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Keywords:

  • heptachlor;
  • heptachlor epoxide;
  • white rot fungi;
  • Phlebia species;
  • hydroxylation;
  • hydrolysis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

White rot fungi of the genus Phlebia have demonstrated a high capacity to degrade organic pollutants, including polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls. In this study, we evaluated the ability of 18 white rot fungi species of genus Phlebia to degrade heptachlor and heptachlor epoxide, and described the metabolic pathways by selected white rot fungi. Phlebia tremellosa, Phlebia brevispora and Phlebia acanthocystis removed about 71%, 74% and 90% of heptachlor, respectively, after 14 days of incubation. A large amount of heptachlor epoxide and a small amount of 1-hydroxychlordene and 1-hydroxy-2,3-epoxychlordene were detected as metabolic products of heptachlor from most fungal cultures. The screening of heptachlor epoxide-degrading fungi revealed that several fungi are capable of degrading heptachlor epoxide, which is a recalcitrant metabolite of heptachlor. Phlebia acanthocystis, P. brevispora, Phlebia lindtneri and Phlebia aurea removed about 16%, 16%, 22% and 25% of heptachlor epoxide, respectively, after 14 days of incubation. Heptachlor diol and 1-hydroxy-2,3-epoxychlordene were produced in these fungal cultures as metabolites, suggesting that the hydrolysis and hydroxylation reaction occur in the epoxide ring and in position 1 of heptachlor epoxide, respectively.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

Over the past few decades, the presence of organochlorine pesticides (OCPs) in the environment has been of great concern due to their persistent, long-range transportable nature and toxic biological effects. Heptachlor is an OCP that was used extensively in the developed world throughout the 1960s and 1970s, mainly against termites and soil insects. Some developed countries banned or restricted the production and usage of heptachlor in the 1970s because animal data suggested that it is carcinogenic in humans (World Health Organization, 1984). Nevertheless, some developing countries continue to use this pesticide in both agriculture and public health programs because of its low cost and versatility in controlling various pests. Heptachlor has not been produced in Japan, but 1500 tons were imported between 1958 and 1972 (Murano et al., 2009). The Japanese government banned the use of heptachlor in 1972.

Heptachlor is likely to remain in the soil for long periods of time (Huber, 1993), albeit at relatively low concentrations (parts per billion). Its reported representative field half-life is 250 days (Augustijn-Beckers et al., 1994). However, traces of heptachlor have been detected in soil even 14 and 16 years after application. A widespread reaction in the environment is heptachlor epoxidation to the more persistent heptachlor epoxide. Heptachlor and heptachlor epoxide are relatively hydrophobic compounds and therefore extensively adsorb onto soil particles, giving these compounds low bioavailability and mobility in soil. Several studies have reported elevated concentrations of heptachlor and heptachlor epoxide in surface water, sediment and soil samples from Asian countries including China, Japan and Thailand (Kim et al., 2007; Gao et al., 2008; Poolpak et al., 2008).

The first evidence that heptachlor is degraded by soil microorganisms came from the experiments of Miles et al. (1969). In their studies, heptachlor is metabolized by soil bacteria and fungi into many different products by many independent metabolic pathways. Heptachlor epoxide, chlordene, chlordene epoxide, 1-hydroxychlordene and 1-hydroxy-2,3-epoxychlordene were the products of the microbial degradation of heptachlor (Fig. 1). Currently, bioremediation conducted on a commercial scale utilizes bacteria; there have been few attempts to use white rot fungi. However, white rot fungi offer advantages over bacteria in the diversity of compounds they can oxidize (Pointing, 2001). These organisms are generally more tolerant to high concentrations of polluting chemicals than bacteria. Therefore, white rot fungi represent a prospective tool for environmental bioremediation. Degradation of heptachlor by white rot fungi was also reported (Arisoy, 1998; Nwachukwu & Osuji, 2007). However, metabolites and metabolic pathways of heptachlor by white rot fungi have not yet been reported.

image

Figure 1.  Pathways of metabolism of heptachlor in soil microorganisms (Miles et al., 1969).

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Recently, we reported on several white rot fungi belonging to the genus Phlebia that are capable of degrading polychlorinated dibenzo-p-dioxins (PCDDs). Mori & Kondo (2002a, b) reported that several white rot fungi could mineralize 2,7-dichlorodibenzo-p-dioxin, and that 2,7-dichlorodibenzo-p-dioxin and 2,8-dichlorodibenzofuran were hydroxylated by Phlebia lindtneri. It was also reported that P. lindtneri and Phlebia brevispora are capable of hydroxylating and methoxylating 2,3,7-trichlorodibenzo-p-dioxin, 1,2,8,9-tetrachlorodibenzo-p-dioxin, 1,2,6,7-tetrachlorodibenzo-p-dioxin and 1,3,6,8-tetrachlorodibenzo-p-dioxin (Kamei & Kondo, 2005; Kamei et al., 2005). Additionally, chloronaphthalene and polychlorinated biphenyls were metabolized to hydroxylated products by P. lindtneri and P. brevispora, respectively (Mori et al., 2003; Kamei et al., 2006). These results suggested that Phlebia species have specific activity in the biotransformation of organohalogen compounds, and led us to pay attention to Phlebia species in selecting heptachlor- and heptachlor epoxide-degrading fungi.

In this paper, we evaluate the ability of genus Phlebia to degrade heptachlor and heptachlor epoxide, and we describe new hydroxylated metabolites of heptachlor epoxide by microorganisms. We also propose metabolic pathways of heptachlor and heptachlor epoxide in this genus. This is the first report describing the metabolites of heptachlor and heptachlor epoxide by white rot fungi.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

Chemicals

Heptachlor, heptachlor epoxide, 1-hydroxychlordene, N,N-dimethylformamide, phenanthrene, acetic anhydride, pyridin and all organic solvents were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Fungi and screening conditions

Eighteen species belonging to the genus Phlebia were used for degradation experiments. Phlebia acanthocystis TMIC34875, Phlebia tremellosa TMIC30511, Phlebia aurea TMIC33908, Phlebia radiata TMIC34599, Phlebia nitidula TMIC32286 and Phlebia tremellosus TMIC31235 were obtained from the Tottori Mycological Institute (Tottori, Japan). Phlebia lindtneri GB1027, Phlebia acerina HHB11146, Phlebia setulosa HHB12067, Phlebia rufa HHB14924, Phlebia ludoviciana HHB9640, Phlebia subochracea HHB8494, Phlebia livida HHB4609, Phlebia subserialis HHB9768, Phlebia bresadolae RLG10795 and Phlebia uda Kropp-1 were obtained from the Forest Products Laboratory of the United States Department of Agriculture (Washington, DC). Phlebia ochraceofulva ATCC96119 was obtained from the American Type Culture Collection (Manassas, VA). Phlebia brevispora TMIC34596 was identified using molecular approach in a previous study (Suhara et al., 2002). These fungi were maintained on 9-cm-diameter potato dextrose agar plates (Difco, Detroit, MI) that had been incubated at 30 °C. The mycelium mats on the agar plate were transferred to a sterilized blender cup containing 50 mL of sterilized water and were homogenized for 30 s. One milliliter of this homogenate was inoculated into 10 mL of liquid medium (pH 4.5) in 100-mL Erlenmeyer flasks containing 1.0% glucose as a carbon source, 1.2 mM ammonium tartrate as a low nitrogen medium, 20 mM sodium acetate, salt solution and trace element solution, as described by Tien & Kirk (1988). The cultures were preincubated statically at 30 °C under ambient atmospheric conditions.

Degradation experiments and metabolite detection

After preincubation for 5 days, 50 μL of substrate (heptachlor or heptachlor epoxide) (5 mM) in N,N-dimethylformamide was added to each inoculated flask (final concentration: 0.25 μmol per flask). The flask was sealed with a glass stopper and sealing tape after the headspace of each flask was flushed with oxygen. As a control, the cultures were killed by adding about 0.2 g of sodium azide after preincubation for 5 days. All experiments were performed in triplicate. After additional incubation for 14 days, cultures were killed by adding about 0.2 g of sodium azide. In order to determine the concentration of each substrate, an internal standard (phenanthrene) was added to the culture and then homogenized with 20 mL of acetone. The biomass was removed by centrifugation at 3000 g for 10 min at room temperature. The resulting supernatant was evaporated at 45 °C for 10 min to remove acetone, and the residue was acidified to pH 2.0 with 0.1 N HCl and extracted three times with 50 mL of ethyl acetate. The organic fraction was dried over anhydrous sodium sulfate and was concentrated to dryness under reduced pressure. The concentrate was analyzed by GC/MS. Acetic anhydride/pyridin was used for acetyl derivatization analysis. GC/MS was performed on an HP 6890 GC system linked to an HP 5973 mass selective detector and a 30-m fused DB-5MS column (0.25 μm inside diameter, J&W Scientific, Folsom, CA). The oven temperature was programmed at 80 °C for 3 min, followed by a linear increase to 320 °C at 20 °C min−1 and held at 300 °C for 5 min.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

The biodegradation of heptachlor by 18 selected Phlebia strains was studied. Table 1 presents the residual concentration of heptachlor by degradation from each fungal strain after 14 days of incubation. Ten strains were each able to remove over 50% of the heptachlor. Several strains exhibiting a high ability to degrade heptachlor; P. tremellosa, P. brevispora and P. acanthocystis degraded about 71%, 74% and 90% of heptachlor, respectively, after 14 days of incubation.

Table 1.   Residues of substrates and production of metabolites from the cultures of Phlebia species with heptachlor or heptachlor epoxide
Phlebia speciesIn heptachlor treatment (%)In heptachlor epoxide treatment (%)
HeptachlorHeptachlor epoxide1-HydroxychlordeneTotalMetabolite BHeptachlor epoxideMetabolite BMetabolite C
  1. Values are means ± SD of triplicate samples.

  2. +, detectable; −, undetectable.

P. bresadolae91.8 ± 4.29.3 ± 1.2101.198.0 ± 4.2
P. livida77.8 ± 4.116.7 ± 1.20.8 ± 0.295.396.9 ± 4.0
P. nitidula77.6 ± 5.020.8 ± 0.90.7 ± 0.199.1+104.2 ± 2.9
P. ludoviciana70.4 ± 2.831.8 ± 7.70.3 ± 0.1102.591.8 ± 5.7
P. ochraceofulva61.2 ± 6.434.5 ± 2.61.4 ± 0.397.1103.2 ± 0.9
P. setulosa59.2 ± 1.932.4 ± 4.01.8 ± 0.293.4+102.1 ± 7.0
P. radiata53.1 ± 4.245.5 ± 3.61.6 ± 0.7100.2107.1 ± 4.6
P. uda53.1 ± 7.944.1 ± 1.91.2 ± 0.598.4+90.0 ± 3.9
P. tremellosus50.0 ± 0.851.8 ± 1.00.9 ± 0.1102.791.3 ± 2.1
P. subochracea45.9 ± 8.052.9 ± 4.61.3 ± 0.4100.1+85.3 ± 4.8
P. acerina43.9 ± 2.750.8 ± 9.61.2 ± 0.295.9+95.8 ± 6.6
P. rufa40.8 ± 2.255.1 ± 2.92.3 ± 1.198.2+99.0 ± 5.9
P. lindtneri32.7 ± 3.452.0 ± 6.72.7 ± 0.787.4+78.4 ± 2.9+
P. aurea30.8 ± 3.856.0 ± 3.43.0 ± 0.289.8+75.5 ± 1.2++
P. subserialis30.6 ± 8.163.7 ± 0.72.9 ± 0.397.2+96.3 ± 1.9
P. tremellosa28.6 ± 2.068.3 ± 7.41.8 ± 0.198.7+105.0 ± 3.2
P. brevispora25.5 ± 7.263.8 ± 4.62.3 ± 0.791.6+83.7 ± 2.3++
P. acanthocystis10.2 ± 3.574.9 ± 5.42.8 ± 0.487.9+84.2 ± 10.4++
Control98.4 ± 8.298.4101.2 ± 3.2

During heptachlor metabolism by each fungal strain, the major metabolic product had a retention time (15.73 min) and mass spectrum identical to authentic heptachlor epoxide. In the cultures of 10 fungal strains,>50% (0.125 μmol per flask) of additional heptachlor was transformed into heptachlor epoxide. Especially, P. acanthocystis transformed 74.9% (0.19 μmol per flask) of heptachlor into heptachlor epoxide after 14 days of incubation, according to quantitative analysis using GC/MS systems. This result indicates that epoxidation of heptachlor is a common metabolic pathway in cultures of all Phlebia fungi studied in these experiments.

Other two metabolic products were detected from the cultures of fungi by GC/MS analysis. Metabolite A was detected from cultures of all fungi, excluding P. bresadolae, which showed the lowest degradation ability (Table 1). The mass spectrum of metabolite A at 14.95 min had a weak molecular ion peak (M+) of m/z 352 (Cl=35). The loss of a chloride ion from this molecular ion peak gives rise to fragment ion at m/z 317, which has a characteristic of five chlorine ions. Other intense fragment ions were observed at m/z 281 (M+-Cl-HCl), 217 (C9H4Cl3), 183 (C9H5Cl2) and 82 (C5H6O) (Fig. 2a). Based on a comparison with an authentic compound, metabolite A was identified as 1-hydroxychlordene, which is a hydroxylated product of heptachlor at the 1 position. In contrast to heptachlor epoxide, only a small amount of 1-hydroxychlordene was detected from all fungal cultures (Table 1). Metabolite B was detected at 15.33 min from 12 fungal cultures. The mass spectrum of metabolite B showed a molecular ion peak (M+) of m/z 368, which has the characteristic of six chlorine ion, and fragment ions at m/z 333 (M+-Cl), 297 (M+-Cl-HCl), 261 (M+-C3H4O2Cl), 235 (M+-C5H6O2Cl) and 97 (C5H5O2) (Fig. 2b). After acetylation, metabolite B disappeared and the compound acetyl B was newly detected at 15.53 min. This compound showed a weak molecular ion peak at m/z 410 (molecular mass of metabolite B[368]+42 mass), and fragment ions at m/z 375 (M+-Cl), 315 (M+-OCOCH3-HCl), 280 (M+-OCOCH3-HCl-Cl) and 235 (M+-C5H6O2Cl) (mass spectrum not shown). Based on these results, metabolite B is thought to be 1-hydroxy-2,3-epoxychlordene. These metabolites were not detected from the azide-killed control culture.

image

Figure 2.  Mass spectra of metabolite A (a) and metabolite B (b) of heptachlor from cultures of Phlebia species.

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The product 1-hydroxy-2,3-epoxychlordene (metabolite B) could conceivably be produced from two alternate pathways: by epoxidation of 1-hydroxychlordene at the 2, 3 positions, or by hydroxylation of heptachlor epoxide at the 1 position. Heptachlor epoxide is known to be rather stable in biological systems (Metcalf & Sanborn, 1975). Thus, the conversion of 1-hydroxychlordene to 1-hydroxy-2,3-epoxychlordene seems to be more probable. In order to understand the ability of fungi to degrade heptachlor epoxide, and to determine the source of the 1-hydroxy-2,3-epoxychlordene, the 18 strains of genus Phlebia were incubated with heptachlor epoxide (0.25 μmol per flask) at 30 °C for 14 days. Table 1 describes the biodegradation of heptachlor epoxide by 18 fungal cultures. In contrast to heptachlor, heptachlor epoxide exhibited lower levels of degradation activity. Phlebia acanthocystis, P. brevispora, P. lindtneri and P. aurea decreased heptachlor epoxide levels by about 16%, 16%, 22% and 25%, respectively, after 14 days of incubation. On the other hand, other Phlebia strains showed less degradation of the substrate. Although P. subserialis and P. tremellosa degraded heptachlor by about 70%, their ability to degrade heptachlor epoxide was not demonstrated in these experiments.

To prove the metabolism of heptachlor epoxide in cultures of the fungi, which were found to reduce heptachlor epoxide levels during 14 days of incubation, the extracts from the cultures with heptachlor epoxide were analyzed by GC/MS. Two metabolic products were detected. The cultures of P. acanthocystis, P. brevispora, P. lindtneri and P. aurea each yielded a small amount of metabolite B product (1-hydroxy-2,3-epoxychlordene). The results show that these fungi can convert heptachlor epoxide into 1-hydroxy-2,3-epoxychlordene via hydroxylation at the 1 position. After acetylation, metabolite C was detected from the cultures of P. acanthocystis, P. brevispora and P. aurea with heptachlor epoxide. The mass spectrum of acetylated metabolite C had an ion peak of m/z 453, which is characteristic of six chlorine ions (Fig. 3). The ion at m/z 453 is considered to arise from the loss of one chlorine ion from the molecular ion (M=488), although the molecular ion peak has not been found. The loss of COOCH2 from the molecular ion gives rise to the fragment ion peak at m/z 430, which has the characteristic of seven chlorine ions. The ion peak at m/z 393 represents the loss of HCl-COOCH3 from the molecular ion. The ion peak at m/z 350 represents the loss of COOCH3 from the major fragment ion at m/z 393. The loss of OH from the peak at m/z 350 gives rise to the peak at m/z 333. The loss of Cl from the peak at m/z 350 produces the peak at m/z 315, which has the characteristic of five chlorine ions. The peaks at m/z 270 and m/z 235 represent fragment ions C5Cl6 and C5Cl5, respectively.

image

Figure 3.  Mass spectrum of metabolite C of heptachlor epoxide from cultures of Phlebia brevispora, Phlebia aurea and Phlebia acanthocystis, after acetylation.

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On the basis of the mass spectrum analysis and the molecular weight of 488 (molecular mass of heptachlor epoxide[386]+2COCH2[84] mass+H2O[18] mass) of metabolite C, we propose that hydrolysis occurs in heptachlor epoxide at the 2 or 3 positions to produce a diol compound, heptachlor diol (metabolite C), which is known as an metabolic intermediate of heptachlor in animals (Feroz et al., 1990).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

In this paper, we examined 18 strains of white rot fungi of the genus Phlebia for their degradation ability against the OCP heptachlor and heptachlor epoxide. We found that most of the strains were able to degrade heptachlor. The proposed metabolic pathways of heptachlor by Phlebia species are presented in Fig. 4. These data clearly indicate two metabolic pathways of heptachlor in most Phlebia species: pathway (1), epoxidation at the 2, 3 positions to heptachlor epoxide; and pathway (2), hydroxylation at the 1 position to 1-hydroxychlordene followed by epoxidation to 1-hydroxy-2,3-epoxychlordene. The former appears to be a major metabolic pathway, because a large amount of heptachlor epoxide was detected in the cultures of most fungi.

image

Figure 4.  Proposed pathways for the metabolism of heptachlor by Phlebia brevispora, Phlebia aurea and Phlebia acanthocystis.

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Miles et al. (1969) showed that heptachlor is metabolized by soil microorganisms into many different products by many independent metabolic pathways, including epoxidation, hydrolysis and reduction (Fig. 1). In addition, the metabolic pathway (2) of heptachlor in our study also has been found in soil by Carter et al. (1971). In our experiments, the dechlorination products of heptachlor, such as chlordene and chlordene epoxide, were not detected from cultures of these Phlebia strains.

Heptachlor epoxide, the most predominant metabolite of heptachlor, is more stable than heptachlor itself and the other metabolites (Lu et al., 1975). Only limited information has been reported on the biodegradation of heptachlor epoxide by microorganisms. Miles et al. (1971) reported that a mixed culture of soil microorganisms obtained from a sandy loam soil could transform heptachlor epoxide to the less-toxic 1-hydroxychlordene, but the mechanism for the conversion of heptachlor epoxide was not determined; the degradation rate was about 1% per week during the 12-week test period. Kataoka et al. (2010) also described that the biodegradation of heptachlor epoxide by a soil fungus, Mucor racemosus strain DDF, which was isolated from a soil with annual endosulfan applications; however, the detection of metabolite is not described in this paper. In contrast to soil microorganisms, white rot fungi such as P. brevispora and P. acanthocystis exhibited higher levels of degradation activity to heptachlor epoxide and two new metabolic pathways of heptachlor epoxide in selected fungi were proposed in this experiments: hydroxylation at the 1 position to 1-hydroxy-2,3-epoxychlordene and hydrolysis at the epoxide ring to heptachlor diol. To our knowledge, heptachlor diol and 1-hydroxy-2,3-epoxychlordene have not been reported previously as a metabolic product from heptachlor epoxide by bacteria or fungi. Feroz et al. (1990) suggested that Daphnia magna, a freshwater microcrustacean, could metabolize heptachlor and that heptachlor was oxidized to heptachlor epoxide, followed by cleavage of the epoxide ring to heptachlor diol, which then can be transformed to trihydroxychlordene. A similar metabolic pathway was found in the metabolism of heptachlor in goldfish, Carassius auratus (Feroz & Khan, 1979). Our results first showed the degradation of heptachlor epoxide via hydrolysis at the epoxide ring to produce heptachlor diol by microorganisms. A comparison between our results and those of the papers describing the degradation mechanism of heptachlor epoxide suggested that, in white rot fungi, the metabolism pathway of heptachlor epoxide seems to be similar to that in mammals, and that heptachlor diol might be further degraded.

Several Phlebia species are known to produce lignin-degrading extracellular enzyme system. Major components of the lignin-degrading extracellular enzyme system include lignin peroxidases, manganese peroxidases and laccase (Vares et al., 1995; Leontievsky et al., 1997). In addition, in our previous report, it is suggested that a cytochrome P450 monooxygenase from P. brevispora and P. lindtneri are involved in the initial degradation of organochlorine compound such as PCDDs (Kamei & Kondo, 2005; Kamei et al., 2005). Although further experiments are needed, it is reasonable to suppose that cytochrome P450 monooxygenase plays some role in the metabolism of OCPs such as heptachlor.

This is the first report on the metabolic pathways of heptachlor and heptachlor epoxide by white rot fungi. Our results suggested that heptachlor and heptachlor epoxide were degraded into several less-toxic products by selected Phlebia species. This result is important because of the possibilities of using fungi for the decontamination and detoxification of organochlorine-polluted environments. The use of microorganisms for bioremediation requires an understanding of all the physiological and biochemical aspects involved in pollutant transformation. Future research includes identification and isolation of an enzyme system involved in the degradation of heptachlor, optimization of the conditions and molecular approaches for application in the organochlorine-polluted soil systems.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference

This work was supported by a grant from Research project for ensuring food safety from farm to table, Ministry of Agriculture, Forestry and Fisheries, Japan (PO-3216).

Reference

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Reference
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