Environ Sci Pollut Res DOI 10.1007/s11356-015-4472-0

RESEARCH ARTICLE

Biodegradation of pesticides using fungi species found in the aquatic environment B. R. Oliveira 1 & A. Penetra 2 & V. V. Cardoso 2 & M. J. Benoliel 2 & M. T. Barreto Crespo 1,3 & R. A. Samson 4 & V. J. Pereira 1,3

Received: 22 December 2014 / Accepted: 30 March 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Relatively limited attention has been given to the presence of fungi in the aquatic environment compared to their occurrence in other matrices. Taking advantage and recognizing the biodegradable capabilities of fungi is important, since these organisms may produce many potent enzymes capable of degrading toxic pollutants. Therefore, the aim of this study was to evaluate the potential ability of some species of filamentous fungi that occur in the aquatic environment to degrade pesticides in untreated surface water. Several laboratory-scale experiments were performed using the natural microbial population present in the aquatic environment as well as spiked fungi isolates that were found to occur in different water matrices, to test the ability of fungi to degrade several pesticides of current concern (atrazine, diuron, isoproturon and chlorfenvinphos). The results obtained in this study showed that, when spiked in sterile natural water, fungi were able to degrade chlorfenvinphos to levels below detection and unable to degrade atrazine, diuron and isoproturon. Penicillium citrinum, Aspergillus fumigatus, Aspergillus Responsible editor: Robert Duran Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-4472-0) contains supplementary material, which is available to authorized users. * V. J. Pereira [email protected] 1

IBET, Apartado 12, 2781-901 Oeiras, Portugal

2

Empresa Portuguesa das Águas Livres, S.A., Avenida de Berlim, 15, 1800-031 Lisbon, Portugal

3

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal

4

CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

terreus and Trichoderma harzianum were found to be able to resist and degrade chlorfenvinphos. These fungi are therefore expected to play an important role in the degradation of this and other pollutants present in the aquatic environment. Keywords Fungi . Biodegradation . Pesticides . Surface water

Introduction The ability to feed the world’s growing population constitutes one of the crucial challenges of this century. The tremendous increase in food production in the last 50 years was possible due to the use of high-yielding crop varieties, fertilization, irrigation and also an extensive use of pesticides (Matson et al. 1997). The projected doubling in global food demand for the next 50 years constitutes an enormous challenge for the sustainability of food production and of terrestrial and aquatic ecosystems (Tilman et al. 2002). The main environmental impacts of agriculture arise from the use of nutrients and pesticides (Tilman et al. 2002). Over 1000 pesticides have been reported to be marketed worldwide that can display a wide variety of chemical structures including chlorinated, aromatic, and nitrogen- and phosphorous-containing compounds (Madigan et al. 2012). Their intense use leads to contamination of surface and groundwaters by drift, runoff, drainage and leaching. Water contamination with pesticides may have ecotoxicological effects for aquatic flora and fauna as well as for human health if these compounds are resilient to treatment and the water is used for public consumption (Castillo et al. 1997; Graymore et al. 2001; Relyea 2009). In recent years, a large group of organic compounds have been labelled as water-emerging contaminants by the Environmental Protection Agency (USA) and the European

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Union. Directive 2008/105/EC sets concentration limits for pollutants such as the pesticides atrazine, diuron, isoproturon, chlorfenvinphos and many other pollutants, with the aim of achieving good surface water chemical quality. The abovementioned pesticides are considered important due to their easy transport in the environment as well as potential for seriously threatening the aquatic environment and human health (Hincapié et al. 2005). These pesticides have been detected in drinking water sources such as surface water and groundwater at levels in the range of 1–52.7 ng/L (Donald et al. 2007; Rodriguez-Mozaz et al. 2004). Consequently, further studies are needed to monitor the occurrence and resilience of these compounds in the aquatic environment. When analyzing different drinking water sources, a vast variety of microorganisms have been detected, including filamentous fungi. Recent studies have reported the occurrence of fungi in different water sources being Aspergillus fumigatus, Aspergillus terreus, Cladosporium sp., Fusarium begoniae, Penicillium citrinum, Penicillium melanoconidium, Penicillium brevicompactum, Phoma glomerata and Trichoderma sp. often reported to occur widely (Al-gabr et al. 2014; Bouchiat et al. 2015; Hageskal et al. 2006, 2009; Kanzler et al. 2007; Oliveira et al. 2013; Pereira et al. 2009; Saravanan and Sivakumar 2013; Schiavano et al. 2014). The biogeochemical capabilities of microorganisms have been reported as almost limitless, and they are known as the BEarth’s greatest chemists^ (Madigan et al. 2012). Contaminant transformations can be facilitated by microorganisms via enzymes that lower the activation energy that must be overcome for a reaction to proceed (Mitchell and Gu 2010). Previous studies have shown the ability of various microbial organisms to remove pollutants from the environment (Jin et al. 2012; Lee et al. 2012; Matsubara et al. 2006; Pothuluri et al. 1996). Most of the research conducted focused on bacteria, with fungal applications only attracting interest within the past two decades. However, characteristics such as a specific bioactivity and/or growth morphology allow filamentous fungi to be better potential degraders than bacteria (Mollea et al. 2005). Moreover, some recent studies have shown that filamentous fungi act synergistically with bacteria to enhance contaminants’ degradation by transforming them into an easier form for bacteria to degrade and, through hyphal growth, they may also help bacteria to reach inaccessible contaminant compounds (Ellegaard-Jensen et al. 2014; Lade et al. 2012). Fungi are known to be robust organisms and were reported to be more tolerant to high concentrations of pollutants than bacteria (Evans and Hedger 2001). Taking advantage and recognizing the biodegradable capabilities of fungi is therefore important, since these organisms have the ability to degrade a wide range of environmental pollutants (Gao et al. 2010; Maiti et al. 2013; Matsubara et al. 2006; Pinto et al. 2012; Purnomo et al. 2013; Uhnáková et al. 2011). Some filamentous fungi

from Aspergillus’ genera have already been reported to degrade organophosphorus compounds (Bhalerao and Puranik 2009) being A. fumigatus and A. terreus two of the most frequently found species isolated from pesticide-contaminated soils (Bordjiba et al. 2001). Moreover, according to Ye et al. (2011) and Silambarasan and Abraham (2012), these species are able to degrade anthracene and chlorpyrifos, respectively. Cladosporium cladosporioides is another species that has also been reported to degrade chlorpyrifos (Gao et al. 2012). P. citrinum, which is known to produce a wide range of secondary metabolites (Malmstrøm et al. 2000), has the ability to degrade coal and hydrocarbons (Polman et al. 1994; Saravanan and Sivakumar 2013). Despite all this research, most of the studies referenced here were performed by inoculating microorganisms into rich growth media spiked with the desirable contaminant to evaluate their biodegradation capability, but very few have addressed this in real environmental situations such as soil (Gao et al. 2012; Mollea et al. 2005). The objective of this study was to understand the ability of different fungi species present in the aquatic environment to achieve biodegradation of different pesticides of concern in water sources. Several experiments were therefore conducted to address whether the presence of fungi in water may contribute to the biodegradation of atrazine, diuron, isoproturon and chlorfenvinphos. The biodegradation experiments were conducted in untreated surface water matrices in the presence of their natural microbial population as well as surface water spiked with selected fungi isolates (A. fumigatus, A. terreus, Cladosporium tenuissimum, C. cladosporioides, F. begoniae, P. citrinum, P. melanoconidium and P. glomerata). The selected fungi were found to occur at high concentrations in water sources with very different compositions, surface water, groundwater and spring water and were isolated in a previous study (Pereira et al. 2009). The concentration of the selected pesticides was followed in different experiments that were monitored over up to a 5-month period. Since biodegradation does not imply mineralization, the formation of breakdown products was also monitored.

Material and methods Chemical reagents The selected pesticides (atrazine, diuron, isoproturon and chlorfenvinphos) and all the other reagents used were purchased as solutions or solids of the highest grade commercially available (Sigma-Aldrich). A standard solution with the mixture of pesticides spiked at a final concentration of 50 mg/L was prepared in HPLC gradient grade methanol. Malt extract agar (Oxoid) was used as culture media and supplemented with 100 mg/L of the antibiotic

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chloramphenicol (Oxoid) to prevent bacterial growth (the concentration was set according to the manufacturer ’s instructions). High performance liquid chromatography (HPLC)-grade methanol (Fisher) was used to prepare concentrated stock solutions and for the chromatographic analysis of the pesticides. The Milli-Q water used was produced by a Milli-Q water system (Millipore). Characterization of the real water sources used Untreated surface water collected from the Tagus River was supplied by the water company Empresa Portuguesa das Águas Livres (EPAL). Samples were collected, stored under dark conditions at 4±2 °C for a maximum of 24 h and analyzed according to described microbiological sampling and examination procedures (Standard Method 9060). This surface water sample was characterized in terms of temperature (22±1 °C), pH (7.5±0.5), total organic carbon (3 mg/L C), turbidity (7±1 NTU), alkalinity (70±5 mg/L CaCO3) and total hardness (140 mg/L CaCO3). The water was also characterized in terms of its fungi population prior to the biodegradation experiments (using the methods detailed in the BIsolation and identification of fungi^ Section). Field and transport blanks accompanied the sample collection. The field blank, a bottle containing sterile Milli-Q water, was opened during sample collection to ensure there were no contaminations in the surrounding environment. The travel blank, an un-open bottle of sterile Milli-Q water, accompanied the samples to ensure there were no contaminations during transport. Growth of fungi was never detected in any of the transport blanks and very infrequently found in the field blanks. Whenever growth was observed in the field blanks, if the same isolate was present in the water sample, it was discarded and not considered to be part of the natural fungi population of the water. Isolation and identification of fungi The natural fungi population present in the untreated surface water collected to perform the biodegradation experiments was evaluated using the membrane filtration technique (Standard Method 9610D) by filtering 100 mL of the matrix (in triplicate) in a Millipore Microfil system, using Millipore S-Pak sterile-gridded membrane filters with a 0.45-μm pore diameter (Millipore). The filters were transferred into dichloran rose-bengal chloramphenicol (DRBC) agar supplemented with 100 mg/L of the antibiotic chloramphenicol (Oxoid). After the biodegradation experiments (detailed below), the fungi population present in the reactors was evaluated by inoculating 100 μL of sample into malt extract agar (MEA, Oxoid) supplemented with 100 mg/L of the antibiotic

chloramphenicol. All the plates were incubated at 25 °C in the dark and checked every 2 days for a maximum of 7 days. The different isolates were grown for 5 to 7 days in MEA at 25 °C. A spore suspension was then prepared according to the procedures described in the European Standard EN 12353. Briefly, the technique consists of scraping the fungi mycelium and filtration with glass wool. After washing with Tween 80 (0.5 g/L) and spin cycles, the resulting pellet is preserved in a cryoprotectant solution consisting of beef extract (3.0 g/L), tryptone pancreatic digest of casein (5.0 g/L) and glycerol (150 g/L). The concentration of the spore suspensions prepared for each fungi species was determined by microscopy (Olympus BH2) using a counting chamber (Neubauer). The selected isolates were spiked in the biodegradation reactors (detailed in the BBiodegradation experiments^ Section) to achieve an initial concentration of 9×105 spores/mL. These isolated and preserved fungi species were then identified at species level. To do so, DNA was extracted from the cells using the Ultra Clean Microbial DNA isolation kit (MoBIO) according to the manufacturer’s instructions. Fragments containing the ITS region were amplified using primers V9G and LS266 (Badali et al. 2005). Amplification of part of the β-tubulin gene was performed using the primers Bt2a and Bt2b (Glass and Donaldson 1994). Also, amplifications of the partial calmodulin (Hong et al. 2005), actin genes (Hong et al. 2005), portions of the nuclear large rRNA subunit (LSU) and translation elongation factor (EF-1α) genes were performed (O’Donnell et al. 2007). PCR reactions were performed in 25-μL reaction mixtures containing 1 μL of genomic DNA (10 ng/μL), 2.5 μL of PCR buffer, 16.45 μL of ultra pure sterile water, 1.95 μL of dNTP (1 mM), 0.5 μL of each primer (50 pmol/μL), 1.25 μL of dimethyl sulfoxide, 0.75 μL of magnesium chloride (50 mM) and 0.1 μL of Taq polymerase (2.5 U/μL DNA) (SpaeroQ). Amplifications were typically performed in a GeneAmp PCR system 9700 (AB Applied Biosystems), programmed for 5 min of denaturation at 94 °C, followed by 35 cycles of 30 s of denaturation at 94 °C, primer annealing for 40 s at 55 °C, extension for 75 s at 72 °C and a final 5-min elongation step at 72 °C. PCR fragments were sequenced directly in both directions with the same primers and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Bioscience). The cycle sequencing reaction mixture had a total reaction volume of 10 μL and contained 1 μL of template DNA, 0.85 μL of BigDye reagent, 3 μL of buffer, 4.75 μL of demineralized water and 0.4 μL of primer (10 mM). Sequencing products were purified according to the manufacturer’s recommendations with Sephadex G-50 superfine columns (Amersham Bioscience) in a multiscreen HV plate (Millipore) and with MicroAmp Optical 96-well reaction plate (AB Applied Biosystems). Samples were analyzed on an ABI PRISM 3700 Genetic Analyzer (AB Applied Biosystems).

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species that were found to resist biodegradation in the experiments above (I and II). The fungi species were spiked individually and as a mixture.

Contigs were assembled using the forward and reverse sequences with the programmes SeqMan and EditSeq from the LaserGene package (DNAStar Inc.) (Samson et al. 2004).

The reactors used in experiments I and II were spiked with a mixture of pesticides: atrazine, diuron, isoproturon and chlorfenvinphos. The spiked concentration (250 μg/L of each pesticide) was slightly higher than reported occurrence levels in untreated matrices, up to 52 μg/L (Donald et al. 2007; Rodriguez-Mozaz et al. 2004), to enable their detection using direct injection over time in the biodegradation experiments. In biodegradation experiment III, chlorfenvinphos was spiked individually (250 μg/L) into sterilized untreated surface water. In biodegradation experiments II and III, the sterile untreated surface water was spiked with fungi isolates (9×105 spores/ mL of each fungi species):

Biodegradation experiments A summary of the three biodegradation experiments conducted in this study is represented in Fig. 1. Three different biodegradation experiments were conducted as depicted in Fig. 1: –

–

–

Biodegradation experiment I—a mixture of the selected pesticides (atrazine, diuron, isoproturon and chlorfenvinphos) was spiked into untreated surface water. This water was not sterilized and thus contained its natural microbial population. Biodegradation experiment II—a mixture of the selected pesticides (same as above) was spiked into sterilized untreated surface water. The sterilized water was also spiked with a mixture of fungi species that were isolated from water in a previous occurrence study (Pereira et al. 2009). Biodegradation experiment III—chlorfenvinphos, the only pesticide that was found to be degraded to levels below detection in the biodegradation experiments I and II, was spiked into sterilized untreated surface water. The sterilized water was also spiked with the identified fungi

–

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A . f u m i g a t u s , A . t e r re u s , C . t e n u i s s i m u m , C. cladosporioides, F. begoniae, P. citrinum, P. melanoconidium and P. glomerata—these fungi were spiked as a mixture in biodegradation experiment II and selected due to their previous isolation and reported occurrence in the aquatic environment (Pereira et al. 2009). Trichoderma harzianum, A. fumigatus, A. terreus and P. citrinum—these fungi were spiked individually and

Fig. 1 Biodegradation experiments conducted in this study

Untreated surface water with its natural populaon

Untreated Surface Water Spiked Pescides

Spiked fungi into sterile untreated surface water

I

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Biodegradaon Experiment I

Biodegradaon Experiment II

resistant fungi aer Biodegradaon Experiment I and Biodegradaon Experiment II

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as a mixture in biodegradation experiment III; they were selected due to their resistance to the target pesticides since they were identified after biodegradation experiments I and II. The three biodegradation experiments took place in a dark room at 27 °C with the reactors (500 mL Erlenmeyer flasks with 250 mL of matrix without external aeration in triplicate) placed in a stir plate set at 100 rpm. Several control experiments were performed and subjected to the same conditions detailed above: –

–

–

Sterile untreated surface water spiked with the target pesticides and sterile Milli-Q water spiked with the pesticides—the objective of these controls was to verify the stability of pesticides in the real water matrix and Milli-Q water; Sterile untreated surface water spiked with the target fungi and sterile Milli-Q water spiked with the fungi—the objective of these controls was to verify the stability of fungi in the real water matrix and Milli-Q water; the plate counts obtained were stable throughout the experimental period; Sterile Milli-Q water spiked with the target pesticides and fungi—the objective of this control was to verify the matrix effect in the degradation efficiency.

Analysis of pesticides Samples (2 mL) were taken twice a week from each biodegradation and control reactors, filtered using a 0.2 μm filter made of regenerated cellulose (Whatman) and directly analyzed in terms of their pesticide composition—atrazine, diuron, isoproturon and chlorfenvinphos—after two injections using a high performance liquid chromatography (HPLC) system (Waters Alliance e2695 Separations Module) equipped with a p hotod iode array dete ctor (2 998 , Wa ters Chromatography) and a Luna 5μ C18 (2) 100A (150 × 3.0 mm) column (Phenomenex Inc.). A gradient mobile phase composition (methanol and laboratory grade water) was used in the chromatographic runs according to Sanches et al. (2013): 0–11 min—55 % methanol; 12.5–19 min—77 % methanol; and 21–28 min—55 % methanol. The mobile phase flow rate used to detect the pesticides was 0.5 mL/min, and the oven temperature was set at 25 °C (biodegradation experiment I and biodegradation experiment II). Under these conditions, the retention times of the target pesticides were 8.5 min (atrazine), 10.4 min (diuron), 9.3 min (isoproturon) and 18.6 min (chlorfenvinphos). The pesticides spiked in the biodegradation experiments were tested but not detected in the collected surface water.

In biodegradation experiment III, the only pesticide to be detected was chlorfenvinphos, so an isocratic method was used (80 % methanol and 20 % laboratory grade water). The mobile phase flow rate used to detect the pesticide was 1 mL/ min, and the oven temperature was set at 25 °C. Under these conditions, the retention time of chlorfenvinphos was 5.6 min. The remaining solution after the biodegradation experiment was analyzed by solid phase extraction using Oasis HLB cartridges (Waters, 6 mL, 200 mg) and gas chromatography (Trace GC ThermoQuest) with mass spectrometry detection (Thermo Finnigan Trace MS) using full scan mode (50–500 m/z) to identify any breakdown products formed. The analysis of breakdown products was not done throughout the experiment due to the large volume needed to concentrate the samples to be able to analyze the formation of breakdown products at low levels of concentration.

Results Biodegradation of pesticides spiked in untreated surface water with its natural microbiological population—biodegradation experiment I Figure 2 shows the variation of the pesticide concentration— atrazine, diuron, isoproturon and chlorfenvinphos—observed over an 82-day experimental period in the reactors where the pesticides were spiked into untreated surface water containing its natural microbial population (biodegradation experiment I depicted in Fig. 1). In the control reactor (sterile Milli-Q water) spiked with 250 μg/L of each pesticide, the concentration of pesticides remained fairly constant over time for atrazine, diuron, isoproturon and chlorfenvinphos. In the biodegradation reactor, the pesticide concentration slowly decreased with approximately 20, 50, 70 and 100 % of degradation obtained after 82 days, as shown in Fig. 2a, b, c and d, respectively. Chlorfenvinphos was the only compound that was degraded to levels below the direct injection detection limit (10 μg/L) after being in contact with the natural microbiological population present in untreated surface water (Fig. 2d). Besides monitoring the degradation of the pesticides over time, samples were also analyzed to characterize the fungi population present in the untreated surface water before the biodegradation experiment and after the 82-day biodegradation period. The identification results are summarized in Table 1. From the six fungi species identified to be present in the beginning of the biodegradation experiment, only two (P. citrinum and T. harzianum) were found to be present in the water samples analyzed after the 82-day biodegradation period.

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control

Fig. 2 Biodegradation of atrazine (a), diuron (b), isoproturon (c) and chlorfenvinphos (d) in untreated surface water containing its natural microbial population. C0 refers to the initial pesticide concentration

while C refers to the concentrations measured along the experimental time. Control reactor contains sterile Milli-Q water spiked with the mix of pesticides—biodegradation experiment I

P. citrinum was the only fungi found in this particular sampling event of untreated surface water that was also one of the fortified species. However, all the introduced strains were found to be present and isolated from different water samples in a previous study (Pereira et al. 2009).

the reactor containing the pesticides and fungi spiked in sterile untreated surface water, only the concentration of chlorfenvinphos started to decrease after approximately 50 days, and it was after 3 more months that the concentration of chlorfenvinphos decreased until levels below the detection limit (Fig. 3d), while the concentration of the three pesticides, atrazine, diuron and isoproturon, remained constant (Fig. 3a–c).

Biodegradation of pesticides in untreated surface water spiked with a selection of fungi—biodegradation experiment II Figure 3 shows the variation of the pesticide concentration— atrazine, diuron, isoproturon and chlorfenvinphos—monitored over a 5-month experimental period in the reactors where the pesticides and the selected fungi isolates (A. fumigatus, A. terreus, C. tenuissimum, C. cladosporioides, F. begoniae, P. citrinum, P. melanoconidium and P. glomerata) were spiked into sterile untreated surface water (biodegradation experiment II depicted in Fig. 1). The concentrations of the pesticides in the control reactor shown in Fig. 3 (sterile Milli-Q water spiked with the mix of fungi and pesticides) remained stable throughout the 5-month experimental period. On the other hand, in

Table 1 Identification of fungi present in biodegradation experiment I

Initial population Final population

Biodegradation of chlorfenvinphos using the identified fungi isolates—biodegradation experiment III A third biodegradation experiment was conducted to identify which of the fungi detected at the end of biodegradation experiments I (conducted in untreated surface water containing its natural microbiological population) and II (sterile untreated surface water spiked with fungi isolates) is responsible for the biodegradation of chlorfenvinphos (Table 2). Figure 4 shows the variation of chlorfenvinphos concentration over a 165-day experimental period in the reactors where only chlorfenvinphos was spiked into sterile untreated surface

Aspergillus brasiliensis, Penicillium citrinum, Rhizopus oryzae, Trichoderma hamatum, Trichoderma harzianum and Trichoderma gamsii Penicillium citrinum and Trichoderma harzianum

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Fig. 3 Biodegradation of atrazine (a), diuron (b), isoproturon (c) and chlorfenvinphos (d) in sterile untreated surface water containing eight fungi species. C0 refers to the initial pesticide concentration while C refers to the concentrations measured along the experimental time.

Control reactor represented in this figure contains sterile Milli-Q water spiked with the mix of fungi and pesticides—biodegradation experiment II

water containing each of the species presented in Table 2 spiked individually and as a mixture. In the control reactor (sterile untreated surface water spiked with the same concentration of chlorfenvinphos), the concentration of chlorfenvinphos remained practically constant over time. In the reactors spiked with fungi, very similar rates of degradation were obtained by the target fungi spiked individually and as mixtures. The obtained results show that P. citrinum, A. fumigatus, A. terreus and T. harzianum are resistant and able to degrade chlorfenvinphos in the aquatic environment. Gas chromatography with mass spectrometry detection was used to detect any breakdown products of chlorfenvinphos formed during the biodegradation experiments since biodegradation does not mean mineralization. The only compounds detected were chlorfenvinphos cis and trans isomers. Figure 5 shows the area of the two peaks

formed in each biodegradation reactor of the biodegradation experiment III.

Table 2 Filamentous fungi resilient to biodegradation experiments I and II that were spiked in biodegradation experiment III Biodegradation experiment I Biodegradation experiment II

Penicillium citrinum and Trichoderma harzianum Aspergillus fumigatus, Aspergillus terreus and Penicillium citrinum

Discussion Biodegradation of pesticides spiked in untreated surface water with its natural microbiological population—biodegradation experiment I When the mix of pesticides was spiked into the reactors containing natural untreated surface water, the concentration of chlorfenvinphos decreased until levels below detection. Since in the environment microorganisms exist all together, chlorfenvinphos is expected to be more prone to biodegradation in the aquatic environment. This conclusion is consistent with a recent study that describes that lightly chlorinated aliphatic and aromatic compounds are prone to biodegradation due to their use as electron donors by microorganisms or by contributing to their growth if they serve as terminal electron acceptors in the dehalorespiration process (Mitchell and Gu 2010). Few studies have been done that report the ability of filamentous fungi to degrade pollutants in the soil matrix. Regarding the fungi species found in the biodegradation

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Fig. 4 Biodegradation of chlorfenvinphos in sterile untreated surface water containing Penicillium citrinum (a), Aspergillus fumigatus (b), Aspergillus terreus (c) and Trichoderma harzianum (d) spiked separately and as a mixture (mix fungi with chlorfenvinphos). C0 refers

to the initial pesticide concentration while C refers to the concentrations measured along the experimental time. Control reactor contains sterile untreated surface water spiked with chlorfenvinphos and without the fungi species—biodegradation experiment III

experiment I, Trichoderma sp. has been described to be genetically diverse, tolerant to several recalcitrant pollutants such as heavy metals, pesticides and polyaromatic hydrocarbons, and associated with a significant impact to agricultural and industrial applications (Tripathi et al. 2013). Specifically, T. harzianum was described by other authors for its ability to degrade various agrochemicals such as dichloro-diphenyltrichloroethane (DDT), dieldrin, endosulfan,

pentachloronitrobenzene and pentachlorophenol in soil (Katayama and Matsumura 1993). P. citrinum, that was also found to be resilient to pesticides in the biodegradation experiment I, has been reported for being able to degrade coal (Polman et al. 1994) and hydrocarbons (Saravanan and Sivakumar 2013) producing a wide range of secondary metabolites (Malmstrøm et al. 2000). To the best of our knowledge, there are no reports about their susceptibility to the

4.E+06 4.E+06 3.E+06 Area of peak

Fig. 5 Area of breakdown products detected after biodegradation experiment III in the different reactors containing Penicillium citrinum, Trichoderma harzianum, Aspergillus fumigatus, Aspergillus terreus and the mixture (mix) of the four fungi species

3.E+06 2.E+06 2.E+06 1.E+06 5.E+05 0.E+00 P. citrinum

T. harzianum trans-chlorfenvinphos

Mix fungi

A. fumigatus

cis-chlorfenvinphos

A. terreus

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pesticides in the study. These two species (T. harzianum and P. citrinum) were found to be able to resist to a continuous exposure of pesticides and may be responsible for the observed chlorfenvinphos degradation. Biodegradation of pesticides in untreated surface water spiked with a selection of fungi—biodegradation experiment II In biodegradation experiment II, a mixture of pesticides and eight selected fungi species (A. fumigatus, A. terreus, C. tenuissimum, C. cladosporioides, F. begoniae, P. citrinum, P. melanoconidium and P. glomerata) were spiked into sterile untreated surface water, and pesticides’ concentration was monitored over 5 months. In Fig. 3d, it can be verified that in the control reactor, which has the same concentration of pesticides and fungi but s p i k e d i n s t e r i l e M i l l i - Q w a t e r, d e g r a d a t i o n o f chlorfenvinphos did not occur showing that the matrix had an important role in this experiment since fungi’s activity was only detected in the reactor that contained untreated surface water. This can be due to the dissolved organic material and nutrients that exist in the water matrix and may serve as carbon and energy sources necessary for fungi growth (Jiao et al. 2010). There is an evident slower degradation of chlorfenvinphos observed in biodegradation experiment II compared to biodegradation experiment I, and this may be due to microbial consortia present in biodegradation experiment I since the untreated surface water used (in biodegradation experiment I) was not sterile, it contained its natural microbial population. Two recent studies support this synergetic relationship between fungi and bacteria reporting that fungi are able to transform the contaminant making it easier for bacteria to degrade (Boyandin et al. 2013; Ellegaard-Jensen et al. 2014; Lade et al. 2012). Even though several studies reported the ability of fungi to degrade different pollutants in other matrices, few research studies have reported the ability of fungi to resist and degrade pesticides in real water matrices (Lew et al. 2013). In this study, fungi were found to be able to degrade chlorfenvinphos in a real surface water matrix. After the biodegradation experiment II, P. citrinum, A. fumigatus and A. terreus were the fungi species identified as the resistant and able to degrade chlorfenvinphos. The microbiological population of biodegradation experiment I and biodegradation experiment II decreased after their exposure to pesticides. A possible reason for this observation is probably the toxicity exerted by the pesticides that may affect the survival of certain species (Lo 2010) or that the reactor population entered into a nutrient shortage enabling only the resistant species to survive (Tripathi et al. 2013). A similar finding was reported by Lew et al. (2013) that reported

that the presence of pesticides can limit the occurrence of fungi and bacteria in the aquatic environment. In terms of the fungi identified, A. fumigatus is the only species in common between the two studies (this study and the study conducted by Lew et al. (2013)). Besides being able to resist to a constant input of atrazine, diuron, isoproturon and chlorfenvinphos (in this study), A. fumigatus was also found to resist to the presence of DDT in the lakes sampled by Lew et al. (2013). Bordjiba et al. (2001) isolated many fungi species from pesticide-contaminated soils being A. fumigatus and A. terreus two of the most frequently encountered species which shows that they are at least resistant to the constant input of pesticides. Furthermore, it was verified that consistently with biodegradation experiment I, P. citrinum was one of the resistant species in biodegradation experiment II. Biodegradation of chlorfenvinphos using the identified fungi isolates—biodegradation experiment III In biodegradation experiment III, the resilient fungi species of biodegradation experiments I and II were spiked individually and as a mixture into sterile untreated surface water with chlorfenvinphos and its concentration was monitored over 165 days. In the control reactor that contained sterile untreated surface water spiked with the same concentration of chlorfenvinphos used in the biodegradation experiments, the concentration of the pesticide remained practically constant over time, showing that the compound is expected to be stable in a real water matrix for at least 165 days. However, when microorganisms are present in the real water matrix, biodegradation of chlorfenvinphos is expected to occur. Specifically, in the reactors spiked with the fungi species found to be resistant to a constant input of pesticides (in the previous biodegradation experiments), very similar rates of degradation were obtained. The four resilient species (P. citrinum, A. fumigatus, A. terreus and T. harzianum) were able to degrade chlorfenvinphos at a similar rate to about 60 % of its initial concentration when spiked in a real matrix individually and as mixtures. This mixture of fungi did not exert a synergistic effect but is compatible and could be expected to be present in a real water source since all the target fungi were isolated from real water sources in a previous study (Pereira et al. 2009). Other fungi and bacteria are thus expected to play an important role in the biodegradation of this compound since degradation to much lower levels (below detection) was observed in the other degradation experiments: biodegradation experiment I (conducted during 2.5 months that also contained other microorganisms) and biodegradation experiment II (conducted during 5 months in the presence of other fungi species). These slower and incomplete levels of degradation observed with these isolates show that there is an enhanced degradation of contaminants when they are exposed to

Environ Sci Pollut Res

a consortia of microorganisms (Ellegaard-Jensen et al. 2014; Lade et al. 2012). Since they need to be in consortia with other species to accelerate and fully degrade the pesticide (observed in biodegradation experiment II and biodegradation experiment III, respectively), these experiments suggest that further studies are needed to identify the best consortia of fungi-bacteria or fungiother microorganisms to promote biodegradation of these pesticides and other emergent contaminants. Rouchaud et al. (1989, 1988) monitored several metabolites from chlorfenvinphos: 2,4-dichlorophenacyl chloride, 2, 4-dichloroacetophenone, α-(chloromethyl)-2,4dichlorobenzyl alcohol, 1-(2′,4′-dichlorophenyl)-ethan-1-ol, 2,4-dichlorobenzoic acid, 2-hydroxy-4-chlorobenzoic acid and 2,4-dihydroxybenzoic acid. These compounds were not detected in this study possibly due to the much lower levels of pesticides spiked in this study and expected to be present in water compared to the levels of pesticides expected in the fields. Regarding the identification of chlorfenvinphos breakdown products obtained in this study, the results obtained show that only both isomers (cis and trans) were found to be present in the biodegradation reactors spiked with the individual fungi species. However, in the reactor spiked with the mixture of the four isolates, trans-chlorfenvinphos was not detected and the area of cis-chlorfenvinphos formed was six to three times lower than in the reactors spiked with the individual isolates. A higher degree of chlorfenvinphos degradation can therefore be expected in nature where a mixture of the fungi isolates is expected to co-exist in the aquatic environment.

Conclusion The results obtained showed that –

–

–

–

Even though the pesticides atrazine, diuron and isoproturon may be degraded by a fungi-bacteria consortium, they were found to be resilient to biodegradation by the fungi targeted in this study; Chlorfenvinphos was degraded to levels below detection in natural untreated surface water and water spiked with several fungi isolates that were reported to occur in different water matrices; From all the fungi species identified, P. citrinum, A. fumigatus, A. terreus and T. harzianum are expected to survive when exposed to a constant input of pesticides in the aquatic environment and to be able to degrade chlorfenvinphos when spiked individually and as mixtures; The mixture of fungi is expected to achieve a higher degree of degradation of the chlorfenvinphos’ isomers.

Acknowledgments Financial support from Fundação para a Ciência e a Tecnologia—through the project PTDC/AAC-AMB/108303/2008, the grant PEst-OE/EQB/LA0004/2011 and the fellowship BPD/26990/ 2006—is gratefully acknowledged. We also thank EPAL as participant institution of the project PTDC/AAC-AMB/108303/2008 for supplying the untreated real water matrices used and for the analysis of the breakdown products. Vanessa J. Pereira thanks the Department of Geography and Environmental Engineering at Johns Hopkins University for hosting her as a Visiting Scholar during the academic year 2012/2013.

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