Bioremedial potential of fenamiphos and chlorpyrifos degrading isolates: Influence of different environmental conditions

Bioremedial potential of fenamiphos and chlorpyrifos degrading isolates: Influence of different environmental conditions

ARTICLE IN PRESS Soil Biology & Biochemistry 38 (2006) 2682–2693 www.elsevier.com/locate/soilbio Bioremedial potential of fenamiphos and chlorpyrifo...

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ARTICLE IN PRESS

Soil Biology & Biochemistry 38 (2006) 2682–2693 www.elsevier.com/locate/soilbio

Bioremedial potential of fenamiphos and chlorpyrifos degrading isolates: Influence of different environmental conditions Brajesh K. Singha,b,, Allan Walkera, Denis J. Wrightb a

Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Department of Biological Sciences, Imperial College London, Silwood Park campus, Ascot, Berks SL57PY, UK

b

Received 4 October 2005; received in revised form 24 March 2006; accepted 6 April 2006 Available online 11 May 2006

Abstract Previously isolated bacterial strains for chlorpyrifos and fenamiphos degradation were used to examine their potential as bioremedial agents in soils and water containing pesticide residues. Both, chlorpyrifos-degrading Enterobacter sp and fenamiphos-degrading consortium rapidly degraded pesticides when inoculated into natural and sterile water and soils. Degradation rate was slower in lower pH soils in comparison with natural and alkaline soils. Soil organic matter had no impact on pesticide degrading ability of isolates. Soil moisture o40% of maximum water-holding capacity slowed down degradation rate. The bacterial isolates were able to rapidly degrade fenamiphos and chlorpyrifos between 15 and 35 1C but their degradation ability was sharply reduced at 5 and 50 1C. Both groups of bacterial systems were also able to remove a range of pesticide degradation. An inoculum density of 104 cells g1 of soil was required for initiating rapid growth and degradation. Ageing of pesticide in soils prior to inoculation produced contrasting results. Ageing of fenamiphos had no impact on subsequent degradation by the inoculated consortium. However, degradation of chlorpyrifos by Enterobacter sp after aging resulted in persistence of 10% of pesticide in soil matrix. Higher Koc value of chlorpyrifos may have resulted in a lack of bioavailability of a smaller percentage of chlorpyrifos to degrading bacteria. Overall, this paper confirms bioremedial potential of a fenamiphos degrading consortium and a chlorpyrifos degrading bacterium under different soil and water characteristics. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bioremediation; Microbial degradation; Organophosphorus compounds; Chlorpyrifos; Fenamiphos

1. Introduction Bioremediation is defined as the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state, or to levels below concentration limits established by regulatory authorities (Vidali, 2001). Bioremediation, which involves the use of microbes to detoxify and degrade pollutants, has received increased attention as an effective biotechnological approach to clean up polluted environments. In general, the approaches to bioremediation are environmental modification, such as through nutrient application (biostimulation,) and aeration (biosparging), or the addition of an appropriate degrader Corresponding author. Present address: Environmental Science, Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK. Tel.: +44 1224 498200; fax: +44 1224 498207. E-mail address: [email protected] (B.K. Singh).

0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.04.019

by seeding (bioaugmentation) (Iranzo et al., 2001; Iwamoto and Nasu, 2001; Vidali, 2001; Atterby et al., 2002). Both success and failure have been reported when species capable of degrading pesticides in liquid culture were introduced into the soil. A strain of Streptomyces was able to grow on eight pesticides and also degraded them in soil (Shelton et al., 1996). Similar results were obtained when an iprodione degrading Arthrobacter strain was inoculated in to the soil (Mercadier et al., 1996). Several chemicals have been successfully removed from soil and aquatic environments using degrading microorganisms such as chlorinated pyridinol (Feng et al., 1997), coumaphos (Mulbry et al., 1996, 1998) and atrazine (Struthers et al., 1998; Topp, 2001). In contrast, MacRae and Alexander (1965) reported the failure of a 4-(2,4-dichlorophenoxy) butyrate utilizing bacteria to degrade the chemical when introduced into a treated soil. Holden and Firestone (1997) and Vidali (2001) suggested that the success of bioaug-

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mentation depends on a number of soil physico-chemical factors such as pH, organic matter, moisture, temperature and nutrient status. Kontchou and Gschwind (1995) reported that a Pseudomonas sp. was less successful in degrading atrazine in soil with lower pH and higher organic matter. Similar results were obtained for ethoprophos bioremediation by Pseudomonas sp. by Karpouzas and Walker (2000a, b). In field bioaugmentation studies, Barles et al. (1979) found that the addition of organic amendments like rice-straw at the time of soil inoculation extended the survival and activity of parathion degrading bacteria in the soil. Moorman et al. (2001) reported rapid degradation of atrazine and metolachlor in organic amended soils. Similar results were obtained by Mishra et al. (2001) for the bioremediation of oil-contaminated soil by inoculation of degrading consortium and nutrient materials. Inoculum size has been identified as a possible reason for the failure of inoculation of contaminated sites with species able to degrade pesticide in cultures (Ramadan et al., 1990). Comeau et al. (1993) suggested that 106–108 cells g1 soil was the recommendable inoculum level to use for the decontamination of pesticide contaminated sites. However in a similar study, Struthers et al. (1998) found that inoculum levels of an Agrobacterim strain as low as 105 cells g1 were adequate to rapidly degrade atrazine. They concluded that a specific bacterium could be an effective bioagumentation agent due to its constitutive expression of degrading enzymes and its broad spectrum of activity against a variety of triazine herbicides. Pesticide concentration has been suggested as another reason for bioagumentation failure (Goldstein et al., 1985). The full potential of bioremediation and its commercialization has not been achieved due to frequent failures in the field (Watanabe, 2001). One of the major reasons for such failure is limited knowledge of changes in microbial communities during bioremediation. The efficient remediation of xenobiotic pollutants by microbial communities remains a major challenge since solutions are based upon biological diversity and functionality (Whiteley and Bailey, 2000). Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) is a widely used insecticide effective against a broad spectrum of insect pests of economically important crops. It is also used for the control of mosquitoes (larvae and adults), flies, various soil and many foliar crop pests and household pests. Additionally, it is used for ectoparasite control on cattle and sheep. Chlorpyrifos persists in soil for 60–120 days, and has very low solubility in water (2 mg l1) but is readily soluble in most organic solvents. It has high soil sorption coefficient (av Koc ¼ 8500 ml g1) (Racke, 1993). It is stable under normal storage conditions, and is defined as a moderately toxic compound having acute oral LD50; for rat of 135–163 mg/kg. Fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate) is an organophosphorate used extensively for the control of soil nematodes. It is systemic, active

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against ecto- and endo-parasitic, cyst forming and root knot nematodes. The half-life in soil for fenamiphos and its metabolites (total toxic residues) varies from 30 to 90 days (Johnson, 1998). It is oxidised rapidly in soil to fenamiphos sulfoxide (FSO) and fenamiphos sulfone (FSO2), both of which have similar nematicidal activity to fenamiphos (Waggoner and Khasawinah, 1974) and degradation studies therefore usually include an estimation of their total toxic residues (TTR), a combination of the amounts of parent compound plus the two oxidation products. It has a high solubility at room temperature (700 mg l1 water), and the acute oral LD50 for rats is 15.3–19.4 mg kg1. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field may lead occasionally to contamination of surface water and ground water (Mason et al., 1999). Reports from the Environmental Protection Agency (EPA) suggest that a wide range of water and terrestrial ecosystems may be contaminated with chlorpyrifos (EPA, 1995). Similarly, contamination of ground water with fenamiphos has been reported in Australia (Bauld, 1996; Cisar and Snyder, 2000). Both of these compounds possess high mammalian toxicity and it is therefore essential to remove them from contaminated environments. Bioremediation may offer an efficient and cheap option for such decontamination. Previously we have reported isolation of a bacterial consortium capable of degrading fenamiphos in liquid medium (Singh et al., 2003a) and a bacterial strain, Enterobacter sp. B-14 which is capable of degrading chlorpyrifos in liquid media and in the soil (Singh et al., 2003b, 2004). Studies on bioremediation of fenamiphos and chlorpyrifos were not attempted before partly due to lack of known degrading microbial isolates. In the present work we investigated for the first time, the potential effectiveness of these isolated bacterial cultures to degrade organophosphorus compounds in soils and water and to determine the main factors that govern chlorpyrifos and fenamiphos biodegradation by these newly isolated bacterial cultures. We also examined the effects of pesticide ageing on the bioremedial activity of the chlorpyrifos- and fenamiphos-degrading bacterial systems. 2. Materials and methods 2.1. Water, soils and pesticides Water samples used for this experiment were taken from the river Dene (Wellesbourne, from the standard tap water supply at Horticultural Research International (HRI) or distilled water from a laboratory source. The soils used were from three sites at HRI (Wellesbourne): deep slade, cottage field and water meadows. Soil properties are listed in Table 1 (Singh et al., 2003a). These soils were never exposed to any organophosphorus compound before. Chlorpyrifos, fenamiphos, parathion, diazinon, coumaphos, ethoprophos, cadusafos, isazofos, and fonofos were

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PH

Organic matter (%)

Moisture contents (%)

Microbial biomass (mg/kg)

Sand (%)

Silt (%)

Clay (%)

Cottage field-1 Cottage field-2 Cottage field-3 Deep slade-1 Deep slade-2 Deep slade-3 Deep slade-4 Deep slade-5 Water meadows

6.5 6.7 7.1 4.7 5.7 6.7 7.7 8.4 6.5

1.47 2.83 5.09 2.33 2.67 2.79 3.02 3.12 0.41

33.12 36.78 38.72 15.9 16.8 16.7 17.7 17.9 27.86

241 275 295 150 172 189 201 200 200

70 71 73 86 83 84 76 85 84

18 17 16 8 10 9 10 8 10

12 12 11 6 7 7 14 7 6

used in this study. Details of analytical grade pesticides used and methods of analysis are described previously (Singh et al., 2002, 2005).

was measured after 24 h by the dilution plate count method (Singh et al., 2004). 2.4. Degradation of chlorpyrifos and fenamiphos in soils

2.2. Bacterial inoculum preparation The inoculum for all the experiments was prepared by growing the appropriate bacterial isolates in 50 ml of either MSMN+chlorpyrifos (chlorpyrifos-degrading bacterium) or MSM+fenamiphos (fenamiphos-degrading consortia) overnight at 25 1C on a rotary shaker at 200 rpm. Cultures were pelleted by centrifugation at room temperature (6000g for 10 min), cells were rinsed twice with 20 ml aliquots of sterilised 0.0125 M phosphate buffer (pH 7.2) and quantified by plate count technique (Singh et al., 2004). All inoculations were made at 1% i.e. the equivalent of 1 ml inoculum to 100 ml water or 100 g soil. 2.3. Degradation of organophosphate in water Samples of river, tap and distilled water were sterilised by autoclaving at 120 1C for 15 min. Triplicate samples (50 ml) of sterile and non-sterile water containing 35 mg l1 chlorpyrifos or fenamiphos were inoculated with 106 cells ml1 (confirmed by the plating of 10 fold dilution on NA plates followed by overnight growth at 25 1C) of chlorpyrifos-degrading bacteria or 106 cells ml1 of overnight grown fenamiphos-degrading consortium (which was isolated from Buchanan East Palmerston (BEP) farm soil). Viable cell counts, prepared at the time of inoculation gave an actual inoculum density of 6.5  106 and 3.8  106 cfu ml1 for chlorpyrifos and fenamiphos isolates, respectively. Triplicate samples without inoculum were kept as controls. Samples were incubated at room temperature on a rotary shaker at 150 rpm. Pesticide residues were measured at time 0, and then after 18, 24, 48 and 72 h. Degradation of the other organophosphorus insecticides, parathion, diazinon and coumaphos and the organophosphate nematicides, FSO, FSO2, ethoprophos, fonofos, cadusafos, and isazofos were measured by adding the respective compounds (35 mg l1) and inoculated as before. Pesticide residues were measured as described previously (Singh et al., 2002, 2005). Microbial growth

The ability of the isolated bacterial systems to degrade chlorpyrifos and fenamiphos and effects of different ecological conditions on their degrading ability were studied in soil samples from the deep slade or cottage field sites. For all experiments, soils (100 g) were treated with chlorpyrifos (35 mg kg1) or fenamiphos (45 mg kg1) and inoculated with the chlorpyrifos-degrading isolate or fenamiphos-degrading consortium to achieve cell density 106 CFU g1. Overnight grown bacterial cultures with cell density 8.4  107 cells for chlorpyrifos-degrading and 9.1  107 for fenamiphos-degrading isolates were inoculated (2 ml) in 100 g of each soil sample which gave an inoculum density of 1.68  106 and 1.82  106 cells g1 of soil for chlorpyrifos and fenamiphos degrading isolates, respectively. Soils were incubated at 40% of maximum water holding capacity in the dark at 20 1C unless stated otherwise. 2.5. Degradation in fumigated and non-fumigated soils Soil samples (600 g) from deep slade field (pH 6.7) were sterilised by fumigation with chloroform for 10 days at 30 1C. Additional soil samples were stored at 4 1C in sealed polyethylene bags. Residual chloroform was removed from the fumigated soils by repeat evacuation in a vacuum desicator. Fenamiphos (45 mg kg1) and chlorpyrifos (35 mg kg1) were aseptically introduced to sub-samples (100 g) of fumigated and non-fumigated soils. Fenamiphostreated soil samples (in triplicate) were inoculated with overnight grown BEP cultures (106 cells g1). One set of fumigated and non-fumigated fenamiphos-treated soils without any bacterial inoculation, were kept as controls. Similarly, one set of fumigated and non-fumigated soils (with chlorpyrifos treatment) was inoculated with chlorpyrifos-degrading bacteria (106 cells g1) and another set without inoculation was kept as controls. Inoculum was thoroughly mixed into soils under sterile conditions. All soil samples were retreated with the appropriate pesticides

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3 and 5 days after the first application to monitor the survival and proliferation of inoculated bacterial systems. Soil samples were incubated at 20 1C and 40% of waterholding capacity in the dark. The effects of an added carbon source on degrading ability of bacterial cultures were studied by adding 1% of glucose (w/w) into pesticide treated soil samples. D-glucose (1 g) was added to 100 g soil and the samples were mixed under aseptic conditions. Triplicate soil samples were inoculated with bacterial cultures as described before. Soils without bacterial inoculation were kept as controls. 2.6. Effect of soil pH and soil organic matter contents Soil samples from deep slade field with similar properties, but differing in soil pH (4.7–8.4), were used to investigate the effect of soil pH on the degrading ability of the isolated bacteria. Triplicate samples (100 g) mixed with fenamiphos or chlorpyrifos were inoculated with the fenamiphos degrading BEP consortium or chlorpyrifos degrading Enterobacter sp. Samples without bacterial inoculation were kept as controls. The incubation conditions were the same as described above. Pesticide residues were measured at 0, 2, 3, 5, 7 and 10 days. To examine the influence of soil organic matter contents on degradation ability of inoculated bacteria, one soil sample from water meadows field with low organic matter content (0.41%) and three samples from cottage field (organic matter, 1.47%, 2.83% and 5.09%) were used in this experiment. Samples from each soil were treated with chlorpyrifos or fenamiphos and triplicate sub-samples (100 g) of each were then inoculated with the chlorpyrifosor fenamiphos-degrading isolates and incubated as described before. Appropriate controls without bacterial inoculation were maintained throughout the experiments. Pesticide residues were measured for 10 days at regular intervals. 2.7. Effect of soil moisture and incubation temperature The effects of soil moisture content on activities of the degrading bacteria were studied in a soil from deep slade (pH 6.7). Triplicate samples (100 g) were inoculated either with the chlorpyrifos-degrading isolate or with the fenamiphos-degrading consortium as described above. Soil moistures were adjusted to 20%, 30%, 40%, 50% or 60% of maximum water-holding capacity by addition of sterile distilled water. Pesticide residues were measured for 10 days. The neutral pH (6.7) deep slade soil was again used for the study on the impact of incubation temperature on degrading ability. Soil samples (100 g) containing chlorpyrifos or fenamiphos were inoculated with chlorpyrifosdegrading bacteria or fenamiphos-degrading consortia (CRF or BEP) as described before. Three treated and three control samples for each treatment were incubated at 5, 15, 25, 35 or 50 1C.

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2.8. Effect of pesticide concentration and inoculum density Soil samples (100 g) from deep slade field (pH 6.7) were treated with either chlorpyrifos or fenamiphos to achieve concentrations of 6.25, 12.5, 25, 50 or 100 mg/kg. Additional samples were treated with 1000 mg kg1 of chlorpyrifos. Triplicate samples were then inoculated with either chlorpyrifos-degrading bacteria or fenamiphos-degrading consortia as appropriate using the methods described before. Triplicate soil samples for each treatment without bacterial inoculation were kept as controls. Effects of inoculum density were measured by a dilution series that was prepared from overnight grown chlorpyrifos-degrading bacteria or fenamiphos-degrading consortium in MSM. Triplicate soil samples (100 g) from deep slade field (pH 6.7) were inoculated either with chlorpyrifos-degrading bacteria or fenamiphos-degrading BEP consortium to achieve cell densities of 108, 107, 106, 105, 104, 103, or 102/g fresh weight. Soil samples also received chlorpyrifos or fenamiphos at the rate of 35 and 45 mg kg1. 2.9. Degradation of aged pesticide residues Triplicate samples of soil from deep slade (pH 6.7) were sterilised by fumigation as previously described. Subsamples (100 g) of fumigated soil were treated with chlorpyrifos or fenamiphos to achieve a concentration of 35 or 45 mg kg1, respectively. Another set of triplicate non-sterile soil samples was treated with same amount of pesticide. One set of fumigated and one set of nonfumigated soil samples were treated with same amount of methanol without pesticides. The soil samples were then incubated at 4 1C for 60 days to minimise microbial mediated degradation but allow pesticide ageing in the soil matrix. After 60 days, pesticide concentrations were measured by HPLC. All initially untreated samples then received an appropriate amount of pesticides. Soil samples with chlorpyrifos were inoculated with the chlorpyrifosdegrading bacterial culture at an inoculum density of 106 cells g1. Appropriate controls for each treatment without bacterial inoculation were maintained. Similarly, triplicate fumigated and non-fumigated soil (100 g) containing aged or freshly added fenamiphos was inoculated with BEP consortium (106 cells g1) and another set without any inoculation was kept as controls. To examine the effect of substrate availability on degradation efficiency of bacterial cultures in aged soil, the concentration of pesticides present in the soil solution was measured. One set of aged soil samples (10 g) was extracted for pesticide residues as described before. The moisture content of another set was raised to 60% of their water-holding capacity by addition of the appropriate amount of distilled water. The sample were then left to equilibrate for 3 days and then extracted with the same solvent (acetonitrile: water; 90:10) with constant shaking for 2 days at room temperature before and after 10 days of inoculation. To examine

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treatment  time interaction to be tested. General Linear Model in Minitab was used with ‘‘treatment’’ and ‘‘treatment  time’’ as terms in the model and ‘‘time’’ as a co-variate.

whether difference in substrate availability affected the degradation efficiency of inoculated bacteria, the concentration of chlorpyrifos and fenamiphos present in the soil solution of the samples was measured as described before (Karpouzas and Walker, 2000b).

3. Results 2.10. Degradation of other organophosphorus pesticides 3.1. Degradation of organophosphate in water Six samples of soil (100 g) from deep slade field (pH 6.7) were treated with chlorpyrifos, parathion, diazinon or coumaphos to achieve a concentration of 35 mg/kg as described. Three samples for each pesticide were inoculated with the chlorpyrifos-degrading bacteria at 1.6  106 cells g1 and other samples were not inoculated. Samples were incubated at 20 1C and pesticide residues were measured at regular interval. A similar experiment was carried out with soil samples treated with fenamiphos, ethoprophos, fonofos, cadusafos or isazofos at the rate of 45 mg kg1. In this experiment, triplicate samples of soil for of each pesticide were inoculated with BEP cultures (1.2  106 cells g1), or remained as non-inoculated controls. One set of soils were also inoculated with the chlorpyrifos-degrading bacterium Enterobacter sp.

Degradation of fenamiphos and chlorpyrifos by the bacterial isolates was rapid following inoculation of sterile and non-sterile water samples (Fig. 1; Table 2), but there was no apparent difference in pesticide degradation between sterile and non-sterile samples of tap and distilled water (Po0:001). Comparatively slower degradation was observed in non-sterile river water but in sterile river water degradation rate was as fast as in other samples. Fenamiphos was completely degraded via fenamiphos phenol. Chlorpyrifos degradation (Fig. 1(b)) was slower than that of fenamiphos (Fig. 1(a)) and degradation was accompanied by the accumulation of the degradation product trichloro pyridinol (TCP). Degradation of pesticides in non-inoculated samples was minimal. The ANCOVA analysis confirmed that the degradation of pesticides in inoculated samples was significantly higher at each sampling time. The chlorpyrifos degrading Enterobacter sp. was able to degrade parathion, diazinon coumaphos and isazofos in all water samples but neither of the fenamiphos-degrading cultures was able to degrade other organophosphorus nematicides or insecticides. Plate counts suggested that there was no bacterial growth in any culture and water combination (data not shown).

2.11. Statistical analysis The data for effects of inoculation on pesticide degradation was analysed by fitting a regression on time to the data from each replicate and analysing the linear slopes using one-way analysis of variance (ANOVA). The analysis was carried out using Minitab (Minitab Inc. PA, USA) and least significant differences from the non-inoculated controls were derived at P ¼ 0:05. In addition, to investigate the difference between treatments with respect to the degradation of pesticide in time an ANCOVA analysis was carried out on the means of the three replicates for each treatment. This allowed the overall significance of the

3.2. Degradation in fumigated and non-fumigated soils Degradation of fenamiphos (TTR) by the BEP consortium in fumigated and non-fumigated soils is presented 40

Chlorpyrifos (mg/l)

Fenamiphos (mg/l)

40

30

20

10

20

10

0

0 0 (a)

30

20

40 Time (Hours)

60

80

0 (b)

20

40

60

80

Time (Hours)

Fig. 1. (a) Degradation of fenamiphos by BEP consortium, and (b) degradation of chlorpyrifos by Enterobacter sp. in different water samples. Symbols: ~—distilled water—steril; }—distilled water; m—tap water—sterile; n—tap water; K—River water—sterile; J—River water. Error bars represent the standard deviation which was within 5% of the mean.

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Table 2 ANOVA table showing the effect of inoculation (statistical significance) on pesticide degradation in inoculated and non-inoculated waters and soils In water samples In fumigated and non-fumigated waters DW-FB Fenamiphos: ControlA A Chlorpryifos: Control DW-FB

DWB DWB

TP-FB TP-FB

TPB TPB

In soil samples In fumigated, non-fumigated and glucose amended soils Fenamiphos: ControlA FumigatedB Non-fumigatedB Chlorpyrifos: ControlA FumigatedB Non-fumigatedB

Soil+glucoseC Soil+glucoseC

In different pH soils Fenamiphos: pH 4.7A Chlorpyrifos: pH 4.7A

pH 6.7A,B pH 6.7B,C

pH 7.7B pH 7.7C

pH 8.4B pH 8.4C

CFFA CFFA

CN-FFC CN-FFC

IFAD IFAD

pH 5.7A,B pH 5.7B

In fresh and aged (with pesticide) soils Fenamiphos: CFAA CN-FAB Chlorpyrifos: CFAA CN-FAB

RP-FB RP-FB

RPB RPB

IN-FAD,E IN-FAD

IFFD,E IFFE

IN-FFE IN-FFE

Within one dataset, sample names followed by the same superscript letter in a row are not significantly different (P ¼ 0:05). Two superscript letters on one sample indicate that it is not significantly different from both. Abbreviation: Water samples (DW-F, distilled water fumigated; DW, distilled water; TP-F, tap water fumigated; TP, tap water: RW-F, river water fumigated; RW, river water), soil samples (CFA, control fumigated aged; CN-FA, control nonfumigated aged; CFF, control fumigated fresh; CN-FF, control non-fumigated fresh; IFA, inoculated fumigated aged; IN-FA; inoculated non-fumigated fresh; IFF, Inoculated fumigated fresh; IN-FF, inoculated non-fumigated fresh).

40

40

Chlorpyrifs (mg/kg)

Fenamiphos (mg/kg)

50

30 20 10 0

20

10

0 0

(a)

30

2

4 Time (Days)

6

8

0 (b)

2

4 Time (Days)

6

8

Fig. 2. Degradation of (a) fenamiphos by BEP consortium and (b) chlorpyrifos by Enterobacter sp. in fumigated, non-fumigated and glucose amended soils. Symbols: ~—control soil—without any inoculation; ’—fumigated soil; &—non-fumigated soil; and n—glucose-amended soil. Error bars represent the standard deviation which was within 5% of the mean.

in Fig. 2(a) and Table 2. Degradation was rapid in all inoculated soil samples with a time to 50% loss (DT50) of about 2 days in the first treatment. The DT50 was reduced to about 1 day for the third treatment. Presence of glucose in the soil samples reduced the rate of degradation in the first treatment (Po0:001) but this inhibitory effect was not apparent after the second and third treatments. Only a small amount of FSO was formed in the initial samples inoculated with BEP consortia. However, in subsequent treatments FSO was not detected, though small amounts of FSO-OH were formed. Fenamiphos phenol was also detected by HPLC as a metabolite of fenamiphos. The addition of Enterobacter sp. to fumigated and nonfumigated soils resulted in rapid degradation of chlorpyr-

ifos (Fig. 2(b); Table 2). The DT50 for the first treatment with chlorpyrifos was 2 days for both fumigated and nonfumigated soils. The DT50 in non-inoculated non-sterile control soil was about 32 days. Addition of glucose significantly inhibited the rate of degradation of the first treatment (Po0:001), but rapid degradation was achieved in subsequent treatments. Repeated treatment with the pesticide in inoculated soil samples resulted in higher rates of degradation and the DT50 of chlorpyrifos was less than 1 day. The degradation of chlorpyrifos was accompanied by accumulation of TCP in the soil (data not shown). The ANCOVA analysis again confirmed that the degradation of pesticides in inoculated samples was significantly higher at each sampling time.

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3.3. Effect of soil pH and soil organic matter contents The rate of fenamiphos degradation was more rapid at pH 5.7 and above than at pH 4.7 (Fig. 3(a); Table 2). The degradation rate of fenamiphos (TTR) in inoculated low pH soils was fast in comparison with non-inoculated soils but was slower than in neutral and alkaline soils (Po0:05). At pH 4.7, less than 2% of the applied pesticide was degraded in non-inoculated soils in 10 days, compared with 15% degradation at high pH (8.4) (data not shown). For each pH, the rate of degradation of chlorpyrifos in soil samples inoculated with the Enterobacter sp. was more rapid than in non-inoculated soils. However, the rate of degradation in inoculated increased with increasing soil pH from 4.7 to 6.7 but there was no significant difference in degradation rate in soil with pH 6.7 to 8.4 (Fig. 3(b); Table 2). Degradation patterns of chlorpyrifos in non-inoculated soils were similar to those for fenamiphos. At low pH (4.7), more than 97% of the pesticide remained after 10 days, while more than 20% of chlorpyrifos was dissipated at high soil pH (data not shown). Degradation of fenamiphos or chlorpyrifos in soils with different organic matter contents had shown that the rate of degradation was similar in all soils tested (data not shown). 3.4. Effect of soil moisture and incubation temperature Effects of moisture contents and incubation temperature on fenamiphos and chlorpyrifos degradation in inoculated soils are shown in Fig. 4. The rate of fenamiphos degradation was slowest at 20% of MWHC in soil inoculated with the BEP consortium. Degradation was most rapid at 40% MWHC and above (Fig. 4(a)). Similar results were obtained for chlorpyrifos degradation with the chlorpyrifos-degrading Enterobacter sp. (Fig. 4(b)) with slower degradation at 20% and 30% of MWHC and rapid degradation at and above 40% MWHC. The rate of fenamiphos and chlorpyrifos degradation was slow at incubation temperatures of 5 and 50 1C in

comparison with the rate at 15, 25 and 35 1C. The slowest rate of degradation was observed at 5 1C in soil inoculated with either BEP consortium (Fig. 4(c)) or Enterobacter sp. (Fig. 4(d)). The most rapid degradation by both bacterial systems was observed at 35 1C. However, the rate of degradation in all inoculated samples was more rapid than that in the non-inoculated control samples, irrespective of temperature (data not shown). 3.5. Effect of pesticide concentration and inoculum density The BEP consortium was able to degrade a wide range of fenamiphos concentrations in soil (Fig. 5(a)). There was an initial phase of slower degradation, which was longer at high concentrations. There was little degradation of fenamiphos at any concentration in the non-inoculated controls during the 10 day incubation period (data not shown). The chlorpyrifos-degrading isolate also degraded all concentrations of chlorpyrifos with a longer lag phase for the higher concentrations (Fig. 5(b)). Rapid degradation of chlorpyrifos was accompanied by the accumulation of TCP (data not shown). Degradation of fenamiphos was rapid in samples inoculated with 103 cells g1 cultures of the BEP consortium (Fig. 5(c)). Degradation was minimal in noninoculated controls and in soil inoculated with a cell concentration less than 102 cells g1 of soil. Degradation of chlorpyrifos was rapid in all soil samples inoculated with 105 cells g1 or greater (Fig. 5(d)). The lag phase was longer in soil inoculated with 103 and 104 cells g1 but rapid degradation was still apparent later in the experiment. No degradation was observed in soil inoculated with densities less than 103 cells g1 of soil. 3.6. Degradation of aged pesticide residues More than 95% of the applied fenamiphos was recovered by vigorous extraction after ageing periods up

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Fig. 3. (a) Degradation of fenamiphos by BEP consortium and (b) chlorpyrifos by Enterobacter sp. when inoculated in soils with different pH value. Symbols: ~—pH 4.7; &—pH 5.7; m—-pH 6.7; n—pH 7.7; K—pH 8.4. Error bars represent the standard deviation which was within 5% of the mean.

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Fig. 4. Effects of soil moisture contents on degradation of (a) fenamiphos by the BEP consortium and (b) on degradation of chlorpyrifos by Enterobacter sp. Symbols: ~—20%; &—30%; m—40%; n—50%; K—60%. Error bars represent the standard deviation which was within 5% of the mean. (c) Degradation of fenamiphos by BEP consortium and (d) degradation of chlorpyrifos by Enterobacter sp. when inoculated soils were incubated at different temperatures. Symbols: ~—5 1C; &—15 1C; m—25 1C; n—35 1C; K—50 1C. Error bars represent the standard deviation which was within 5% of the mean.

to 60 days at 4 1C. The degradation pattern of aged and fresh fenamiphos in soil samples inoculated with BEP consortium is presented in Fig. 6(a) and Table 2. There was little difference in degradation rate between aged and freshly applied fenamiphos, if any. The rate of degradation for both fresh and aged fenamiphos was faster in all inoculated samples compared with non-inoculated controls (Po0:001). About 91% of applied chlorpyrifos was recovered by solvent extraction after ageing for 60 days at 4 1C. The rate of degradation for both aged and fresh chlorpyrifos was faster in all inoculated samples relative to the corresponding controls (Fig. 6(b)). Initially, the pattern of degradation for aged and fresh chlorpyrifos was similar. However, after incubation for 7 days, less than 1 mg kg1 chlorpyrifos was left in the fresh soil compared with 3.7 mg kg1 in aged soils and was statistically significant. Ageing of fenamiphos in soil did not have significant effect on the amount of fenamiphos present in the water phase in the uninoculated and inoculated samples. However the impact of ageing on the presence of chlorpyrifos in water phase was apparent. Less than 10% of chlorpyrifos was present in the water phase in aged soil as compared to 18% in the freshly treated soils.

3.7. Degradation of other organophosphorus pesticides The BEP consortia was not able to degrade other organophosphorus nematicides, although, they did degrade FSO and FSO2 rapidly (data not shown). The Enterobacter sp. degraded all tested insecticides but none of the nematicides except, isazofos (data not shown). 4. Discussion Success or failure of bioremediation depends on several factors, such as the competitive ability of the bioremedial agents (Goldstein et al., 1985; Gunalan and Fournier, 1993), bioavailability of pollutants (Geer and Shelton, 1992; Alexander, 2000) and abiotic factors such as soil moisture, pH, and temperature (Van Veen et al., 1997). Successful removal of pesticides by the addition of bacteria (bioagumentation) has been previously reported for many compounds including, parathion (Barles et al., 1979), coumaphos (Kearney et al 1986; Mulbry et al., 1996), ethoprophos (Karpouzas and Walker, 2000b) and atrazine (Struthers et al., 1998; Topp, 2001). Results from the present study confirm that the isolated fenamiphos-degrading

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Fig. 5. Effects of initial concentration of pesticide on degradation by inoculated bacteria (a) degradation of fenamiphos by BEP consortium (b) degradation of chlorpyrifos by Enterobacter sp. Symbols: ~—6.25 mg/kg; &—12.5 mg/kg; m—25 mg/kg; n—50 mg/kg; K—100 mg/kg; J—1000 mg/kg. (c) Degradation of fenamiphos by BEP consortium and (d) degradation of chlorpyrifos by Enterobacter sp when soils were inoculated with different initial inoculum density. Symbols: X—control ~—101;, ’—102; m—103; K—104; }—105; &—106; n—107; J—108. Error bars represent the standard deviation which was within 5% of the mean.

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Fig. 6. Effects of pesticide ageing on (a) fenamiphos degradation by the BEP consortium and (b) chlorpyrifos degradation by Enterobacter sp. Symbols: ~—control—fumigated aged; ’—control—non-fumigated aged; m—control—fumigated fresh; K—control—non-fumigated fresh; }—inoculated— fumigated aged; &—inoculated—non-fumigated aged;n—inoculated—fumigated fresh;J—inoculated—non-fumigated fresh. Error bars represent the standard deviation which was within 5% of the mean.

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consortia and chlorpyrifos-degrading Enterobacter sp. could be used successfully for the removal of these pesticides from contaminated water and soil. Chlorpyrifos and fenamiphos were degraded rapidly in all inoculated water samples. However, no growth of inoculated bacterial systems was observed in any of the water samples in the present experiment, which may have resulted from an absence of sufficient mineral elements in the water samples. In the present work, the bacterial isolates successfully degraded fenamiphos or chlorpyrifos in fumigated and non-fumigated soils, suggesting that these bacterial isolates can compete and survive with the local microflora. The addition of glucose to the soil samples significantly reduced the initial degradation rate (Po0:001) but this lag phase was followed by a log phase. This result contrasts with previous findings of Karpouzas and Walker (2000a, b), where addition of glucose stimulated the degradation rate of ethoprophos. However, initial inhibition of pesticide degradation in the presence of glucose can be attributed to the environmental adaptation of bacterial isolates where easily available and rich carbon sources are preferentially utilised. Once the readily available carbon source is depleted, the bacteria begin to utilise the pesticides. This approach gives the bacterial isolates a competitive advantage since they are able to utilise both readily available and less available carbon sources. When inoculated soils were treated with pesticides after 3 months of inoculation, degradation rate was still rapid suggesting that degrading bacteria can survive in soils at least for 3 months even in the absence of pesticides. Soil pH had a marked influence on pesticide degradation by the bacterial isolates used for inoculation. Fenamiphos degradation was slow at soil pH 4.7. Slowing of degradation was more pronounced for chlorpyrifos at this pH. Degradation rate was similar at neutral and alkaline pH. Karpouzas and Walker (2000b) reported similar results for ethorprophos degradation by Pseudomonas putida. Vidali (2001) reported that pH 5.5–8.8 is required for activities of most bacteria in soil with an optimum value for degrading properties often between pH 6.5 and 8. The present results are in agreement with this observation. In the present study, the organic matter content of the soils had no apparent effect on fenamiphos or chlorpyrifos degradation. This contrasts with previous reports where high organic matter resulted into reduced degradation (Karpouzas and Walker, 2000b; Morrison et al., 2000). Weber and Huang (1996) suggested that high organic matter lead to reduced bioavailability of substrate to the degrading microorganisms, especially when the compounds have high Koc value. Hydrophobic compounds become non-available because they get entrapped in the solid phase of organic matter and also in nanopores at specific sites. However, many degrading microorganisms produce surfactants or other emulsifiers that desorb chemical compounds from soil and make them bioavailable (Aronstein et al., 1991). Both fenamiphos and chlorpyrifos were degraded rapidly in inoculated soils at 40%, 50% and 60% of

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water-holding capacity of the soil. Soil moisture is important for availability of chemicals and for movement and proliferation of microorganisms (Barles et al., 1979). Degradation was slowest at low moisture contents. Pesticide degradation was most rapid at 35 1C, which is consistent with the studies in liquid media studies (Singh et al., 2004) and with the degradation of ethoprophos (Karpouzas and Walker, 2000b). Concentration of fenamiphos or chlorpyrifos had no apparent effect on the degradation rate except the longer initial lag phase. This longer lag phase for higher concentrations of pesticides may be due to the need for greater numbers of bacteria to initiate rapid degradation of higher concentrations of the pesticides (Karpouzas and Walker, 2000b). Inoculum density had a marked effect on degradation of both fenamiphos and chlorpyrifos. No degradation of fenamiphos was observed in soils inoculated with o104 cells g1. Similarly, Enterobacter sp. was not able to degrade chlorpyrifos below an inoculum density 103 cells g1. Similar results were cited by Ramadan et al. (1990) who found that a Pseudomonas sp. did not mineralise p-nitrophenol at a density less than 104 cells/ ml of lake water. The authors explained that when lower inoculum densities were used, the small number of bacteria was not able to survive the initial competition and population decline that usually occurs following inoculation. Similar results were obtained by Comeau et al. (1993) for 2,4-D degradation by Pseudomonas (now Burkholderia) cepacia. The bioavailability of the pollutant is one of the most important factors, which determines the effectiveness of bioremediation. In the present study, there was no effect of ageing for 60 days on subsequent fenamiphos degradation. In contrast, while chlorpyrifos was degraded rapidly in all inoculated soil samples, at the end of the 10 days incubation more than 3.5 mg kg1 (410% of initial recovery) chlorpyrifos remained in the soil samples containing aged chlorpyrifos while no chlorpyrifos was detected in soils treated freshly with chlorpyrifos. Similar results were obtained by Cullington and Walker (1999) following inoculation of soil with a diuron-degrading bacterium and by Karpouzas and Walker (2000b) with an ethoprophos-degrading Pseudomonas sp. Previous studies with a range of pollutants have demonstrated that increased soil residence time of the compound leads to a reduction in bioavailability (Blair et al., 1990; Chung and Alexander, 1998; Alexander, 2000). The above findings may explain why bioremediation by microorganisms often does not result in total elimination of target contaminants. This has been explained by diffusion of solute molecules to sites in the soil matrix that are inaccessible to microorganisms (Scribner et al., 1992). Nevertheless, a time-dependent decline in bioavailability does not always occur. This may be related to properties of the soil (Hatzinger and Alexander, 1995) or of the compounds (Alexander, 2000). Also the amount of compound available to different organisms is different (Tang et al., 1998). One of the

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possible explanations for differential effect of ageing on fenamiphos and chlorpyrifos degradation in the present study could be that the high Koc value of chlorpyrifos resulted in non-reversible binding to the solid phase or movement into the nanopore regions of the organic matrix. Such residues could be extracted by solvent extraction but could not be available for biodegradation. In conclusion, the results confirmed that the newly isolated chlorpyrifos and fenamiphos degrading isolates can be successfully used for bioremediation of contaminated water and soils. It also confirmed that bioremedial potential of the bacterial isolates could be influenced by a range of abiotic factors such soil pH, temperature, inoculum density. Acknowledgement We are very grateful to Dr. Jackie Potts (Biomathematics and Statistics Scotland; BIOSS, Aberdeen) for her help in statistical analyses of our data. Part of this project was supported by the Biotechnology and Biological Sciences Research Council. BKS work at the Macaulay Institute is funded by the Scottish Executive Environment and Rural Affairs Department through their grant-in-aid to the Macaulay Institute. References Alexander, M., 2000. Aging, bioavailability and overestimation of risk from environmental pollutants. Environmental Science and Technology 34, 4259–4265. Aronstein, B.N., Calvillo, Y.M., Alexander, M., 1991. Effect of surfactants at low concentration on the desorption and biodegradation of sorbed aromatic compounds. Environmental. Science and Technology 25, 1728–1731. Atterby, H., Smith, N., Chaudhry, Q., Stead, D., 2002. Exploiting microbes and plants to clean up pesticide contaminated environment. Pesticide Outlook 13, 9–13. Barles, R.W., Daughton, C.G., Hsieh, D.P.H., 1979. Accelerated parathion degradation in soil inoculated with acclimated bacteria under field conditions. Archive of Environmental Contamination and Toxicology 8, 647–660. Bauld, J., 1996. Ground water quality: human impact on a hidden resource. In: Proceedings of the Hydrology and Water Resources Symposium on Water and the Environment, Hobart, 1996. pp. 143–147. Blair, A.M., Martin, T.D., Walker, A., Welch, S.J., 1990. Measurement and prediction of isoproturon movement and persistence in three soils. Crop Protection 9, 289–294. Chung, N., Alexander, M., 1998. Differences in sequestration and bioavailability of organic compounds aged in dissimilar soils. Environmental Science and Technology 32, 855–858. Cisar, J.L., Snyder, G.H., 2000. Fate and management of turfgrass chemicals. ACS Symposium Series 743, 106–126. Comeau, Y., Greer, C.W., Samson, R., 1993. Role of inoculum preparation and density on the bioremediation of 2,4-D contaminated soil by bioagumentation. Applied and Microbial Technology 38, 681–687. Cullington, J.E., Walker, A., 1999. Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biology & Biochemistry 31, 677–686. EPA, 1995. Review of chlorpyrifos poisoning data. US EPA. 1-46.

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Struthers, J.K., Jauachandran, K., Moorman, T.B., 1998. Biodegradation of atrazine by Agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil. Applied and Environmental Microbiology 64, 3368–3375. Tang, J., Carroquino, M.J., Robertson, B.K., Alexander, M., 1998. Combined effect of sequestration and bioremediation in reducing the bioavailability of polycyclic aromatic hydrocarbons in soil. Environmental Science and Technology 32, 3586–3590. Topp, E., 2001. A comparison of three atrazine-degrading bacteria for soil bioremediation. Biology and Fertility of Soils 33, 529–534. Van Veen, J.A., Van Overbeek, L.S., Van Elas, J.D., 1997. Fate and activity of microorganisms introduced into soils. Microbiology and Molecular Biology Review 61, 121–135. Vidali, M., 2001. Bioremediation. An overview. Pure Applied Chemistry 73, 1163–1172. Waggoner, T.B., Khasawinah, W., 1974. New aspects of organophosphorus pesticides, VII. Metabolisms, biochemical, and biological aspects of nemacur and related phosphoramidate compounds. Residue Review 53, 79–97. Watanabe, M.E., 2001. Can bioremediation bounce back? Nature Biotechnology 19, 1111–1115. Weber Jr., W.J., Huang, W., 1996. A distributed reactivity model for sorption by soil and sediments 4. Intraparticle heterogeneity and phase-distribution relationships under nonequilibrium conditions. Environmental Science and Technology 30, 881–888. Whiteley, A.S., Bailey, M.J., 2000. Bacterial community structure and physiological state within an industrial phenol bioremediation system. Applied and Environmental Microbiology 66, 2400–2407.