Method data of the SIM-mode analyses.
Pesticides play an important role in modern agriculture. Synthetic pesticides are recognized as a cost-effective method of controlling pests, improving productivity and food quality. However, while pesticides may have a beneficial effect on agricultural productivity, their indiscriminate use causes many serious problems to the environment and human health, since these compounds are toxic to non-target species (Diez, 2010; Coutinho et al., 2005).
The fate of pesticides in the environment is influenced by many processes (biological, chemical and physical) that determine their persistence and mobility (Gravilescu, 2005). Millions of tons of pesticides are applied annually, but it is believed that only a small fraction of these products effectively reaches the target organisms, and the remainder are deposited on the soil, contaminating non-target organisms and moving into the atmosphere and water (Eerd
Detoxification of pesticides
Effective techniques for soil bioremediation are bioaugmentation, biostimulation, phytoremediation and enzymatic bioremediation. However, the three first techniques are limited by their dependence upon the growth rate of the remediating plants and microbes, which will vary with nutrients, aeration, pH and other factors relating to the contaminated soil (Scott et al., 2008; Sutherland et al., 2004). A successful bioremediation technique requires efficient organisms that can degrade pollutant to a minimum level. In the case of pesticides, an adequate rate of biodegradation is required to attain the acceptable level of a pesticide residue or its metabolites at the contaminated site in a limited period of time (Singh, 2008).
Organophosphate pesticides (OPs) are used worldwide in agriculture, municipal hygiene, disease vector control and against household pests; they were also a group of compounds used historically as chemical warfare agents (Yang et al., 2008; Zheng et al., 2007; Edwards and Tchounwou, 2005). OPs are phosphorus-containing pesticides whose insecticidal qualities were first observed in Germany during World War II (Edwards and Tchounwou, 2005). The principal types are phosphotriesters, thiophosphotriesters, and phosphorothiolesters. Phosphotriesters contain a phosphate center with three
This chemical class of pesticides has been used to replace the organochlorine pesticides, banned in the United States since the 1970s (Jauregui et al., 2003). However, the OPs are also highly toxic pesticides, since they are potent irreversible acetylcholinesterase (AChE) inhibitors that have a profound effect on the nervous system of exposed organisms, including human beings (Edwards and Tchounwou, 2005).
The hydrolysis mechanism normally catalyzed by AChE depends on the attack of a serine residue at the active site on the carbonyl group in ACh, but in the presence of organophosphates, this residue is readily phosphorylated, as follows: a histidine residue at the active site captures a proton from the serine residue, increasing its nucleophilic character, so that it readily attacks the electrophilic phosphorus atom, releasing the leaving group (X) (Figure 2). Unlike the acetylated enzyme, the phosphorylated enzyme reacts slowly with water, allowing the dealkylation of the alkoxy substituent (R2) attached to the phosphorus atom. The organophosphate compounds thus inactivate acetylcholinesterase by phosphorylation of the serine at the enzyme active site. The result is the formation of a strong hydrogen bond between a protonated histidine residue of the catalytic site and the negatively charged oxygen atom of the inhibitor. Therefore, the protonated histidine cannot function as a general base catalyst for the hydrolysis of the phosphorylated enzyme, which is a necessary step for the reactivation of AChE (Figure 2) (Mileson et al., 1998; Santos et al., 2007)
According the Brazillian Food, Drug and Sanitary Surveillance Agency (ANVISA), analysis of pesticide waste in food showed that OPs are those with the greatest number of occurrences in unsatisfactory samples. Among then, chlorpyrifos, methamidophos and acephate are the main active ingredients responsible for food contamination. Profenofos appeared to be the 12th commonest active ingredient in irregular samples of food, being found in samples of orange, strawberry and pepper (ANVISA, 2012).
Classified as a moderately hazardous (Toxicity class II) pesticide by the World Health Organization (WHO) (Abass et al., 2007; Malghani et al., 2009), profenofos has a moderate order of acute toxicity following oral and dermal administration (McDaniel and Moser, 2004; Abass et al., 2007). According to US Environmental Protection Agency (EPA), profenofos was first registered in the United States in 1982 and about 775,000 pounds (lbs.) of active ingredient are applied to cotton each year (EPA, 2012).
Chemical decontamination of organophosphates relies on bleach treatment, alkaline hydrolysis or incineration, but these conditions are harsh and the byproducts can be toxic (Ghanem and Raushel, 2005). Specific bioremediation of OPs requires highly specialized enzymes, so genetic engineering has been used to improve the properties of enzymes from various sources to enhance catalytic rates, stability and substrate range (Sutherland et al., 2004).
A number of enzymes capable of detoxifying OPs have been discovered and the majority of them belong to the class of phosphotriesterases (PTE). Various PTEs have been identified: organophosphate hydrolase (OPH), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1) (Bigley and Raushel, 2013). All of these enzymes are found to promote the hydrolysis of organophosphate compounds. The most frequently cited enzyme in the literature, OPH, isolated from the bacteria
Some degradation pathways are described in the literature for profenofos. The metabolic pathway of profenofos in cotton plants involves the cleavage of the phosphorothioate ester bond to yield 4-bromo-2-chlorophenol, followed by conjugation with glucose (Capps et al., 1996). In the literature there are some cases of (bio)degradation of organophosphate pesticides that occur by subsequent biotransformations, yielding novel polar metabolites, such as glycosylated and sulfated derivatives. According to the Food and Agriculture Organization of the United Nations (FAO), in aerobic soil conditions, profenofos degraded rapidly, with mineralization and formation of unextracted residues. In sterilized soil, cleavage of the phenol-phosphorus ester bond in profenofos proceeded via chemical hydrolysis, with accumulation of 4-bromo-2-chlorophenol and formation of unextracted residues. The metabolic biotransformations of profenofos in plants and animals are similar and occur via hydrolysis to 4-bromo-2-chlorophenol which is then conjugated by several enzymatic reactions (Figure 4) (FAO, 2012).
Fungi degrade a wide variety of compounds, a process known as mycodegradation. This process involves degradation to smaller molecules which may be toxic or non-toxic, as well as the removal of the pesticide molecule through a simple absorption or adsorption mechanism (Ramadevi et al., 2012).
The ability of bacterial species to degrade organophosphates is well established and researches have even proposed possible degradation mechanisms for the OPs (Van Eerd et al., 2003). However, the mechanisms of fungal degradation of these compounds are less established than those used by bacteria, since there are few studies on fungal degradation of OPs.
There are few studies on the biodegradation of profenofos by microorganisms. Malghani
Marine enzymes have a great potential for use in biocatalytic reactions, as in biodegradation of pesticides, due to the peculiar characteristics of the marine environment. As the sea covers more than three quarters of the Earth's surface and provide abundant resources for biotechnological research and development (Rush et al., 2007), marine organisms offer a dramatically different environment for the biosynthesis of molecules than terrestrial organisms, and are a vast untapped source of enzymes (Venter et al., 2004; Venter et al., 2010). In recent years, a variety of new enzymes with specific activities have been isolated from bacteria, fungi and other marine organisms; moreover, some can produce a considerable number of molecules with potential to be transformed into commercial drugs (Ghosh et al., 2005; Haefner, 2003). In fact, the marine environment is a very rich source of extremely potent compounds exhibiting significant activities in anti-tumor, anti-inflammatory, analgesic, immunomodulatory, allergic and anti-viral assays (Newman and Cragg, 2004; San-Martín et al., 2008).
Marine organisms in general (fungi, bacteria, algae, sponges, fish, prawns and other crustaceans) can be rich sources of novel enzymes, but most of the current bioprospecting activity focuses on microbial ones. A marine enzyme is a protein molecule with unique properties as it is derived from an organism whose natural habitat is saline or brackish water (Trincone, 2010; Sarkar et al., 2010). These enzymes can be biocatalysts with properties such as high salt tolerance, hyperthermostability, barophilicity and cold adaptability. Microorganisms isolated from ocean sediment and seawater are the most widely studied sources of marine enzymes, especially proteases, carbohydrases and peroxidases (Ghosh et al., 2005).
Enzymatic reactions catalyzed by marine fungi can be used when the fungi are cultured in media based on artificial seawater. The filamentous marine fungi
In this chapter, the first results obtained in the biodegradation of profenofos by whole cells of marine fungi are presented. Marine fungi were selected by us, with high potential to biodegrade profenofos and its main metabolite. The results presented in this chapter explore the potential of marine fungi in biotransformation and biodegradation of a xenobiotic (pesticide profenofos). The fungal biodegradation of OPs is still underexplored by researches, especially with regard to the biodegradation of profenofos, making this work extremely relevant. The 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 conditions, where they may suffer different biodegradation processes.
2. Materials and methods
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).
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
2.4. Composition of marine fungi growth media
The culture media were sterilized in autoclave for 20 minutes (at 121 °C, 1.5 kPa). All manipulations 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.
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.
2.7. 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. sydowiiCBMAI 935 and P. raistrickiiCBMAI 931 in liquid medium
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. sydowiiCBMAI 935 and P. raistrickiiCBMAI 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. sydowiiCBMAI 935 and P. raistrickiiCBMAI 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
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. sydowiiCBMAI 935 and P. raistrickiiCBMAI 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. raistrickiiCBMAI 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
2.10. GC-MS analyses
The GC-MS system was a Shimadzu GC2010plus gas chromatograph coupled to a mass-selective 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 temperature 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 biodegradation of profenofos. Table 1 shows the retention time and selected ion for each compound, used in the SIM-mode analyses.
3. Results and discussion
3.1. Screening marine fungi on solid medium
The strains studied were the filamentous marine fungi
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 concentrations 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 measurement of colonies had no quantitative purpose, and the test was done only to estimate the fungal growth.
|4.0 x 3.0||1.0 x 1.5||1.0 x 1.0||1.0 x 1.0|
|Whole plate*||3.0 x 2.5||3.0 x 2.5||3.0 x 2.5|
|3.5 x 2.5||3.0 x 2.5||3.0 x 2.5||2.0 x 2.0|
|2.5 x 2.5||1.0 x 1.5*||3.0 x 2.0||1.0 x 1.0*|
|3.5 x 3.0||3.0 x 3.0||3.0 x 2.5||2.5 x 2.5|
|4.0 x 3.0||2.0 x 2.0||1.0 x 1.0||1.5 x 1.0|
|Whole plate*||3.5 x 2.5||3.0 x 2.0||2.0 x 2.0|
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
There was a difference between the growth on the plate with 10.0 µL of pesticide and the other amounts, for the fungus
Finally, after this screening on solid culture medium, the strains of
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 hydrolysis 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
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 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. raistrickiiCBMAI 931 and A. sydowiiCBMAI 935
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
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
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. raistrickiiCBMAI 931 and A. sydowiiCBMAI 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
The duplicate reactions in the experiments with the fungus
|Reaction 1 (Extraction of liquid medium)||2.1||2.7c||53.0|
|Reaction 1 (Extraction of mycelium)||0.96||8.5||20.8|
|Reaction 2 (Extraction of liquid medium)||1.4||2.4c||45.0|
|Reaction 2 (Extraction of mycelium)||0.64||7.7||25.1|
|Reaction 1 (Extraction of liquid medium)||8.4||1.1||81.4|
|Reaction 1 (Extraction of mycelium)||0.29||1.7||8.2|
|Reaction 2 (Extraction of liquid medium)||9.3||3.0||48.0|
|Reaction 2 (Extraction of mycelium)||0.36||1.6||23.0|
The concentrations of profenofos in the extracts of the liquid culture medium from the
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
3.4.2. Biodegradation of profenofos by marine fungi
P. raistrickiiCBMAI 931 and A. sydowiiCBMAI 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
The results for both marine fungi showed a good percentage of biodegradation of the pesticide profenofos at 20 days of reaction. However,
|Reaction 1 (Extraction of liquid medium and mycelium)||0.36||5.5||13.0||74.0|
|Reaction 2 (Extraction of liquid medium and mycelium)||0.33||4.9||16.3||67.4|
|Reaction 1 (Extraction of liquid medium and mycelium)||0.19||13.8||11.0||78.0|
|Reaction 2 (Extraction of liquid medium and mycelium)||0.21||11.0||7.4||85.2|
3.4.3. Biodegradation of profenofos by marine fungi
P. raistrickiiCBMAI 931 and A. sydowiiCBMAI 935 after 30 days of reaction
Finally, profenofos biodegradation reactions using marine fungi
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);
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 source, and nitrate as the nitrogen source (Table 7);
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 medium (Table 8);
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 without the pesticide influence (Table 9).
|Reaction 1 (Extraction of liquid medium and mycelium)||0.38||12.0||12.5||75.0|
|Reaction 2 (Extraction of liquid medium and mycelium)||0.39||11.0||16.0||68.0|
|Reaction 1 (Extraction of liquid medium and mycelium)||0.22||17.8||2.3*||95.4|
|Reaction 2 (Extraction of liquid medium and mycelium)||0.20||21.4||0.6||98.8|
|Reaction 1 (Extraction of liquid medium and mycelium)||0.39||0.8||98.4|
|Reaction 2 (Extraction of liquid medium and mycelium)||0.37||0.9||98.2|
|Reaction 1 (Extraction of liquid medium and mycelium)||0.30||1.8||96.4|
|Reaction 2 (Extraction of liquid medium and mycelium)||0.35||1.2||97.6|
Profenofos concentrations for reaction in liquid medium with 2% malt extract and reaction in mineral medium, in the presence of
|Reaction 1 (Extraction of liquid medium and mycelium)||*||39.5||3.7||92.6|
|Reaction 2 (Extraction of liquid medium and mycelium)||*||38.0||5.7||88.6|
|Reaction 1 (Extraction of liquid medium and mycelium)||*||46.3||2.2c||95.6|
|Reaction 2 (Extraction of liquid medium and mycelium)||*||44.8||1.8c||96.4|
|Reaction 1 (Extraction of liquid medium and mycelium)||3.3||30.2||39.6|
|Reaction 2 (Extraction of liquid medium and mycelium)||4.2||31.0||38.0|
It was observed that the biodegradation of profenofos by
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
The final concentrations of profenofos in 30 days biodegradation reactions with
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 percentage 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 biodegradation 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
3.4.4. Biodegradation of the pesticide profenofos by
P. raistrickiiCBMAI 931 at several concentrations
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 biodegradation 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.
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
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 phosphotriesterases enzymes. The fungus also promoted the degradation or conversion of the part of the metabolite.
The growth of fungal strains on profenofos was promising, even at the highest tested concentration. The fungi
Liquid medium reactions using
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 Tecnológico (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).
Abass K, Reponen R, Jalonen J, Pelkonen O. In vitro metabolism and interaction of profenofos by human, mouse and rat liver preparations. Pesticide Biochemistry and Physiology 2007; 87: 238–247.
ANVISA, 2011 - Agência Nacional de Vigilância Sanitária. http://www4.anvisa.gov.br/base/visadoc/CP/CP[4708-1-0].PDF. (Accessed 2011/09).
ANVISA, 2012 - Agência Nacional de Vigilância Sanitária. http://portal.anvisa.gov.br/wps/wcm/connect/ b380fe004965d38ab6abf74ed75891ae/Relat%C3%B3rio+PARA+2010+-+Vers%C3%A3o +Final.pdf?MOD=AJPERES (Accessed 2012/08).
Ahmed RMAH. Chemical and biochemical studies on the residuals of the organophosphorus insecticide (prothiofos) in potato plant. Thesis. Department of Chemistry Faculty of Science Zagazig University. http://lib.bioinfo.pl/files/theses/thesis_11.pdf. (Accessed 2012/08)
Ali OA, Badawy MI. Hydrolysis of organophosphate insecticides in aqueous media. Environmental International 1982; 7:373-377.
Bigley, A. N, Raushel, F. M. Catalytic mechanisms for phosphotriesterases. Biochimica et Biophysica Acta (BBA)-Proteins & Proteomics (2013). , 1, 443-453”
Bonugli-Santos RC, Durrant LR, Da Silva M, Sette LD. Production of laccase, manganese peroxidase and lignin peroxidase by brazilian marine-derived fungi. Enzyme and Microbial Technology 2010; 46: 32-37.
Capps TM, Barringer VM, Eberle WJ, Brown DR, Sanson DR. Identification of a unique glucosylsulfate conjugate metabolite of profenofos in cotton. Journal of Agricultural and Food Chemistry 1996; 44:2408-2411.
Chen W, Mulchandani A . The use of live biocatalysts for pesticide detoxification. Tibtech 1988; 16:71-76.
Chowdhury A, Pradhan S, Saha M, Sanyal N. (2008) Impact of pesticides on soil microbiological parameters and possible bioremediation strategies. Indian Journal of Microbiology 2008; 48:114-127.
Coutinho CFB, Tanimoto ST, Galli A, Garbellini GS, Takayama M, Amaral RB, Mazo LH, Avaca LA, Machado SAS. Pesticidas: mecanismo de ação, degradação e toxidez. Pesticidas: Revista de Ecotoxicologia e Meio Ambiente 2005; 15:65-72.
Diez MC. Biological aspects involved in the degradation of organic pollutants. Journal of Plant Nutrition and Soil Science 2010;10: 244-267.
Edwards FL, Tchounwou PB. Environmental toxicology and health effects associated with methyl parathion exposure – A scientific review. International Journal of Environmental Research and Public Health 2005; 2:430-441.
Eerd LAV, Hoagland RE, Zablotowicz RM, Hall JC. Pesticide metabolism in plants and microorganisms. Weed Science 2003; 51: 472–495.
Madigan, M. T, Dunlap, P.V, Clark, D.P. Microbiologia de Brock. Porto Alegre: Artmed (2010).
EPA, 2012 - Environmental Protection Agency Reregistratione eligibility decision for profenofos. http://www.epa.gov/ oppsrrd1/REDs/profenofos_red.pdf (Accessed 2012 /08).
FAO, 2012 - Food and Agriculture Organization of the United Nations (2012) http://www.fao.org/fileadmin/templates/ agphome/documents/Pests_Pesticides/JMPR/Evaluation08/Profenofos.pdf. Accessed 2012/08.
Gavrilescu M. Fate of pesticides in the environment and its bioremediation. Engineering in Life Sciences 2005; 5:497-525.
Ghanem E, Raushel FM. Detoxification of organophosphate nerve agents by bacterial phosphotriesterase. Toxicology and Applied Pharmacology 2005; 207:S459 – S470.
Ghosh D, Saha M, Sana B, Murkherjee J. Marine enzymes. Advances in Biochemical Engineering/Biotechnology 2005; 96:189-218.
Haefner B. Drugs from the deep: Marine natural products as drug candidates. Drug Discovery Today 2003; 8:536-544.
Hasan HA. Fungal utilization of organophosphate pesticides and their degradation by Aspergillus flavusand A. sydowiiin soil. Folia Microbiology (Praha) 1999; 44:77-84.
Jauregui J, Valderrama B, Albores A, Vazquez-duhalt R. Microsomal transformation of organophosphorus pesticides by white rot fungi. Biodegradation 2003;14:397–406.
Kogure K. Bioenergetics of marine bacteria. Current Opinion in Biotechnology 1998; 9:278-282.
MacLeod RA. The question of the existence of specific marine bacteria. Bacteriological Review 1965; 29: 9-23.
Eivazi, F, Tabatabai, M. Phosphatases in soils. Soil Biology and Biochemistry (1977). , 9,167-172.
Malghani S, Chatterjee N, Yu HX, Luo Z. Isolation and identification of profenofos degrading bacteria. Brazilian Journal of Microbiology 2009;40: 893-900.
Martins MP, Mouad AM, Boschini L, Seleghim MHR, Sette LD, Porto ALM. Marine fungi Aspergillus sydowiiand Trichodermasp. catalyze the hydrolysis of benzyl glycidyl ether. Marine Biotechnology 2011; 13:314-320.
McDaniel KL, Moser VC. Differential profiles of cholinesterase inhibition and neurobehavioral effects in rats exposed to fenamiphos or profenofos. Neurotoxicology and Teratology 2004; 26:407–415.
Mileson BE, Chambers JE, Chen WL, Dettbarn W, Enrich M, Eldefrawi AT, Gaylor DW, Hamernik K, Hodgson E, Karczmar AG, Padilla S, Pope CN, Richardson RJ, Saunders DR, Sheets LP, Sultatos LG, Wallace KB. Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicological Sciences 1998; 41:8-20.
Newman DJ, Cragg GM. Marine natural products and related compounds in clinical and advanced preclinical trials. Journal of Natural Products 2004; 67: 1216-1238.
Ortega SN, Nitschke M, Mouad AM, Landgraf MD, Rezende MOO, Seleghim MHR, Sette LD, Porto ALM. Isolation of Brazilian marine fungi capable of growing on DDD pesticide. Biodegradation 2011; 22:43-50.
Pelczar MJ, Chan ECS, Krieg NR. Microbiologia. 2ªEd. São Paulo: Pearson Makron Books; 1997.
Ramadevi C, Nath MM, Prasad MG. Mycodegradation of malathion by a soil fungal isolate, Aspergillus niger International Journal of Basic and Applied Chemical Sciences 2012; 2:2277-2073.
Reddy NC, Rao JV. Biological response of earthworm, Eisenia foetida(Savigny) to an organophosphorous pesticide, profenofos. Ecotoxicology and Environmental Safety 2008; 71:574–582.
Ribani M, Bottoli CBG, Collis CH, Jardim ICSF, Melo LFC. Validação em métodos cromatográficos e eletroforéticos. Quimíca Nova 2004; 27:771-780.
Rissato SR, Libânio M, Giafferis GP, Gerenutti M. Determinação de pesticidas organoclorados em água de manancial, água potável e solo na região de Bauru (SP). Quimíca Nova 2004; 27:739-743.
Rocha LC, Ferreira HV, Pimenta EF, Berlinck RGS, Seleghim MHR, Javaroti DCD, Sette LD, Bonugli RC, Porto ALMBioreduction of α-chloroacetophenone by whole cells of marine fungi. Biotechnology Letters 2009; 3:1559-1563.
Rogers K R. Organophosphorus hydrolase-based assay for organophosphate pesticides. Biotechnology Progress 1999;15:517-521 .
Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S, Wu D, Eisen JA, Hoffman JM, Remington K, Beeson K, Tran B, Smith H, Baden-Tillson H, Stewart C, Thorpe J, Freeman J, Andrews-Pfannkoch C, Venter JE, Li K, Kravitz S, Heidelberg JF, Utterback T, Rogers YH, Falcón LI, Souza V, Bonilla-Rosso G, Eguiarte LE, Karl DM, Sathyendranath S, Platt T, Bermingham E, Gallardo V, Tamayo-Castillo G, Ferrari MR, Strausberg RL, Nealson K, Friedman R, Frazier M, Venter JC. The sorcerer ii global ocean sampling expedition: Northwest Atlantic through eastern tropical pacific. PLoS Biology 2007; 5:398-431.
San-Martín A, Rovirosa J, Astudillo L, Sepúlveda B, Ruiz D, San-Martín C. Biotransformation of the marine sesquiterpene pacifenol by a facultative marine fungus. Natural Product Research 2008; 22:1627-1632.
Santos VMR, Donnici CL, Dacosta JBN, Caixeiro JMR. Compostos organofosforados pentavalentes: histórico, métodos sintéticos de preparação e aplicações como inseticidas e agentes antitumorais. Quimíca Nova 2007; 30:159-170.
Sarkar S, Pramanik A, Mitra A, Mukherjee J. Bioprocessing data for the production of marine enzymes. Marine Drugs 2010; 8:1323-1372.
Scott C, Pandey G, Hartley CJ, Jackson JC, Cheesman MJ, Taylor MC, Pandey R, Khurana JL, Teese M, Coppin CW, Weir KM, Jain RK, Lal R, Russell RJ, Oakeshott JG. The enzymatic basis for pesticide bioremediation .Indian Journal of Microbiology 2008; 48: 65–79.
Singh DK. Biodegradation and bioremediation of pesticide in soil: concept, method and recent developments. Indian Journal of Microbiology 2008; 4:35–40.
Sogorb MA, Vilanova E. Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis. Toxicology Letters 2002; 128:215-228.
Sutherland T, Russell R, Selleck M. Using enzymes to clean up pesticide residues. Pesticide Outlook 2002; 13:149-151.
Sutherland TD, Horne I, Weir KM, Coppin CW, Williams MR, Selleck M, Russell RJ, Oakeshott JG. Enzymatic bioremediation: from enzyme discovery to applications. Clinical and Experimental Pharmacology and Physiology 2004; 31: 817–821.
Syngenta, 2012. http://www.syngenta.com/country/br/pt/produtosemarcas/protecao-de-cultivos/Pages/POLYTRIN.aspx (Accessed 2012/08).
Tortora, G. J, Funke, B. R, Case, C. L, Johnson, T. R. Microbiology: an introduction. San Francisco: Benjamin Cummings ( 2004).
Trincone A. Potential biocatalysts originating from sea environments. Journal of Molecular Catalysis B: Enzymatic 2010; 66: 241-256.
Van Eerd LL, Hoagland RE, Zablotowicz RM, Hall JC. Pesticide metabolism in plants and microorganisms. Weed Science 2003; 51:472–495.
Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson W, Fouts DE, Levy S, Knap AH, Lomas MW, Nealson K, White O, Peterson J, Hoffman J, Parsons R, Baden-Tillson H, Pfannkoch C, Rogers Y-H, Smith HO. Environmental genome shotgun sequencing of the Sargasso sea. Science 2004; 304:66-74.
Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson, Trincone A. Potential biocatalysts originating from sea environments. Journal of Molecular Catalysis B: Enzymatic 2010; 66:241-256.
Yang C, Cai N, Dong M, Jiang H, Li J, Qiao C, Mulchandani A, Chen W Surface display of MPH on Pseudomonas putidaJS444 using ice nucleation protein and its application in detoxification of organophosphates. Biotechnology and Bioengineering 2008; 99:30-37.
Zamy, C, Mazellier, P, Legube, B. Analytical and kinetic study of the aqueous hydrolysis of four organophosphorus and two carbamate pesticides. International Journal of Environmental Analytical Chemistry (2004). , 84, 1059-1068.
Zheng YZ, Lan WS, Qiao CL, Mulchandani A, Chen W Decontamination of vegetables sprayed with organophosphate pesticides by organophosphorus hydrolase and carboxylesterase (B1). Applied Biochemistry and Biotechnology 2007; 136:233-241.