Biodegradation kinetics of dicofol by selected microorganisms

Pesticide Biochemistry and Physiology 91 (2008) 180–185

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Biodegradation kinetics of dicofol by selected microorganisms Khaled A. Osman a,*, Gamal H. Ibrahim b, Ahmad I. Askar b, Abdul Rahman A. Aba Alkhail b a b

College of Agriculture & Veterinary Medicine, Buraidah, P.O. Box 1482, Al-Qassim, Saudi Arabia College of Science Al-Qassim University, Buraidah, P.O. Box 1482, Al-Qassim, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 21 October 2007 Accepted 31 March 2008 Available online 29 April 2008 Keywords: Bacteria Fungi Dicofol Bioremediation Solid phase extraction HPLC Half-life

a b s t r a c t The capacity of some plant growth promoters (seven species of bacteria namely, Azospirillium barasilense, Azotobacter chroococcum, Klebsilense pneumoneae, Pseudomonas cepacia, Bacillus subtilis, Pseudomonas fluorescens and Bacillus polymyxa) and some control agents for plant pathogens (two species of fungi namely, Trichoderma viride and Trichoderma harzianum) to degrade dicofol pesticide (DCF) in liquid culture media was investigated. The recovered amount of DCF was extracted based on the solid phase extraction (SPE) with methylene chloride and then analyzed by HPLC. Contrary to published reports, no intermediate or final degradation metabolites of DCF could be observed. About 26–33% of DCF degradation was observed after 3 days of incubation with the tested bacteria which increased to 61–80%, 74–85%, 77–87% and 75– 94% after 7, 14, 21 and 28 days of incubation, respectively. On the other hand, the tested fungi removed roughly 35% of available DCF within the first three days of incubation which increased to 84–87%, 89– 95%, 91–95% and 92–96% after 7, 14, 21 and 28 days of incubation, respectively. A biphasic model was assumed to explore the disappearance of DCF from media enriched with either bacteria or fungi, where the rate of disappearance in the first phase was faster than the second. This is clearly reflected in the halflife (t1/2) for the first and second phases, where the t1/2 values of DCF ranged from 2.82–4.42 to 19.32– 29.73 days, respectively. The results have implications for the development of a bioremediation strategy. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Pesticides are widely used in agriculture to control a variety of pernicious organisms that spoil the crops. More than 600 kinds of agrochemicals are used around the world [1]. They provide unquestionable benefit for agricultural production, even though, as a consequence, low amounts of some residues may persist in the food supply, air, water and soil and could constitute a significant exposure pathway for humans. Exposure to these residues has created uncertainty for potential chronic toxicity and in some cases, acute toxicity [2–4]. Residue of organochlorine pesticides in the environment is still a world-wide problem although the use of chlorinated hydrocarbon insecticides has been sharply curtailed or banned, but they are still the active ingredients of some pest control products [5]. For example, dicofol (DCF) is used world-wide as a pre-harvest miticide on cotton, citrus, vegetable, nuts, date palm and other crops [6–9]. DCF is structurally similar to DDT, which is used as the starting material for synthesis for DCF [10]. The US-EPA became concerned about the continued use products containing DCF because they also contained DDT and related compounds [5].

* Corresponding author. Fax: +966 6 380 1360. E-mail address: [email protected] (K.A. Osman). 0048-3575/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2008.03.012

Soil receives various pesticides and microbial population could either be adapted to these pesticides or capable of degrading it through existing enzymes and may serve as a source of nutrients [11–14]. Several studies were conducted to examine the degradation of pesticides in soil [11,13,15,16]. Bioremediation is a pollution control technology that uses biological systems to catalyze the degradation or transformation of various toxic chemicals. The general approaches to bioremediation are to enhance natural biodegradation by native organisms (intrinsic bioremediation), to carry out environmental modification by applying nutrients or aeration (biostimulation) or through addition of microorganisms (bioaugmentation). Fungi are unique among microorganisms in that they secrete a variety of extracellular enzymes, free radical and nonspecific mode of degradation, which allow them to degrade both soluble and insoluble contaminants [17,18]. On the other hand, several species of bacteria are good degraders of toxic pesticides [19–21]. The bioremedial potential of the bacterial isolates could be influenced by a range of abiotic factors such as pH, temperature and inoculum density [22]. DCF is released to the environment through its manufacture and use as a nonsystemic acaricide [23]. If released to soil, it will be expected to bind to the soil strongly but under some circumstances it may reach groundwater and threat human health. It is classified as slightly toxic compound having acute oral LD50 for rat of 595 mg/ kg [24] and environmental endocrine-disrupting chemical [25].

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Also, DCF has a high acute toxicity in the aquatic environment, with a LC50 of 15 lg/l for eastern oyster and of 120 lg/l for rainbow trout, can affect eggshell quality in birds, and in falcon, feminized male embryos have been found from females given 5 mg/kg [25]. Such concerns have heightened the need for innovative and advanced technology for effective removal of DCF from a variety on contaminated environmental sources including water, sediments and soils. Unfortunately, limited data are available on in vitro biodegradation of DCF by pure cultures. Therefore, the present investigation was carried out to compare between capability of some bacteria such as Bacillus subtilis (Ehrenberg) Cohn culture BI, Pseudomonas fluorescens, Pseudomonas cepacia (Burkholderia ceppacia ex Burkholder), Bacillus polymyxa, Azospirillium barasilense, Azotobacter chroococcum, and Klebsilense pneumoneae and two isolates of fungi namely, Trichoderma viride and T. harzianum to degrade DCF, the widely used in Saudi Arabia as a pre-harvest miticide on date palm to control mites [9].

its optical density at A650 nm to 0.01 (107 cell/ml). The pH of the either PDA or NA media was adjusted to 7.0 using phosphate buffer. Bacterial suspension (1 ml) or one disc of the fungus (5 mm, diameter 1.6  106 spore/disk) was added to 250 ml Erlenmeyer flasks containing 100 ml of either NA broth medium containing (in g/l) beef extract, 3; peptone, 5; and glucose, 2.5 yielding dissolved organic carbon of 0.999 g/l or Czapek-Dox containing (in g/l) NaNO3, 2; K2HPO4, 1; MgSO4.7H2O, 0.5; KCl, 0.5; FeSO4, 0.01 and sucrose, 30 yielding dissolved organic carbon of 12.62 g/l, respectively, and spiked with 0.1 ml of stock solution of DCF dissolved in acetone providing a final concentration of 100 ppm (the final acetone concentration in media was 0.10%). Flasks were kept at 27 ± 1 °C for a period of 28 days without shaking. Aliquots (20 ml each) of each media were withdrawn by micropipette after 0, 3, 7, 14, 21 and 28 days of incubation and subjected to chromatographic analysis. A separate set of uninoculated flasks was maintained as reference. All experiments were performed in triplicates.

2. Materials and methods

2.4. Extraction procedure

2.1. Chemicals and standard

Three 20-ml aliquots of media from each time interval were used, filtered under vacuum through Whatmann No. 2 filter paper and then subjected to solid phase extraction (SPE). Each cartridge was conditioned with two times of 5 ml of methanol and then with two times of 5 ml of ultra-pure deionized water of 15 MX cm resistivity and slowly aspirated. Aliquots were loaded and vacuumed at rate of 5 ml/min. Subsequently, the elution took place with two times of 5 ml of methylene chloride into glass vials (12 ml), followed by evaporation to dryness under vacuum. The dry residues of DCF were redissolved in 1 ml of methanol, agitated by vortex and then subjected to analysis by HPLC.

Technical dicofol (DCF), [2,2,2-trichloro-1,1-bis-(4-chlorophenyl) ethanol], was provided by Environmental Protection Agency (EPA, USA) with purity of 98%. Certified HPLC-grade of methylene chloride, methanol and acetonitrile (ACN) and granular AR anhydrous sodium sulfate were purchased from BDH Company, while the Baker spe-12G Column Processor designed vacuum manifold capable of processing up to 12 solid phase extraction columns and solid phase extraction column (BAKERBOND speTM, C18, 500 mg per column) was purchased from J.T. Baker, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA. Ultra-pure deionized water of 15 MX cm resistivity was obtained from a water purification system (PURELAB Option-R, ELGA, UK) and used throughout this study. All other chemicals used in this study were of the highest grade available. 2.2. Source of microorganisms Bacillus subtilis (Ehrenberg) Cohn culture BI (B.S.I.) and P. fluorescens were originally isolated from soil of the wheat fields of the Experimental Farm of Sabahia Research Station, Alexandria, Egypt [26]. P. cepacia (B. ceppacia ex Burkholder) isolate was obtained from the Plant Pathology Department, North Carolina State University, USA, type culture collection (ATCC55344). B. polymyxa was kindly provided by Dr. M.A. El-Meleigi, Plant Production and Protection Department, College of Agriculture & Veterinary Medicine, Al-Qassim University, KSA. This isolate was originally isolated from wheat roots of Al-Qassim fields, Central Saudi Arabia. Three growth promoting nitrogen fixing rhizobacteria, namely A. barasilense, A. chroococcum, and K. pneumoneae were kindly provided by Dr. M.G. Hassonna, Plant Pathology Department, College of Agriculture, Alexandria University, Egypt. T. viride and T. harzianum were obtained from Plant Pathology & Weed Science Department, Colorado State University, Fort Collins, Colorado, USA. 2.3. Degradation experiments Fungi culture, 7 days old, were maintained at 27 ± 1 °C on potato dextrose agar (PDA) medium containing (in g/l sterilized distillated water) peeled potato 200, dextrose 20 and agar 15 and served as a source of inoculum. Bacteria isolates were grown at 27 ± 1 °C for 48 h on nutrient agar (NA) medium containing (in g/l sterilized distillated water) beef extract, 3; peptone, 5; agar, 15 and glucose, 2.5, then suspended in sterilized distillated water and adjusted to

2.5. Preparation of standard solutions The HPLC system was standardized on the same day as the samples were analyzed by injecting 20 ll of freshly prepared DCF in methanol with concentrations ranging from 0.0 to 10 ppm from a stock solution of 1000 ppm. Areas under the peak (lV s) versus concentrations (lg) were plotted and fit by simple linear regression to obtain an equation for the standard curve. The amount of DCF in each sample was thus calculated based on the slope of the standard curve. 2.6. High performance liquid chromatography (HPLC) analysis Extracts were chromatographed on a Perkin Elmer HPLC system model 200 equipped with a degasser, quaternary LC pump model 2000Q/410, 20 ll loop, Spheri-5 RP-18 column (15 cm  4.6 mm i.d., 5 mm, Perkin-Elmer), oven column, a LC200 UV detector. The Turbochrom Workstation Software package was used for instrument control, data acquisition, and data analysis. The column temperature was kept at 25 °C. DCF was eluted using an isocratic gradient of ACN:H2O in ratio of 78:22 (v/v) and detected at wavelength (k) of 220 nm with flow rate of 1.1 ml/min. The retention time for DCF under these conditions was 3.2 min. Half-life of DCF was estimated by regression of the recovered amounts of DCF against time of incubation. 2.7. Limits of detection and quantification The limits of detection (LOD) and limits of quantification (LOQ) with this procedure were defined as the concentration (expressed as ppm) that gave signals of 3 and 10 times the noise, respectively, within its retention time (tR) window [27]. The HPLC response was linear (r2 = 0.934) for DCF in the range of 0.0 to 10 ppm with corre-

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lation coefficient (R) of 0.967. The LOD and LOQ of DCF were 0.54 and 1.80 ppm, respectively. 2.8. Statistical analysis Data were calculated as mean ± standard deviation (SD) analyzed using analysis of variance (ANOVA). Probability of 0.05 or less was considered significant. The statistical package of Costat Program [28] was used for all chemometric calculations.

of DCF (Table 2). There were considerable variations between the two fungi with respect to their abilities to degrade DCF during the periods of 7–28 days of incubation. Both T. viride or T. harzianum removed roughly 35% of available within the first three days of incubation which increased to 84–87%, 89–95%, 91–95% and 92–96% after 3, 7, 14, 21 and 28 days of incubation, respectively. Steady state was reached after 14 days of inoculation with either T. viride or T. harzianum and the rate of DCF dissipation was very slow. At the end of experiment the maximum removal of DCF was observed with T. harzianum (96%) followed by T. viride (92%).

3. Results and discussion 3.3. Kinetic studies It was found that the peak of DCF on HPLC chromatogram gradually decreased with increasing incubation time, however, no other intermediate metabolites or dead-end product could be observed with different periods of incubation (Fig. 1). 3.1. Recovered and removal of DCF from media inoculated with bacteria In the present study, DCF was used at concentration of 100 ppm (Initial concentration) without inhibitory effects to either bacteria or fungi (data not shown). In normal agriculture concentration DCF did not exhibit measurable effect on soil bacteria populations [29]. The culture of Aspergillus niger could tolerate 400 ppm of technical grade of endosulfan [30]. On the other hand, complete degradation of DDT at concentrations up to 15 ppm in flasks, with shaking, had been achieved but inhibitory effects were observed at 50 ppm [31]. Data in Table 1 illustrate that the recovered amount of DCF significantly declined from the initial concentration with increasing the incubation period in the media amended with all the tested bacteria, while media without any amendment (i.e., uninoculated control) showed less dissipation of DCF. About 26–33% of DCF degradation was observed after 3 days of incubation which increased to 61–80%, 74–85%, 77–88% and 75–94% after 7, 14, 21 and 28 days of incubation with the tested bacteria, respectively. Steady state was reached after 7 days with A. barasilense, 14 days with K. pneumoneae and P. cepacia, and 21 days with B. subtilis, P. fluorescens and B. polymyxa. In case of A. chroococcum, the degradation was slow from day 7 to day 21. After 4 weeks of incubation the highest dissipation (94%) was found with A. chroococcum followed by K. pneumoneae (88%), B. subtilis (85%), B. polymyxa (84%), P. fluorescences (82%) and then A. barasilense (75%). 3.2. Recovered and removal of DCF from media inoculated with fungi The recovered amount of DCF significantly declined from the initial concentration with increasing the incubation period in the media amended with the tested fungi, while media without any amendment (i.e., uninoculated control) showed less dissipation

A biphasic model was assumed in order to carry out the statistical study of the loss of DCF according to the Eq. (1). R ¼ A0 eat þ B0 ebt

ð1Þ

where R is the recovered amount of DCF at t days, A0 and B0 are the concentrations of DCF at t = 0 and a and b are the disappearance rate constants for the first and second phase model, respectively. The half-life (t1/2) of the exponential decay was calculated according to the Eq. (2). t1=2 ¼ ð2:303 log 2Þ=rate constant

ð2Þ

The biphasic model is characterized by a rapid phase which appears to be over in a few hours or days, and a much slower phase which may continue over weeks or even months [32–34]. The remaining residues are often quite resistant to degradation [17]. The data indicated that there was a faster rate of DCF disappearance in the first phase than the second one (Table 3). This is clearly reflected in the t1/2 values, where the half-lives of DCF were 2.86, 4.42, 3.36, 3.79, 3.19, 3.61 and 2.82 days in media amended with A. chroococcum, K. pneumoneae, P. cepacia, B. subtilis, P. fluorescences, B. polymyxa and A. barasilense, while they were 3.08 and 3.18 days in culture amended with T. viride and T. harzianum, respectively. However, the half-life values (t1/2) values for DCF in the second phase model were 24.67, 26.94, 21.35, 25.54, 29.73, 19.32 and 40.76 days in culture amended with A. chroococcum, K. pneumoneae, P. cepacia, B. subtilis, P. fluorescens, B. polymyxa and A. barasilense, while they were 19.93 and 24.19 days in media amended with T. viride and T. harzianum, respectively. This finding are in accordance with many investigators who reported that the kinetics of pesticides degradation in soil is commonly biphasic with a very rapid degradation rate at the beginning followed by a very slow prolonged dissipation [32–34]. The relative importance of the phases depends on the availability of the pollutants, hydrophobicity and affinity for organic mater. DCF was found to be more easily degradable than DDT and other organochlorine insecticides [19] showing the lowest accumulation hazard. Indeed, the trichloromethyl group of DCF is

Fig. 1. HPLC profile for the biodegradation of DCF by either bacteria or fungi.

Table 1 Levels (ppm)a and percentages of dicofol removalb after biodegradation by selected bacteria at different time intervals Time (day)

a b

A. chroococcum

K. pneumoneae

P. cepacia

B. subtilis

P. fluorescences

B. polymyxa

A. barasilense

DCF level

DCF level

% Of removal

DCF level

% Of removal

DCF level

% Of removal

DCF level

% Of removal

DCF level

% Of removal

DCF level

% Of removal

DCF level

% Of removal

99.50 ± 0.92a 97.70 ± 1.20c 92.00 ± 1.43g 88.20 ± 2.49e 83.40 ± 3.55g 76.11 ± 1.79g

99.3 ± 1.10a 65.70 ± 0.28a 18.47 ± 0.49a 14.37 ± 0.49a 10.89 ± 0.29a,b 4.90 ± 0.113a

0.20 33 80 84 87 94

99.3 ± 1.10a 72.30 ± 0.42b 35.46 ± 0.45f 13.31 ± 0.35a 12.69 ± 0.33b,c 8.87 ± 0.23b

0.20 26 61 85 85 88

99.3 ± 1.10a 68.50 ± 0.42a,b 24.80 ± 3.66b,c 13.47 ± 0.35a 10.16 ± 0.27a 10.21 ± 0.12c

0.20 30 73 85 88 87

99.3 ± 1.10a 69.25 ± 0.35a,b 29.08 ± 0.76d 18.61 ± 0.49b 11.90 ± 0.32a,b 11.12 ± 0.29c,d

0.20 29 68 79 86 85

99.3 ± 1.10a 67.80 ± 0.52a,b 23.84 ± 0.64b 22.67 ± 0.60d 15.70 ± 0.42d 14.03 ± 0.44e

0.20 31 74 74 81 82

99.3 ± 1.10a 69.75 ± 0.35a,b 27.98 ± 0.74c,d 20.18 ± 0.52b 14.55 ± 0.14c,d 12.47 ± 0.33d

0.20 29 70 77 83 84

99.3 ± 1.10a 66.50 ± 0.71a 21.70 ± 0.51b 19.11 ± 1.54b 19.01 ± 0.50f 18.85 ± 0.49f

0.20 32 76 78 77 75

Each value is the mean ± SD of the remaining DCF in culture of three replicates. Means in the same row followed by the same letters are not significantly different (P 6 0.05, Duncan’s multiple-range test). Biodegradation (%) was calculated relative to the remaining DCF in uninoculated control.

Table 2 Levels (ppm)a and percentages of dicofol removalb after biodegradation by selected fungi at different time intervals Time (day)

0 3 7 14 21 28 a b

Uninoculated control

T. viride

DCF level

DCF level

% Of removal

T. harzianum DCF level

% Of removal

99.60 ± 0.88a 96.40 ± 1.21b 91.22 ± 1.87c 87.30 ± 1.34c 80.06 ± 2.40c 76.40 ± 1.67c

98.20 ± 1.22a 62.25 ± 0.35a 15.05 ± 0.40 b 9.20 ± 0.24b 7.46 ± 0.20b 5.76 ± 0.16b

1.40 35 84 89 91 92

98.20 ± 1.22 a 62.75 ± 0.35a 12.04 ± 0.32a 4.56 ± 0.12a 4.22 ± 0.11a 3.28 ± 0.08a

1.40 35 87 95 95 96

K.A. Osman et al. / Pesticide Biochemistry and Physiology 91 (2008) 180–185

0 3 7 14 21 28

Uninoculated control

Each value is the mean ± SD of the remaining DCF in culture of three replicates. Means in the same row followed by the same letters are not significantly different (P 6 0.05, Duncan’s multiple-range test). Biodegradation (%) was calculated relative to the remaining DCF in uninoculated control.

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51.33 ± 1.88a 16.92 ± 0.66a 0.218 ± 0.06a 0.029 ± 0.008a 3.18 ± 0.41a,b 24.19 ± 2.60b 0.974 Each value is the mean ± SD of four replicates. Means in the same row followed by the same letters are not significantly different (P 6 0.05, Duncan’s multiple-range test).

97.66 ± 2.67c 21.00 ± 1.10a 0.225 ± 0.08a 0.035 ± 0.006a 3.08 ± 0.23a 19.93 ± 1.67a 0.980 90.00 ± 2.70b,c 42.67 ± 1.88f 0.192 ± 0.04a 0.036 ± 0.003a 3.61 ± 0.60c,d 19.32 ± 1.70a 0.982 95.60 ± 1.59c 20.00 ± 1.11b 0.157 ± 0.02a 0.026 ± 0.006a 4.42 ± 0.34e 26.94 ± 1.60b 0.990 A0 (ppm) B0 (ppm) a (days-1) b (days-1) t1/2a (days) t1/2b (days) Regression coefficient

95.70 ± 2.22c 24.67 ± 1.20c 0.242 ± 0.05a 0.02 ± 0.009a 2.86 ± 0.22a 24.67 ± 1.33b 0.974

94.58 ± 0.88bc 22.00 ± 2.05b 0.195 ± 0.01a 0.032 ± 0.006a 3.36 ± 0.30b,c 21.35 ± 2.05a 0.982

91.96 ± 2.40bc 38.62 ± 1.34e 0.183 ± 0.01a 0.027 ± 0.004a 3.79 ± 0.29d 25.54 ± 3.80b 0.987

97.17 ± 3.10c 34.67 ± 2.50d 0.217 ± 0.02a 0.023 ± 0.009a 3.19 ± 0.11a,b 29.73 ± 2.99c 0.972

85.79 ± 4.33b 25.33 ± 1.09c 0.246 ± 0.03a 0.017 ± 0.001a 2.82 ± 0.19a 40.76 ± 2.44d 0.967

T. viride B. polymyxa K. pneumoneae A. chroococcum

Bacteria Statistical parameters

Table 3 Statistical parameters of DCF dissipation by selected microorganisms

P. cepacia

B. subtilis

P. fluorescences

A. barasilense

Fungi

T. harzianum

8.90 2.20 0.11 0.02 0.30 2.60

LSD0.05

184

extraordinary susceptible to carbon–carbon bond cleavage to form 4,40 - dichlorobenzophenone [19,35,36]. The biodegradation of DCF yielded nearly equal chloride (3.3 lg from 20 lg) than in the case of methoxychlor (2 lg from 20 lg) when calculated on total substituted chloride [19]. Also, the pathway between DCF and 4,40 dichlorobenzophenone in fungus Phanerochaete chrysosporium might proceed in a manner similar to that observed in bacterial systems, in which trichloromethyl carbon undergoes successive reductive dechlorinations followed by oxidation to form the carboxylic acid, which then undergoes decarboxylation to form dichlorobenzophenone [36]. In our experiment dichlorobenzophenone could not be detected using the HPLC during the biodegradation of DCF either by bacteria or fungi. Also, the absence of DCF metabolites may suggest that these microorganisms may degrade DCF by another pathway different from previous reports [19,35,36]. The lack of DCF metabolites may support its complete degradation. Complete disappearance of endosulfan was seen on day 12 of incubation with A. niger with evolution of CO2 and change in pH of medium to acid side indicating microbial transformation of endosulfan [30], while removal of endosulfan by Pseudomonas aeruginosa and Flaviminas oryzihabitans was lower than that of DDT, indicating that both bacteria preferentially degrade aromatic compounds [31]. In the present study DCF could biodegraded by P. fluorescens, while it was found to bioaccumulate in the P. fluorescens [29] and in Gram-negative nitrogen soil bacterium Azospirillum lipoferum [37], resulting in enhanced persistence of DCF. In general organochlorine pesticides possess halogen electron withdrawing groups which make these compounds resist aerobic degradation [38]. However, in some cases the degradation of other organochlorine pesticides such as lindane has also been successfully conducted under aerobic conditions by white-rot fungi [39]. 4. Conclusion Degradation of DCF proceeded rapidly in pure media amended with A. barasilense and A. chroococcum with t12 values of <3 days compared with >4 days in case of K. pneumoneae. For the remaining isolates, the t12 values ranged from >3 to <4 days. The relatively fast rate of DCF degradation may be attributed to the adaptation of the tested microorganisms to degrade DCF. By the end of the experiment A. chroococcum degraded roughly 94% of DCF, while K. pneumoneae, P. cepacia, B. subtilis, P. fluorescens and B. polymyxa degraded 82–88%. On contrary, A. barasilense was less degrader of DCF, with 75% of the compound degraded. In case of the fungi, T. viride and T. harzianum were also effective degraders of DCF, with 92, 96% of the compound degraded by the end of the experiment, respectively. During the incubation of DCF with these microorganisms, no known intermediate or dead-end product could be detected using HPLC. These results demonstrate that fungi and bacteria can reduce the persistence of DCF in liquid media culture. The results have implications for the development of a bioremediation strategy by mixing bacterial and/or fungal-inoculated substrates with the contaminated soil. References [1] Y. Miyake, K. Koji, H. Matsuki, R. Tajima, Fate of agrochemical residues, associated with malt and hops, J. Am. Soc. Brew Chem. 57 (1999) 46–54. [2] D.S. Saunders, C. Harper, Pesticides, in: A.W. Hays (Ed.), Principles and Methods of Toxicology, Taylor & Francis, 1994, p. 389. [3] G. Ekström, H. Hemming, M. Palborg, Swedish pesticide risk reduction. 1985– 1995: food residues, health hazard and reported poisoning, Rev. Environ. Contam. Toxicol. 147 (1996) 119–139. [4] K.A. Osman, S. Al-Rehiayani, Risk assessment of pesticide to human and the environment, Saudi J. Biol. Sci. 10 (2003) 81–106. [5] J.A. Moore, Dicofol: intent to cancel registrations of pesticide products containing dicofol: denial of applications for registration of pesticide

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