Degradation of terbuthylazine, difenoconazole and pendimethalin pesticides by selected fungi cultures

Science of the Total Environment 435–436 (2012) 402–410

Contents lists available at SciVerse ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Degradation of terbuthylazine, difenoconazole and pendimethalin pesticides by selected fungi cultures A.P. Pinto a, b,⁎, C. Serrano a, T. Pires a, E. Mestrinho a, L. Dias a, d, D. Martins Teixeira a, b, d, A.T. Caldeira a, c, d a

Chemistry Department, Évora University, Rua Romão Ramalho 59, 7000-671 Évora, Portugal ICAAM — Institute of Mediterranean Agricultural and Environmental Sciences, Évora University, Portugal CQE — Évora Chemistry Centre Évora University, Rua Romão Ramalho 59, 7000-671 Évora, Portugal d Hercules Laboratory, Évora University, Portugal b c

H I G H L I G H T S ► ► ► ► ►

Biodegradation of terbuthylazine, difenoconazole and pendimethalin was evaluated. Fungi capable to degrade the pesticides were isolated from a soil and a biomixture. All the fungi tested were very efficient in the biodegradation of the pesticides. L. saksenae (isolated fungi) could remove 99.5% of the pendimethalin. The biosorption of pesticides proved to be a process dependent on the fungi species.

a r t i c l e

i n f o

Article history: Received 27 April 2012 Received in revised form 9 July 2012 Accepted 9 July 2012 Available online 9 August 2012 Keywords: Biodegradation Difenoconazole Fungi Pendimethalin Terbuthylazine

a b s t r a c t Contamination of waters by xenobiotic compounds such as pesticides presents a serious environmental problem with substantial levels of pesticides now contaminating European water resources. The aim of this work was to evaluate the ability of the fungi Fusarium oxysporum, Aspergillus oryzae, Lentinula edodes, Penicillium brevicompactum and Lecanicillium saksenae, for the biodegradation of the pesticides terbuthylazine, difenoconazole and pendimethalin in batch liquid cultures. These pesticides are common soil and water contaminants and terbuthylazine is considered the most persistent triazine herbicide in surface environments. P. brevicompactum and L. saksenae were achieved by enrichment, isolation and screening of fungi capable to metabolize the pesticides studied. The isolates were obtained from two pesticide-primed materials (soil and biomixture). Despite the relatively high persistence of terbuthylazine, the results obtained in this work showed that the fungi species studied have a high capability of biotransformation of this xenobiotic, comparatively the results obtained in other similar studies. The highest removal percentage of terbuthylazine from liquid medium was achieved with A. oryzae (~80%), although the major biodegradation has been reached with P. brevicompactum. The higher ability of P. brevicompactum to metabolize terbuthylazine was presumably acquired through chronic exposure to contamination with the herbicide. L. saksenae could remove 99.5% of the available pendimethalin in batch liquid cultures. L. edodes proved to be a fungus with a high potential for biodegradation of pesticides, especially difenoconazole and pendimethalin. Furthermore, the metabolite desethyl-terbuthylazine was detected in L. edodes liquid culture medium, indicating terbuthylazine biodegradation by this fungus. The fungi strains investigated could prove to be valuable as active pesticide-degrading microorganisms, increasing the efficiency of biopurification systems containing wastewaters contaminated with the xenobiotics studied or compounds with similar intrinsic characteristics. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author at: Chemistry Department, Évora University, Rua Romão Ramalho 59, 7000-671 Évora, Portugal. Tel.: +351 266745310. E-mail address: [email protected] (A.P. Pinto). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2012.07.027

Throughout the world, environmental contamination through anthropogenic and industrial activities is a widespread and serious problem. The extensive and massive use of pesticides in agriculture activities has serious impacts on the environment, compromising soil and water quality (Köck-Schulmeyer et al., 2012; Pino and

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410

Peñuela, 2011; Roca et al., 2009; Younes and Galal-Gorchev, 2000). Several pesticides are used simultaneously on most agricultural crops, which lead to a higher risk and increased pollution (Pino and Peñuela, 2011; Roca et al., 2009). Emissions of pesticides in the environment are generally divided into diffuse and direct losses. Diffuse contamination via percolation, runoff, drainage and drift, explains only a part of the pesticides that reach surface and groundwater. Point-source contamination by pesticides has been identified as a major concern contributing significantly to the deterioration of natural water resource quality. Indeed, several studies demonstrated that 40–90% of surface water contamination is attributable to point-source contamination produced by improper pesticide handling before or after their field application (De Wilde, 2009; Spliid et al., 2006). Given the public concern for environmental pollution by pesticides, there is increasing attention towards the development of biopurification systems for reducing the risk from point source contamination of water resources (Karanasios et al., 2010). Biological systems are being developed all over European Union countries to protect water-bodies from pesticide contamination at farm level (Coppola et al., 2011). They are an efficient and sustainable way to treat pesticide contaminated wastewater and consist of a biologically active matrix that retains pesticides into the organic matter and enhances their microbial degradation (De Wilde et al., 2007, 2010). Biobeds are an example of a biological system used to reduce point source water contamination by pesticides (Coppola et al., 2007; Vischetti et al., 2007). They are simple to operate and cost effective on-farm systems in which the efficiency is based on their increasing capacity to retain and microbially degrade pesticides (Karanasios et al., 2010). Agricultural polluted wastewaters are discharged in the biobed, where, by leaching through a biomixture (mixture of different organic biomaterials and soil in different percentages), they are decontaminated, due to the adsorption process and the action of microbial communities (grown and selected within the biomass), able to degrade and/or metabolize pesticide residues. The system efficiency is high and it has been demonstrated that it can decrease some residues in the water by more than 90% (Coppola et al., 2011). The biomixture composition is a key factor controlling the efficacy of biobeds (Castillo et al., 2008; Karanasios et al., 2010). According to previous studies, the selection of organic material to be used as a biofilter is critical. The biofilter efficiency in water decontamination depends on the adsorption capacity of the material and on the presence of a microbial biomass active, genotypic and phenotypic versatile for the degradation of different residues even at high concentrations (Coppola et al., 2011). Indeed, microbial degradation of pesticides is the most important and effective way to remove these compounds from the environment. Microorganisms have the ability to interact, both chemically and physically, with substances leading to structural changes or complete degradation of the target molecule (Diez, 2010). Among the microbial communities, bacteria and fungi are the main transformers and pesticide degraders (Diez, 2010). Fungi generally biotransform pesticides and other xenobiotics by introducing minor structural changes to the molecule, rendering it nontoxic and released the biotransformed pesticide into the soil for further degradation by bacteria (Diez, 2010). The main oxidative enzyme producers to the remediation of polluted environments are white-rot fungi (Rao et al., 2010). Indeed, white-rot fungi have received extensive attention due to their powerful lignin-degrading enzyme system (Michel et al., 1991; Wu et al., 2005). These organisms are very effective, because they are robust organisms and may tolerate higher concentrations of pollutants than bacteria. Fungal degradation occurs extra-cellularly in two steps: firstly by the action of the hydrolytic system, that produces hydrolases responsible for degradation of macromolecular substances, and subsequently by the action of the oxidative, lignolytic system.

403

The oxidative lignolytic system is a complex, non-specific, very powerful extracellular enzymatic system and, under nutrient limiting conditions, is capable of degrading lignolytic compounds, dyes and several environmental pollutants that cannot be degraded by other microorganisms (Pointing, 2001; Rao et al., 2010; Sánchez, 2009). Many fungi, including the white-rot fungi, have been tested for their ability to degrade pesticides. Studies on pesticide metabolism by fungi such as A. oryzae degrading organophosphorus pesticide monocrotophos (Bhalerao and Puranik, 2009), Aspergillus niger degrading pyrethroids (Liang et al., 2005), endosulfan (Bhalerao and Puranik, 2007), carbaryl (Zhang et al., 2003) and dimethoate (Liu et al., 2001) and F. oxysporum degrading pendimethalin (Kulshrestha et al., 2000), are reported as results for pesticide biodegradation, emphasizing the enormous potential of soil fungi for bioremediation. The objectives of the present study were to investigate the ability of Penicillium brevicompactum PP0021 (Pinto et al., 2011), Lecanicillium saksenae PP0011 (Caldeira et al., 2011), Fusarium oxysporum CCMI 866, Aspergillus oryzae CCMI 125 and Lentinula edodes EL1 fungi strains on the degradation of the pesticides terbuthylazine, difenoconazole and pendimethalin. The strains P. brevicompactum PP0021 (Pinto et al., 2011) and L. saksenae PP0011 (Caldeira et al., 2011), were isolated from a two pesticide primed-material (soil and biomixture), therefore the isolation and characterization of these strains are also described. The pesticide-primed biomixture used in this study simulates the biologically active matrix of a biopurification system for further use in field application (e.g. biobeds). The pesticide-degrading microbial strains, able to mineralize the xenobiotics selected, may be used as inoculation sources in on-farm sustainable biopurification system, in order to increase its degradation efficiency, explained by the proliferation of an adapted population. 2. Material and methods 2.1. Chemicals Difenoconazole, pendimethalin and terbuthylazine of analytical standard grade (99%) were purchased from Sigma-Aldrich. Table 1 displays the three pesticides and formulated products used in the study. The pesticide aqueous solutions used in batch degradation studies were prepared in sterile conditions, by dilution from commercial suspensions with Milli-Q water. 2.2. Microorganisms The microorganisms used in this study were F. oxysporum CCMI 866, A. oryzae CCMI 125, L. edodes EL1, from the Culture Collection of the Industrial Microorganisms (Lisbon) and two strains isolated in our laboratory from a contaminated soil and a biomixture, P. brevicompactum PP0021 and L. saksenae PP0011 (see 2.3), respectively. 2.3. Isolation and identification of adapted soil indigenous fungal strains Native populations of fungi were isolated from samples of a loamy sand soil and a biomixture made under controlled laboratory conditions. Biomixture consisted of 10 kg of soil and granulated cork (25 g kg−1 soil), an organic substrate with high adsorption capacity and easily available in Portugal. The soil and biomixture used were contaminated six months earlier, with terbuthylazine (60 mg kg−1); difenoconazole (5 mg kg −1); and pendimethalin (60 mg kg−1). The physico-chemical characteristics of soil and cork used are given in Table S1 of the Supporting information section. Soil and biomixture were moistened to 80% of field capacity, covered with aluminum foil and incubated in the dark at room temperature during the period before the isolation and cultivation of more adapted microbial communities. During the incubation period the

404

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410

Table 1 Physicochemical characteristics of the pesticides studied. Formulated product

Aspect®

Score250EC®

Stomp33E®

Manufacturer Active ingredient Pesticide composition and formulation Pesticide type Molecular formula Chemical class Molecular weight (g mol−1) pH Density (g cm−3 at 20 °C) Water solubility (mgL−1 a pH = 7 e 20 °C)a Kow (log P) Henry's law constant 25 °C (Pa m3 mol−1) Vapor pressure 25° (mPa) LD50 (mammals — acute oral) mg kg−1 Bioaccumulation potential

Bayer Terbuthylazine 333 g a.i. l−1 SC Herbicide C9H16ClN5 Triazine 229.71 4–7 1.18 8.5 (20 °C) 3.20a,b 3.24 × 10−03 0.12 1590–2000 Low

Syngenta Difenoconazole 250 g a.i. l−1 EC Fungicide C19H17Cl2N3O3 Triazole 406.26 4–8 1.40 15 (25 °C) 4.20a 9.0 × 10−07 3.33 × 10−05 1453 Moderate

BASF Pendimethalin 330 g a.i. l−1 EC Herbicide C13H19N3O4 Dinitroaniline 281.31 6–7 1.17 0.3 5.18a,c 2.73 × 10−03 1.94 1250 High

a.i., active ingredient; EC, emulsifiable concentrate; Kow, octanol–water partition coefficient. a Pesticide Properties Database, University of Hertfordshine, 2011. b Bending et al., 2002. c Kimmo A. Mäenpää et al., 2003.

soils were thoroughly mixed every week to ensure adequate distribution of the pesticides. The isolation and cultivation of more adapted microbial communities were performed using samples (from soil or biomix) with 10 g, to which was added 90 ml of NaCl 0.9% solution and shaken mechanically for 1 h. Serial dilutions in saline solution were used to prepare Malt Extract Agar (MEA), Potato-Dextrose Agar (PDA) and Cook Rose Bengal (CRB) plates. Fungal colony isolation was done successively, using standard MEA, PDA and CRB mycological medium. All cultures were grown for 7 days at 28 °C. Macroscopic and microscopic characteristics of the obtained isolates were examined. The identification of fungi was based on the macroscopic features of colonies grown on agar plates, and the micro-morphology of the reproductive structures, identified by optical microscopy. The strains P. brevicompactum PP0021 and L. saksenae PP0011 were also characterized by partial sequence of 28S rDNA and ITS region. The genomic DNA extraction was carried out by using a kit (NucleoSpin Tissue-Macherey–Nagel, Germany). The DNA was precipitated with ethanol and maintained at 4 °C. DNA was amplified using the primers ITS 4 and ITS 5. The temperatures for denaturation annealing and extension were 94, 50 and 70 °C, respectively. The PCR products were purified by a kit (Nucleo Spin Extract II, Macherey– Nagel, Germany) and nucleotide sequences determined by using an automatic sequencer (ABI PRISM 310 Genetic Analyses), according to the manufacturer's protocol, using the Kit BDT v1.1 (Applied Biosystems). The results were processed by using the program Bio Edit 7 Sequence Alignment Editor. DNA partial sequences were published on GenBank, NCBI, with the accession number JQ282680 (P. brevicompactum PP0021) and JQ282679 (L. saksenae PP0011).

2.4. Growth conditions Growth took place in 1 l shake flasks, each containing 400 ml of defined medium with (NH4)2SO4 (1.5 g l −1); KH2PO4 (1.7 g l −1); Na2HPO4 (1.7 g l −1); yeast extract (0.5 g l −1); glucose (10 g l −1) and Vishniac solution sterilized separately (2 ml l −1). The cultures were shaken in the dark at room temperature on an orbital shaker at 150 rpm. Fungal growth period before batch degradation experiments was 8 days.

2.5. Batch degradation experiments After growth period, 600 μl of an aqueous suspension containing each pesticide separately, was added to the respective fungal batch cultures described at the above section. The final pesticide concentrations used were 25 mg l −1 of terbuthylazine and pendimethalin, and 19 mg l −1 of difenoconazole. Control experiments were performed in the same conditions. Each fungal culture containing the respective pesticide was incubated during 10 days at room temperature on a shaker at 150 rpm and sampled over time (12, 24, 36, 48, 72, 84, 96, 120, 144, 168, 192 and 240 h). Erlenmeyer flasks were covered with aluminum foil and all experiments carried out in triplicate. Furthermore, for each pesticide, blank assays were performed to discard abiotic degradation. After collecting, all samples were filtered by 0.45 μm PTFE filter (Macherey–Nagel, Germany). Samples were re-dissolved (1:1, v/v) in 80:20 (v/v) methanol: 0.1% H3PO4 water solution, and analyzed by high performance liquid chromatography with ultraviolet detection (HPLC–UV), to quantify the pesticides. At the end of the experiments, biomass was separated by centrifugation at 10,000 rpm for 10 min and the pesticide concentration in the supernatant was determined. The pesticide metabolites were also investigated. Microbial biomass separated by centrifugation was shaken with 20 ml of 80:20 (v/v) methanol: 0.1% H3PO4 water solution, during 48 h at 150 rpm and room temperature. At the end of the incubation period, the supernatant was separated by centrifugation at 10,000 rpm during 10 min, filtered by 0.45 μm PTFE filter and analyzed with HPLC– DAD or LC–ESI-MS. 2.6. Pesticide analysis 2.6.1. HPLC–UV analysis An Elite LaChrom HPLC system with UV detection (Hitachi, Japan) was used for quantification of pesticides. The analytical column was a reversed-phase Zorbax Eclipse XDB-C18, 250× 4.6 mm (length× I.D.) and 5 μm particle size (Agilent Technologies, Germany). The mobile phase was Solvent A: acetonitrile; Solvent B: 0.1% H3PO4 water solution. Gradient program was adopted as follows: linear from 60 to 85% of Solvent A (0–10 min) and isocratic at 85% of Solvent A (10–15 min). LC analyses were performed at room temperature with a flow-rate of 1.0 ml min−1 and an injection volume of 20 μl. The chromatographic profile was recorded at 225 nm for the three pesticides studied. Five replicate injections were made for each sample. Calibration curves were derived for standard solutions prepared in sterile

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410

conditions by dilution from commercial suspensions of terbuthylazine, difenoconazole and pendimethalin, separately. Three replicates were made for each standard solution and each solution was injected five times. The average areas of the compound peaks were plotted against the standard concentrations resulting in linear correlations with R 2 equal to or higher than 0.999 in every calibration curves. 2.6.2. LC–ESI-MS analysis Samples collected from liquid cultures of L. edodes, before and after the addition of the pesticides (at time 0 and 5 days), were analyzed by LC–ESI-MS in order to investigate the presence of fungal pesticide degradation products. These analyses were carried out in a LCQ Advantage ThermoFinnigan mass spectrometer equipped with an electrospray ionization source and using an ion trap mass analyzer. The conditions of the MS analysis were: capillary temperature of 350 °C; source voltage of 5.0 kV, source current of 100.0 μA and capillary voltage of 8.0 V in positive ion mode. The mass spectrometer equipment was coupled to an HPLC system with autosampler (Surveyor ThermoFinnigan) and diode array detector (DAD). The analytical column was a reversed phase Zorbax Eclipse XDB (C18, particle size 3.0 μm, 150 mm × 2.1 mm). The chromatographic separation was performed with mobile phase at a flow rate of 0.2 ml min−1, by injecting 20 μl of each sample and the elution program was equal to the one used in the HPLC–UV analysis. The DAD detector was scanned from 200 to 500 nm and the chromatographic profile was recorded at 225 nm. 2.7. Statistical analysis Statistical comparisons among treatments were made by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Levene's test was used to check homogeneity of variances. 3. Results and discussion 3.1. Pesticide removal from liquid culture In this work, liquid cultures of F. oxysporum CCMI 866, A. oryzae CCMI 125, L. edodes EL1, P. brevicompactum PP0021 and L. saksenae PP0011, were performed in the presence of the s-triazine herbicide terbuthylazine, the triazole fungicide difenoconazole and the dinitroaniline pre-emergence herbicide pendimethalin. The HPLC– UV analysis showed that all fungi were able to efficiently remove the xenobiotics from the liquid culture medium (Fig. 1). However, there was considerable variation between tested strains with respect to their abilities to degrade the pesticides. Observing Fig. 1, we can conclude that L. edodes was able to remove appreciable amounts of the three pesticides from liquid culture, although the greatest removal has occurred with pendimethalin. The fungi F. oxysporum and A. oryzae were also able to remove significant amounts of all pesticides, with both proving to be effective degraders of pendimethalin. For difenoconazole, the highest pesticide removal rate was obtained with P. brevicompactum, followed by A. oryzae, L. edodes, L. saksenae and finally F. oxysporum. The evolution of the pesticide removal rate from liquid culture (Fig. 1), shows that the highest removal rate occurred in the first hours of exposure whereas during the remaining period, much lower increases were observed for all pesticides. This can be explained, probably, by the simultaneous occurrence of biodegradation of the pesticide accumulated in cells. Indeed, following uptake, xenobiotic must be biodegraded in the microbial cells (although this process appears to be slow), otherwise toxic intracellular accumulation of the xenobiotic would lead to cell death. The results of the batch degradation studies led to the remaining percentages of terbuthylazine, difenoconazole and pendimethalin in liquid cultures, shown in Table 2. The pesticides were removed

405

in the following order of efficiencies, by all studied fungi, pendimethalin > difenoconazole > terbuthylazine. The lower removal percentages were exhibited with terbuthylazine, by all fungi. Triazine herbicides including terbuthylazine, form a wide group of compounds used for pre- and post-emergence weed control. They are very persistent in soil, water, plant and animals. Among s-triazines, terbuthylazine is the most persistent in environmental compartments (Carafa et al., 2007; Navarro et al., 2004). The main degradation process is microbial and its half-life in the soil has been reported to vary between 5 and 331 days depending on the soil characteristics and temperature (Navarro et al., 2004). However, despite the persistence characteristics of terbuthylazine, the results obtained in this study showed remaining percentages of terbuthylazine in the liquid culture by the end of the assays, much lower than the results published by other authors (Bending et al., 2002). Regarding difenoconazole (Table 2), it was found that all fungi removed higher amounts of this compound from solution, when compared with the results obtained for terbuthylazine. Remaining percentages of difenoconazole as low as 7.3%, were obtained in degradation experiments with P. brevicompactum. P. brevicompactum was isolated from a long term contaminated soil with high concentration of difenoconazole. This removal capability was presumably acquired through chronic exposure to contamination, which can promote the emergence of microorganisms consequently better-adapted and more capable of pesticide biotransformation. As far as we know, this work is the first study where P. brevicompactum was used to remove these pesticides, and where the special ability of this fungus to biotransform difenoconazole was strongly evidenced. L. edodes and A. oryzae have been successfully used to perform a difenoconazole remaining percentage as low as 11.9 and 13.1%, respectively. In fact, the white-rot fungus L. edodes belongs to an ecological group of white-rot basidiomycetes, which possesses an extracellular, radical-based ligninolytic enzyme system capable of degrading a wide variety of pollutants (D'Annibale et al., 2004; Pointing, 2001; Rao et al., 2010; Sánchez, 2009). L. edodes is one of the most widely cultivated mushrooms worldwide and the interest in this edible mushroom cultivation is increasing because of its high nutritional value and medicinal properties (Ngai and Ng, 2003). Pendimethalin is a dinitroaniline pre-emergence herbicide, used in various formulations, in terrestrial agro-ecosystem to destroy or prevent the growth of weeds. It is a selective herbicide absorbed by roots and leaves, inhibiting cell division and cell elongation (Tomlin, 2011). Widespread use of this herbicide led to its detection as a contaminant in soil (≥0.3 mg kg−1) and water (0.1–6 μg l−1) by means of evaporation, drift, leaching, and runoff (Roca et al., 2009). It adsorbs strongly to organic matter and clay minerals and is thus not mobile in soil. It is moderately persistent with a field half-life of approximately 90 days (Mohan et al., 2007). Pendimethalin was normally degraded through photo-degradation, volatilization or by biodegradation (Mohan et al., 2007; Zhang et al., 2000). In this work, pendimethalin removal percentages of 99.5% from liquid cultures were achieved in batch experiments with L. saksenae. This fungus was isolated from a long term contaminated soil with the pesticide, as occurred with P. brevicompactum. With this fungus, a remaining percentage of pendimethalin close to 1% was also achieved. Soil microorganisms that are repeatedly exposed to pesticide may develop new capabilities to degrade such chemicals. In fact, repeated use of certain compounds over a number of seasons can result in enhanced degradation. This is probably due to adaptation and proliferation of specific microbial communities which utilize the compound as an energy source and thus degrade it more easily (De Wilde, 2009; Kulshrestha et al., 2000). However, there are very few reports on the degradation of pendimethalin by soil microorganisms (Veena et al., 2010). L. edodes, A. oryzae and F. oxysporum were able to remove appreciable amounts of pendimethalin with 3.9, 0.9 and 0.6% (Table 2),

406

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410 Pendimethalin Difenoconazole Terbuthylazine

24

L. edodes

22 20 18 16 14 12 10 8 6 4 2

Pendimethalin Difenoconazole Terbuthylazine

26

Pesticide concentration (mgL-1)

Pesticide concentration (mgL-1)

26

24 22 20 18 16 14 12 10 8 6 4 2 0

0 0

50

100

150

200

250

0

50

Time (hours)

A. oryzae

150

22 20 18 16 14 12 10 8 6 4 2

Pendimethalin Difenoconazole Terbuthylazine

26

Pesticide concentration (mgL-1)

Pesticide concentration (mgL-1)

24

100

200

250

Time (hours)

Pendimethalin Difenoconazole Terbuthylazine

26

24

P. brevicompactum

22 20 18 16 14 12 10 8 6 4 2 0

0 0

50

100

150

200

250

0

50

Time (hours) Pendimethalin Difenoconazole Terbuthylazine

26

Pesticide concentration (mgL-1)

F. oxysporium

24

100

150

200

250

Time (hours)

L. saksenae

22 20 18 16 14 12 10 8 6 4 2 0 0

50

100

150

200

250

Time (hours) Fig. 1. Concentration (mg l−1) of pesticides terbuthylazine (Δ), difenoconazole (□) and pendimethalin (◊), remaining in liquid culture, after 10 days of batch assays.

remaining in liquid cultures by the end of batch assays, respectively. Kulshrestha et al. (2000), obtained similar results using F. oxysporum, isolated from a field soil treated with pendimethalin, in pure culture laboratory experiments. Moreover, several studies using A. oryzae, isolated and screened from contaminated soils, had already demonstrated its high capacity to biodegrade other pesticides as endosulfan and monocrotophos (Bhalerao and Puranik, 2007, 2009). The latest study even suggests that this fungus could be used for bioaugmentation of contaminated soils and/or treatment of aqueous wastes.

However, in this work, we assessed the impact of the adsorption process on the mycelial surface (see Section 2.5), in addition to the action of microbial communities able to degrade and/or metabolize pesticide residues. Fig. 2 presents the total removal percentage of each pesticide, resultant from the sum of the percentage removed through the adsorption process and the percentage removed by biodegradation.

Table 2 Remaining percentages of terbuthylazine, difenoconazole and pendimethalin in liquid cultures by the end of the assays. Average ± SE.

3.2. Adsorption versus biodegradation In most studies carried out under similar conditions to those described in this work, the authors did not evaluate the significance of adsorption process, performed by microbial biomass, on the total pesticide removal from the contaminated aqueous medium.

L. edodes F. oxysporum A. oryzae P. brevicompactum L. saksenae

Terbuthylazine (%)

Difenoconazole (%)

Pendimethalin (%)

25.8 ± 1.2 22.9 ± 0.7 21.4 ± 0.8 29.2 ± 1.0 22.6 ± 0.8

13.1 ± 0.6 17.1 ± 0.5 11.9 ± 0.4 7.3 ± 0.3 14.8 ± 0.5

3.9 ± 0.1 0.6 ± 0.02 0.9 ± 0.03 1.2 ± 0.04 0.5 ± 0.02

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410

In this figure it is assumed that the xenobiotic amount, which was not in solution and/or adsorbed at the end of the batch experiments, was biodegraded by the microorganisms. However, to ensure that some extension of biodegradation occurs would be necessary to prospect the presence of intermediate metabolites from pesticide metabolic pathways. In order to find evidence for the occurrence of pesticide biotransformation, an LC–ESI-MS and MS 2 analysis of some samples was conducted to enable a preliminary identification of some compounds (see Section 3.3). The results obtained for terbuthylazine (Fig. 2A) shows that the adsorption process can be very different depending on the fungal species

(A)

Terbuthylazine

100

% Removal

a

a

80

a

a

b

60 40 20 0

m

riu

y

F.

ox

ae

yz

r .o

o sp

m

A

ico

od

en

L.

ev

es

ae

um

ct

pa

s ak

d .e

s

L

r .b

P

(B)

Difenoconazole

100

b

a

a

a

a

% Removal

80 60 40 20 0

e

m

riu

po

F.

s xy

o

za

pa

om

de

na

se

L.

vic

re

s

e

um

ct

ry

o A.

k sa

do

e L.

b P.

(C)

Pendimethalin a

a

a

100

a

a

% Removal

80 60 40 20 0

m

riu

po

F.

o

s xy

ae

yz

A.

or

vic

re

es

ae

um

ct

pa

om

od

en

ks

L

a .s

L.

ed

b P.

Fig. 2. Removal of terbuthylazine (A), difenoconazole (B) and pendimethalin (C) (%). Removal due to pesticide biodegradation ( ) and removal due to adsorption process on the microbial biomass surface ( ). In each figure, values followed by the same letter are not significantly different at p b 0.05.

407

present. It was found that the fungus with the highest terbuthylazine adsorption was L. saksenae (1020.0±47.2 μg g−1 of biomass), much higher than the value observed with P. brevicompactum (33.0±1.5 μg g−1 of biomass), which had the lowest amount adsorbed. Moreover, the higher pesticide adsorption by L. saksenae has led to a lower biodegradation of the pesticide compared to P. brevicompactum, that showed the highest amount metabolized. This increased adsorption was also reflected in the development of the microbial biomass, which has been reduced to L. saksenae by more than 40% in the presence of the pesticide, possibly due to the increased toxicity of terbuthylazine to the microbial community in these conditions. P. brevicompactum was the fungus that showed higher biodegradation of terbuthylazine, being the fungus that increased biomass production in the presence of the pesticide in relation to the absence, in approximately 45%. In general, for terbuthylazine, higher adsorption inhibited the biodegradation by fungi, demonstrating that the pesticide molecules initially bind to the cell surface followed by pesticide accumulation to toxic intracellular levels. These results can help to demonstrate the importance of intracellular and extracellular competitive interactions in determining the toxicity of xenobiotics. The initial surface binding is considered to occur with proteins, lipids and different polysaccharides like glucan, mannan, chitin, and chitosan present on the cell wall through ion exchange, complexation, precipitation, crystallization and/or physical forces within the multilaminate, microfibrillar cell wall structure which contains a large number of functional groups, e.g. carboxyl, amino, hydroxyl, phosphate, etc. (Das et al., 2008). For difenoconazole a similar behavior to that recorded for terbuthylazine was verified, i.e., the lowest adsorption on the mycelial surface (10.4 ± 0.5 μg g −1 of biomass) was observed with L. edodes, which was the fungus that had a higher rate of biodegradation (82.6%) (Fig. 2B). The results obtained allowed to conclude that with difenoconazole, similarly as observed for terbuthylazine, higher adsorption rate implies lower biodegradation. L. edodes was the only fungus that significantly increased its microbial biomass in the presence of the pesticide (>50%) vs absence, being this result probably associated with an increased tolerance and ability to metabolize this pesticide by this fungus. The biodegradation rates obtained for difenoconazole were always higher to those observed for terbuthylazine, for all five fungi. The results obtained with pendimethalin (Fig. 2C), generally showed higher biodegradation rates and lower amounts of pesticide retained on the microbial biomass, comparatively to previous pesticides. This allowed to conclude that the fungi studied showed the highest mineralization capacity with pendimethalin, resulting in biodegradation percentages of 92.7, 94.4 and 98.8%, with L. saksenae, L. edodes and F. oxysporium, respectively. Although the results found in our study for F. oxysporium confirm those published by other authors (Kulshrestha et al., 2000), the findings obtained with the other fungi are completely innovative showing the potential of the strains studied for biodegradation of this xenobiotic. L. edodes was the fungus with a result closer to that obtained by F. oxysporium, showing his great potential to biodegrade pendimethalin. L. edodes showed again a significant increase on his microbial biomass development (approximately 50% more), in the presence of this pesticide vs absence. The fungus with the highest retained pesticide amount (371 ± 17 μg g −1 of biomass) was A. oryzae. This fungus also registered the highest decrease on microbial biomass in the presence of the pesticide, in relation to the absence of the xenobiotic. The amounts of pesticide retained on microbial biomass were 2.1 ± 0.1, 7.7 ± 0.4, 34.0 ± 1.6 and 287.0 ± 13.3 μg g −1 for F. oxysporium, L. edodes, L. saksenae and P. brevicompactum, respectively. Sorption is dependent on Kow (partition coefficient between octanol and water) values while the biodegradation depends on the availability of xenobiotic, the capability of the microbes to degrade

408

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410

it and the population density of the degraders (Kengara et al., 2010). In fact, compounds with high log Kow, being hydrophobic, would partition more into the microbial phase while compounds with low log Kow, being more hydrophilic and soluble, would not be sorbed considerably by the organic phase (Kengara et al., 2010). Our results however contradicted this fact, since higher partition into the organic (microbial) phase was observed with L. saksenae (1020.0 ± 47.2 μg g−1) and terbuthylazine (log Kow = 3.2), whereas for pendimethalin (log Kow = 5.18) the highest value of adsorption obtained with A. oryzae, was only 371 ± 17 μg g−1. The pesticide sorption process to microbial biomass seems to be more dependent on the fungi species, influenced perhaps by fungal morphology, than on pesticide physical parameters such as partition coefficient. This study encourages the use of these selected fungi for a biodepuration system (e.g. biobeds), containing the pesticides studied. In fact, the lack of a proper indigenous population of microbial degraders can be overcome by inoculating foreign microorganisms into the system (Kengara et al., 2010). This strategy, usually referred to as “bioaugmentation” is based on the inoculation of a pollutant-degrading microbial strain or a microbial consortium into the contaminated system (Wang et al., 2010). Beyond that, studies in liquid culture are a good approach to assess an organism's ability to utilize a target compound (Juhasz and Naidu, 2000) and field application that confirms the viability of these fungi in real conditions is necessary and will provide valuable information to properly develop decontamination procedures to apply in situ; moreover, P. brevicompactum PP0021 and L. saksenae PP0011 used in this study, were isolated from a soil and a biomixture contaminated with the pesticides in a long term microcosm experiment, which indicates the viability of these strains in heterogeneous environment as natural soil. Recently a number of studies have focused on biomaterials that are capable of biodegrading and biosorbing for example, dyes from wastewaters (Srinivasan and Viraraghavan, 2010). All fungi evaluated in our work showed a relatively high biodegradation and sorption capacity for the studied xenobiotics, pointing to the possibility of using these fungi strains in similar environmental applications. 3.3. Degradation of terbuthylazine Among s-triazines, terbuthylazine is the most persistent in surface environments (Carafa et al., 2007; Grenni, 2011). Metabolite formation is mainly due to biochemical processes such as dealkylation, dechlorination and hydroxylation, deamination and ring cleavage of the parent compounds (Grenni, 2011). According to studies by Dousset et al. (1997), the prominent dealkylation product of terbuthylazine is desethyl-terbuthylazine. In order to evaluate the presence of terbuthylazine metabolites, we analyzed by LC–ESI-MS and MS 2 some samples collected from liquid cultures of L. edodes. (see Section 2.6.2). In Fig. 3 the total ion current (TIC) chromatogram of a sample collected after 5 days of the pesticide addition to the liquid culture is presented. The major peaks present in the TIC chromatographic profile of Fig. 3 (rt: 12,29; 12,99; 34,06; 40,18; 44.75) were also present in samples of the fungal cultures, collected before and after the addition of the pesticides (at time 0), allowing to conclude that they are probably compounds associated to the culture medium. In the chromatogram obtained in Fig. 3 it was possible to identify two other peaks. The peak with retention time (rt) 9.22 min was identified as terbuthylazine, since the full MS and MS 2 spectra are coincident with the ESI-MS spectra reported for this triazine herbicide (peaks at m/z 230.1, 174.1 and 104.0) (Baglio et al., 1999; Hernández et al., 2005; Singer et al., 2010). The peak obtained at rt 6.86 min corresponds probably to the one of the most common terbuthylazine metabolites, desethyl-terbuthylazine. The full MS spectra show an ion signal at m/z 202.1, with abundance of

Fig. 3. Total ion current chromatogram (TIC) obtained for the samples of Lentinula edodes culture medium, collected after a five day period. Compounds with rt 9.22 and 6.86 min were identified as terbutylazine (TBZ) and desethyl-terbuthylazine (DTBZ), respectively.

100%, corresponding to the [M+ H]+ parent ion, and at m/z 224.9 the peak corresponding to the sodium aduct [M+ Na]+ (data not shown). The MS2 spectrum of the parent ion at m/z 202.1 is presented in Fig. 4. This fragmentation yielded ions at m/z 202.1 [M + H] + and m/z 146.1 [M + H‐buthyl] + (Hernández et al., 2005). The fragment pattern obtained for the peak with m/z 202.1 is in agreement with the fragmentation pathways proposed by Baglio et al. (1999) for triazine herbicides. There are no reports in the literature about the metabolic pathways of terbuthylazine by L. edodes fungus. However, the eventual presence of the dealkylated product desethyl-terbuthylazine in the liquid culture media seems to indicate that dealkylation is one of the possible metabolic pathways of this fungus. To ensure that terbuthylazine metabolite was not initially present in the fungal liquid culture, and to prove that the presence of other matrix compounds did not interfere with the metabolite detection, samples collected at time zero, and the corresponding to the blank without fungi and other without contaminant, were used and previously analyzed by LC–ESI-MS. In those samples, and as expected, the peak obtained at rt 6.86 min (attributed to desethyl-terbuthylazine) was not detected, proving that this peak can be attributed to a product of the pesticide biodegradation by fungi. The presence of intermediate metabolites resulting from the other pesticide biotransformation was also investigated in batch liquid cultures of L. edodes fungus. Despite the evidence of the possible presence of difenoconazole and pendimethalin metabolites, its unequivocal identification was not possible to achieve by LC–ESI-MS. However, more studies are being conducted, with this and other fungi, to understand the metabolic pathways of the three pesticides by the studied fungi.

Fig. 4. LC–ESI-MS2 spectrum obtained for parent ion with m/z 202, i.e., desethylterbuthylazine (DTBZ).

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410

4. Conclusions All studied fungi demonstrated high ability to degrade the pesticides, terbuthylazine, difenoconazole and pendimethalin. The lower removal percentages and biodegradation rates were exhibited with terbuthylazine, by all fungi. The evolution of the pesticide removal rate from liquid culture was characterized by a fast initial stage occurring within the first period of 48 h, probably due to a rapid adsorption to the cell surface followed by uptake. Moreover, we believe that fungal cells use the initial high adsorption as a defense mechanism to block the xenobiotic uptake, and prevent its accumulation to toxic intracellular levels. Following uptake, xenobiotics can potentially be metabolized in the microbial cells, being this partly responsible for the pesticide net reduction from liquid culture. The eventual presence of desethyl-terbuthylazine in samples of L. edodes liquid cultures seems to indicate that terbuthylazine dealkylation is one of the possible metabolic pathways of this xenobiotic by this fungus. The biosorption of studied pesticides, attributed to fungi relatively high surface area and high binding affinity, proved to be a process more dependent on the fungi species, influenced probably by fungal morphology, than on pesticide physical parameters such as partition coefficients. Thus, these results may be a very important data for future studies involving these pesticides and the microorganisms studied. Acknowledgments The authors wish to acknowledge Maria do Carmo Romeiras, Avelino Manuel A. Balsinhas and Bayer CropScience Portugal, for their help given on the product supply and technical support. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.scitotenv.2012.07.027. References Baglio D, Kotzias D, Larsen BR. Atmospheric pressure ionisation multiple mass spectrometric analysis of pesticides. J Chromatogr A 1999;854:207–20. Bending GD, Friloux M, Walker A. Degradation of contrasting pesticides by white rot fungi and its relationship with ligninolytic potential. FEMS Microbiol Lett 2002;212:59–63. Bhalerao TS, Puranik PR. Biodegradation of organochlorine pesticide, endosulfan, by a fungal soil isolate, Aspergillus niger. Int Biodeterior Biodegrad 2007;59: 315–21. Bhalerao TS, Puranik PR. Microbial degradation of monocrotophos by Aspergillus oryzae. Int Biodeterior Biodegrad 2009;63:503–8. Caldeira AT, Teixeira DM, Pinto AP., 2011. Lecanicillium saksenae strain PP0011 28S large subunit ribosomal RNA gene, partial sequence. GenBank, NCBI, JQ282679. Carafa R, Wollgast J, Canuti E, Ligthart J, Dueri S, Hanke G, et al. Seasonal variations of selected herbicides and related metabolites in water, sediment, seaweed and clams in the Sacca di Goro coastal lagoon (Northern Adriatic). Chemosphere 2007;69:1625–37. Castillo MDP, Torstensson L, Stenstrom J. Biobeds for environmental protection from pesticide use — a review. J Agric Food Chem 2008;56:6206–19. Coppola L, Castillo MP, Monaci E, Vischetti C. Adaptation of the biobed composition for chlorpyrifos degradation to Southern Europe conditions. J Agric Food Chem 2007;55(2):396–401. Coppola L, Comitini F, Casucci C, Milanovic V, Monaci E, Marinozzi M, et al. Fungicides degradation in an organic biomixture: impact on microbial diversity. New Biotechnol 2011;29:99-109. D'Annibale A, Casa R, Pieruccetti F, Ricci M, Marabottini R. Lentinula edodes removes phenols from olive-mill wastewater: impact on durum wheat (Triticum durum Desf.) germinability. Chemosphere 2004;54:887–94. Das SK, Ghosh P, Ghosh I, Guha AK. Adsorption of rhodamine B on Rhizopus oryzae: role of functional groups and cell wall components. Colloids Surf B Biointerfaces 2008;65:30–4.

409

De Wilde T., 2009. Sorption and degradation of pesticides in biopurification systems. PhD Thesis. Ghent University, Belgium. De Wilde T, Spanoghe P, Debaer C, Ryckeboer J, Springael D, Jaeken P. Overview of onfarm bioremediation systems to reduce the occurrence of point source contamination. Pest Manag Sci 2007;63:111–28. De Wilde T, Spanoghe P, Sniegowksi K, Ryckeboer J, Jaeken P, Springael D. Transport and degradation of metalaxyl and isoproturon in biopurification columns inoculated with pesticide-primed material. Chemosphere 2010;78:56–60. Diez MC. Biological aspects involved in the degradation of organic pollutants. J Soil Sci Plant Nutr 2010;10:244–67. Dousset S, Mouvet C, Schiavon M. Degradation of [14C]terbuthylazine and [14C]atrazine in laboratory soil microcosms. Pestic Sci 1997;49:9-16. Grenni P., 2011. Effects of pesticides and pharmaceuticals on soil and water bacterial communities. PhD thesis in Environmental Science. University of Milano-Bicocca, Italy. Hernández F, Pozo OJ, Sancho JV, López FJ, Marín JM, Ibánez M. Strategies for quantification and confirmation of multi-class polar pesticides and transformation products in water by LC–MS2 using triple quadrupole and hybrid quadrupole time-offlight analyzers. Trends Anal Chem 2005;24:597–612. Juhasz A, Naidu R. Extraction and recovery of organochlorine pesticides from fungal mycelia. J Microbiol Methods 2000;39:149–58. Karanasios E, Tsiropoulos NG, Karpouzas DG, Menkissoglu-Spiroudi U. Novel biomixtures based on local Mediterranean lignocellulosic materials: evaluation for use in biobed systems. Chemosphere 2010;80:914–21. Kengara FO, Schramm KW, Doerfler U, Munch JC, Henkelmann B, Welz G, et al. Degradation capacity of a 1,2,4-trichlorobenzene mineralizing microbial community for traces of organochlorine pesticides. Sci Total Environ 2010;408:3359–66. Köck-Schulmeyer M, Ginebreda A, González S, Cortina JL, López de Alda M, Barceló D. Analysis of the occurrence and risk assessment of polar pesticides in the Llobregat River Basin (NE Spain). Chemosphere 2012;86:8-16. Kulshrestha G, Singh SB, Lal SP, Yaduraju NT. Effect of long-term field application of pendimethalin: enhanced degradation in soil. Pest Manag Sci 2000;56: 202–6. Liang WQ, Wang ZY, Li H, Wu PC, Hu JM, Luo N, et al. Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11. J Agric Food Chem 2005;53:7415–20. Liu YH, Chung YC, Xiong Y. Purification and characterization of dimethoate-degrading enzyme of Aspergillus niger ZHY256, isolated from sewage. Appl Environ Microbiol 2001;67:3746–9. Mäenpää KA, Sormunen AJ, Kukkonen JVK. Bioaccumulation and toxicity of sediment associated herbicides (ioxynil, pendimethalin, and bentazone) in Lumbriculus variegates (Oligochaeta) and Chironomus riparius (Insecta). Ecotoxicol Environ Saf 2003;56:398–410. Michel Jr FC, Dass SB, Grulke EA, Reddy CA. Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent. Appl Environ Microbiol 1991;57(8):2368–75. Mohan SV, Krishna MR, Muralikrishna P, Shailaja S, Sarma PN. Solid phase bioremediation of pendimethalin in contaminated soil and evaluation of leaching potential. Bioresour Technol 2007;98:2905–10. Navarro S, Vela N, Gimenez MJ, Navarro G. Persistence of four s-triazine herbicides in river, sea and groundwater samples exposed to sunlight and darkness under laboratory conditions. Sci Total Environ 2004;329:87–97. Ngai PH, Ng TB. Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells. Life Sci 2003;73:3363–74. Pesticide Properties Database. University of Hertfordshine. http://sitem.herts.ac.uk/ aeru/footprint/en/2011. (accessed 15.02.12, last updated 03.04.12). Pino N, Peñuela G. Simultaneous degradation of the pesticides methyl parathion and chlorpyrifos by an isolated bacterial consortium from a contaminated site. Int Biodeterior Biodegrad 2011;65:827–83. Pinto AP, Teixeira DM, Caldeira AT., 2011. Penicillium brevicompactum strain PP0021 28S large subunit ribosomal RNA gene, partial sequence. GenBank, NCBI, JQ282680. Pointing SB. Feasibility of bioremediation by white-rot fungi. Appl Microbiol Biotechnol 2001;57:20–32. Rao MA, Scelza R, Scotti R, Gianfreda L. Role of enzymes in the remediation of polluted environments. J Soil Sci Plant Nutr 2010;10(3):333–53. Roca E, D'Errico E, Izzo A, Strumia S, Esposito A, Fiorentino A. In vitro saprotrophic basidiomycetes tolerance to pendimethalin. Int Biodeterior Biodegrad 2009;63: 182–6. Sánchez C. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol Adv 2009;27:185–94. Singer H, Jaus S, Hanke I, Lück A, Hollender J, Alder AC. Determination of biocides and pesticides by on-line solid phase extraction coupled with mass spectrometry and their behaviour in wastewater and surface water. Environ Pollut 2010;158:3054–64. Spliid NH, Helweg A, Heinrichson K. Leaching and degradation of 21 pesticides in a fullscale model biobed. Chemosphere 2006;65:2223–32. Srinivasan A, Viraraghavan T. Decolorization of dye wastewaters by biosorbents: a review. J Environ Manage 2010;91:1915–29. Tomlin CDS. The pesticide manual. fifteenth ed. Surrey: British Crop Protection Council; 2011. Veena BM, Preezi NT, Robercyril SH, Skandar IM, Harichandra ZN. Biodegradation of pendimethalin by Bacillus circulans. Indian J Biotechnol 2010;9:173–7. Vischetti C, Coppola L, Monaci E, Cardinali A, Castillo MP. Microbial impact of the pesticide chlorpyrifos on Swedish and Italian biobeds. Agron Sustainable Dev 2007;27: 267–72. Wang F, Dörfler U, Schmid M, Fischer D, Kinzel L, Scherb H. Homogeneous inoculation vs. microbial hot spots of isolated strain and microbial community: what is the

410

A.P. Pinto et al. / Science of the Total Environment 435–436 (2012) 402–410

most promising approach in remediating 1, 2, 4-TCB contaminated soils? Soil Biol Biochem 2010;42:331–6. Wu J, Xiao YZ, Yu HQ. Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm. Bioresour Technol 2005;96(12):1357–63. Younes M, Galal-Gorchev H. Pesticides in drinking water — a case study. Food Chem Toxicol 2000;38(1):87–90.

Zhang C, Hughes JB, Nishino SF, Spain J. Slurry-phase biological treatment of 2,4dinitrotoulene and 2,6-dinitrotoulene: role of bioaugmentation and effects of high dinitrotoulene concentrations. Environ Sci Technol 2000;34:2810–6. Zhang Q, Liu Y, Liu YH. Purification and characterization of a novel carbaryl hydrolase from Aspergillus niger PY168. FEMS Microbiol Lett 2003;228:39–44.