkaline medium it undergoes hydrolysis, resulting in substituted phenol and dialkyphosphoric acid

promote the degradation of the metabolite, as does the enzymatic system (Table 8).

compounds (Figure 12) (Ali and Badawy, 1982; Ahmed, 2012).

scarce.

product.

for profenofos degradation.

a c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

b4-bromo-2-chlorophenol

**Table 8.** Quantitative degradation of profenofos by spontaneous hydrolysis, at 30 days in liquid medium (32 °C, 130 rpm, pH 7).


**Table 9.** Quantitative mycelia mass produced by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, in absence of profenofos, at 30 days in liquid medium (32 °C, 130 rpm, pH 7).

It was observed that the biodegradation of profenofos by *A. sydowii* CBMAI 935 exhibited stagnation between 20 and 30 days of reaction, as can be seen by comparing Tables 4 and 5 data. However, between 20 and 30 days, there was a significant increase in the metabolite concentration. According to literature data, the organophosphates hydrolysis proceeds by breaking the bond between the phosphorus atom and the leaving group (Sogorb and Vilanova, 2002; Bigley and Raushel, 2012). Thus, the concentration of profenofos degraded should be about the same as the concentration of metabolite formed, where there is total degradation of the pesticide to the metabolite. However, according the United Nations Food and Agriculture Organization (FAO), after the hydrolysis and formation of 4-bromo-2-chlorophenol, the latter can be conjugated with another molecule, can react with a molecule from the fungal metabo‐ lism or even be metabolized (Figure 4) (FAO, 2012).

So, a possible explanation for the incomplete conversion of reactants to products may be the 4-bromo-2-chlorophenol further degradation or the conversion to other metabolites by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931. However, at 30 days of reaction, the biode‐ gradation of the pesticide was stagnant (compared to 20 days), so that stagnation in the degradation or conversion of the metabolite may also have occurred, causing a slight accu‐ mulation at 30 days of reaction. This stagnation may be caused either by fungal death or reaching its stationary growth phase, after one of the nutrients in the liquid medium became scarce.

**Duplicate reaction ca metaboliteb ca profenofos**

**Table 8.** Quantitative degradation of profenofos by spontaneous hydrolysis, at 30 days in liquid medium (32 °C, 130

*A. sydowii CBMAI 935*

*P. raistrickii CBMAI 931*

**Table 9.** Quantitative mycelia mass produced by *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, in absence of

It was observed that the biodegradation of profenofos by *A. sydowii* CBMAI 935 exhibited stagnation between 20 and 30 days of reaction, as can be seen by comparing Tables 4 and 5 data. However, between 20 and 30 days, there was a significant increase in the metabolite concentration. According to literature data, the organophosphates hydrolysis proceeds by breaking the bond between the phosphorus atom and the leaving group (Sogorb and Vilanova, 2002; Bigley and Raushel, 2012). Thus, the concentration of profenofos degraded should be about the same as the concentration of metabolite formed, where there is total degradation of the pesticide to the metabolite. However, according the United Nations Food and Agriculture Organization (FAO), after the hydrolysis and formation of 4-bromo-2-chlorophenol, the latter can be conjugated with another molecule, can react with a molecule from the fungal metabo‐

**Duplicate reaction Fungal dry**

Reaction 1 (Extraction of liquid medium and

170 Applied Bioremediation - Active and Passive Approaches

Reaction 2 (Extraction of liquid medium and

c (ppm) = concentration (data duplicates) determined by GC-MS-SIM

Reaction 1 1.50 1.49 Reaction 2

Reaction 1 1.40 1.23 Reaction 2

profenofos, at 30 days in liquid medium (32 °C, 130 rpm, pH 7).

lism or even be metabolized (Figure 4) (FAO, 2012).

mycelium)

mycelium)

b4-bromo-2-chlorophenol

a

rpm, pH 7).

**% of profenofos degraded**

3.3 30.2 39.6

4.2 31.0 38.0

**mass (g)**

The final concentrations of profenofos in 30 days biodegradation reactions with *P. raistrickii* CBMAI 931 were estimated because of the very low concentration obtained, as previously mentioned. The biodegradation of the pesticide in the presence of this fungus was almost complete, suggesting that this has a greater biocatalytic potential than *A. sydowii* CBMAI 935 for profenofos degradation.

The results for the biodegradation of the metabolite were satisfactory, since there was an almost complete degradation (or conversion) of 4-bromo-2-chlorophenol by both fungi (Table 6). However, it was not possible to identify the other metabolites formed in this degradation.

Complementing the biodegradation studies, an experiment was conducted in liquid mineral medium supplemented with potassium nitrate that demonstrated, through the high percent‐ age of degradation of profenofos, that the fungi could be using the pesticide as a source of carbon, since this was the sole carbon source present in the reaction medium. The concentration of pesticide in the mineral medium was 100.0 ppm, higher than in the earlier tests, since the pesticide was the sole source of carbon, it was needed a high concentration for the fungi growth. There was a greater final concentration of the metabolite, which could be partially degraded / converted in other molecules, since only 50% of the pesticide was converted to this product.

Through the control reaction of profenofos, in the absence of the fungus (and hence without enzymes), the spontaneous hydrolysis of the pesticide in the medium was assessed. This experiment revealed degradation of about 40%, indicating that this pesticide is not persistent in the environment, since a half-life of about a month is relatively low compared to other pesticides, such as organochlorines. However, approximately 60% of the pesticide was not degraded, showing that the enzymatic process is highly effective for promoting the biodegra‐ dation of profenofos. It should be noted, also, that the spontaneous hydrolysis does not promote the degradation of the metabolite, as does the enzymatic system (Table 8).

Aly and Badway discussed the hydrolysis of profenofos at 20°C with buffered solutions at pH 5, 7 and 9. A loss of 50%occurred in 106 days at pH5, 43 days at pH7 and 0.7 days at pH 9. Rate constants and half-lives (t1/2) revealed that this insecticide was relatively stable in acid medium and its stability decreased in higher pHs. The studies showed that the mode of decomposition of profenofos in acidic and neutral media is dealkylation, but in an alkaline medium it undergoes hydrolysis, resulting in substituted phenol and dialkyphosphoric acid compounds (Figure 12) (Ali and Badawy, 1982; Ahmed, 2012).

In the control reaction of *A. sydowii* CBMAI 935 and *P. raistrickii* CBMAI 931, in the absence of the pesticide, there was a much greater mycelia growth indicating that the pesticide can partially inhibit the growth of fungi, as noted previously in the screenings carried out on solid medium (Table 9).

**Figure 12.** Degradation of profenofos in aqueous medium

#### *3.4.4. Biodegradation of the pesticide profenofos by P. raistrickii CBMAI 931 at several concentrations*

Figure 12. Degradation of profenofos in aqueous medium

Aiming to evaluate the biodegradation of the pesticide profenofos at various concentrations, this test was carried out with variations of the initial concentration under standard biodegra‐ dation reaction (liquid culture medium with malt + profenofos + fungal inoculum for 30 days). The reactions were performed in duplicate, at concentrations of 15.0, 30.0, 50.0 and 65.0 ppm, and also a pesticide control (liquid culture medium + profenofos) was carried out and average results are shown in Table 10.


a c (ppm) = concentration (data in duplicates) determined by GC-MS-SIM

b4-bromo-2-chlorophenol

**Table 10.** Quantitative biodegradation of profenofos by *P. raistrickii* CBMAI 931 in different concentrations, at 30 days in liquid medium (32 °C, 130 rpm, pH 7).

7

Almost complete biodegradation was observed at lower initial concentrations of the pesticide (15.0 and 30.0 ppm). At higher concentrations (50.0 and 65.0 ppm), the results were also satisfactory, with 95% degradation of profenofos. This test proved that in liquid medium, as well as on solid media, the fungus *P. raistrickii* CBMAI 931 was resistant to higher concentra‐ tions of pesticide and showed an excellent potential for biodegradation of profenofos.

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In the profenofos control, in the absence of the fungus, at a concentration of 30.0 ppm was observed a degradation of only 50% in a period of 30 days, confirming that the presence of fungi accelerates degradation reaction, possibly through the action of phosphotriesteras‐ es enzymes. The fungus also promoted the degradation or conversion of the part of the

The growth of fungal strains on profenofos was promising, even at the highest tested concen‐ tration. The fungi *P. raistrickii* CBMAI 931, *A. sydowii* CBMAI 935, *A. sydowii* CBMAI 1241 and *Trichoderma* sp. CBMAI 932 may have a good biocatalytic potential in the presence of profe‐ nofos, according to the results of screening. Marine fungi should be further explored as sources of enzymes capable of degrading OPs, since studies in fungal bioremediation of pesticides has

The fungi *P. raistrickii* CBMAI 931 and *A. sydowii* CBMAI 935 were efficient in profenofos biodegrading in liquid medium, as well as promoting the transformation or degradation of the metabolite, 4-bromo-2-chlorophenol, and showing that the enzymatic system of these fungi effectively expressed the necessary enzymes for a complete degradation process. Further research is under way to assess the biodegradation of methyl parathion and chlorpyrifos.

Liquid medium reactions using *P. raistrickii* CBMAI 931 with increasing concentration of the pesticide leaded to the almost complete biodegradation (99.0 to 95.0%) of the pesticide profenofos at all concentrations (15.0, 30.0, 50.0 and 65.0 ppm), showing that this fungus is resistant to high concentrations of this pesticide. The fungus *P. raistrickii*-CBMAI 931 may be a good source of phosphotriesterases, which could be isolated and purified for applications in biotechnology, biodegradation reactions in soil and water contaminated with pesticides.

NAS thanks FAPESP and WGB thanks CNPq for the scholarships. The authors wish to thank Prof. R.G.S. Berlinck (Instituto de Química de São Carlos - USP) for providing the marine fungal strains. ALMP is grateful to the Conselho Nacional de Desenvolvimento Científico e Tecno‐ lógico (CNPq) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) for financial support. The authors also wish to thank Professor Marcos Roberto de Vasconcellos Lanza (IQSC/USP, São Paulo, Brazil) for donating the commercial pesticides (profenofos).

shown great potential, albeit much less explored than bacterial bioremediation.

metabolite.

**4. Conclusion**

**Acknowledgements**

Almost complete biodegradation was observed at lower initial concentrations of the pesticide (15.0 and 30.0 ppm). At higher concentrations (50.0 and 65.0 ppm), the results were also satisfactory, with 95% degradation of profenofos. This test proved that in liquid medium, as well as on solid media, the fungus *P. raistrickii* CBMAI 931 was resistant to higher concentra‐ tions of pesticide and showed an excellent potential for biodegradation of profenofos.

In the profenofos control, in the absence of the fungus, at a concentration of 30.0 ppm was observed a degradation of only 50% in a period of 30 days, confirming that the presence of fungi accelerates degradation reaction, possibly through the action of phosphotriesteras‐ es enzymes. The fungus also promoted the degradation or conversion of the part of the metabolite.
