Mineralization of hydroxylated isoproturon metabolites produced by fungi

ARTICLE IN PRESS

Soil Biology & Biochemistry 39 (2007) 1751–1758 www.elsevier.com/locate/soilbio

Mineralization of hydroxylated isoproturon metabolites produced by fungi Stig Rønhedea,b, Sebastian R. Sørensena, Bo Jensenb, Jens Aamanda, a

Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Øster Voldgade 10, DK-1350 Copenhagen K, Denmark b Department of Microbiology, Institute of Biology, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen K, Denmark Received 21 November 2006; received in revised form 19 January 2007; accepted 31 January 2007 Available online 9 March 2007

Abstract When exposed to the herbicide isoproturon, some soil fungi in pure culture metabolize the substance to hydroxylated metabolites. Hydroxylated metabolites of isoproturon have also been detected in soil studies. In an agricultural soil not previously exposed to isoproturon we found that the hydroxylated isoproturon metabolite N-(4-(2-hydroxy-1-methylethyl)phenyl)-N0 ,N0 -dimethylurea mineralized faster than both isoproturon and its N-demethylated metabolite N-(4-isopropylphenyl)-N0 -methylurea (MDIPU), thus indicating that mineralization of isoproturon is stimulated by fungal hydroxylation in this soil. In soils previously treated with isoproturon, in contrast, isoproturon and both its hydroxylated and demethylated metabolites mineralized at almost the same rate with up to 52% of the 14C-ring-carbon being degraded to 14CO2 within 63 days. Thus hydroxylated metabolites of isoproturon do not seem to be more persistent than isoproturon, and hence may degrade before they can leach from topsoil and contaminate the aquatic environment. While an isoproturon-mineralizing bacterium Sphingomonas sp. SRS2 and a MDIPU-mineralizing mixed bacterial culture were able to deplete the medium of hydroxylated metabolites, little or no mineralization took place. This indicates that other bacteria must be present in the soil that are able to benefit from isoproturon being made available to mineralization by fungal hydroxylation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Phenylurea herbicide; Degradation; Bioconversion; Hydroxylation; Sphingomonas sp. SRS2; Phoma eupyrena

1. Introduction Cereal farmers in many parts of the world use the herbicide isoproturon (Fig. 1). In Europe, isoproturon has since the sixties been one of the most heavily used herbicides in winter wheat and barley, where it is used as a pre-emergence herbicide in autumn (Sørensen et al., 2003). Isoproturon is relatively mobile in soil, and its extensive use has resulted in the frequent contamination of isoproturon in surface water and groundwater in Europe. The European Union has accordingly placed isoproturon on a list of 33 priority substances that present a significant risk to or via the aquatic environment (European Council, 2001). Microbial degradation of isoproturon in the topsoil plays an important role in the environmental fate of isoproturon. In some soils bacteria use isoproturon for Corresponding author. Tel.: +45 3814 2326; fax: +45 3814 2050.

E-mail address: [email protected] (J. Aamand). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.01.037

growth, and completely mineralize the compound to CO2 and biomass. In other soils, in contrast, isoproturon is transformed into metabolites that may be more mobile than isoproturon itself and therefore a threat to surface water and groundwater (Sørensen et al., 2003). Bacterial strains that completely mineralize the isoproturon ring carbon into CO2 and biomass have been isolated from soils that have been exposed to isoproturon for long periods (Sørensen et al., 2001; Bending et al., 2003; Sebai et al., 2004). Sphingomonas sp. strain SRS2, a bacterium isolated from a British soil, degrades isoproturon by initial N-demethylation to MDIPU (MonoDesmethyl-IsoProtUron) and DDIPU (DiDesmethyl-IsoProtUron) (Fig. 1). The DDIPU is then hydrolyzed to 4-isopropyl-aniline, after which the ring carbon is mineralized (Sørensen et al., 2001). An Arthrobacter sp. has been shown to hydrolyze the carbonyl group of the urea side chain of isoproturon, mineralize the side chain and accumulate 4-isopropylaniline in the medium (Cullington and Walker, 1999;

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O R3

R1

H3C

N N H

R4 H3C

NH2

R2 H3C

General structure

4-isopropyl-aniline

R1

R2

R3

R4

IUPAC name

Isoproturon

CH3

CH3

CH3

H

N-(4-isopropylphenyl)-N´,N´-dimethylurea

1-OH-IPU

CH3

CH3

CH2OH

H

N-(4-(2-hydroxy-1-methylethyl)phenyl)-N´,N´-dimethylurea

2-OH-IPU

CH3

CH3

CH3

OH

N-(4-(1-hydroxy-1-methylethyl)phenyl)-N´,N´-dimethylurea

MDIPU

H

CH3

CH3

H

N-(4-isopropylphenyl)-N´-methylurea

1-OH-MDIPU

H

CH3

CH2OH

H

N-(4-(2-hydroxy-1-methylethyl)phenyl)-N´-methylurea

2-OH-MDIPU

H

CH3

CH3

OH

N-(4-(1-hydroxy-1-methylethyl)phenyl)-N´-methylurea

DDIPU

H

H

CH3

H

N-(4-isopropylphenyl)urea

1-OH-DDIPU

H

H

CH2OH

H

N-(4-(2-hydroxy-1-methylethyl)phenyl)urea

2-OH-DDIPU

H

H

CH3

OH

N-(4-(1-hydroxy-1-methylethyl)phenyl)urea

Fig. 1. Chemical structure and names of the herbicide isoproturon and the metabolites included in this study.

2. Materials and methods

GmbH (Augsberg, Germany). The [14C-U-phenyl]-labeled isoproturon (2.15 MBq mg1) and MDIPU (4.42 MBq mg1) were purchased from Izotop (Budapest, Hungary). As evaluated by thin-layer chromatography (TLC), the radiochemical purity of our stocks of 14C-isoproturon and 14C-MDIPU were 99% and 90%, respectively. Buffer 1 (per liter): 1.33 g Na2HPO2  2H2O, 1.38 g KH2PO4 and 9.0 g NaCl. The pH was 6.6. Medium A (per liter): 2.14 g Na2HPO2  2H2O, 0.572 g K2HPO4, 0.179 g (NH4)2SO4, 66.5 mg KNO3, 66.5 mg MgSO4  7H2O, 13.3 mg CaCl2, 3.80 mg H3BO4, 2.05 mg MnSO4  H2O, 50 mg CuSO4  5H2O, 27 mg ZnCl2, 54 mg CoCl2  6H2O, and 33 mg Na2MoO4  2H2O. After autoclaving, 1.33 ml of a sterile filtered solution of FeCl3  6H2O was added to yield a final concentration of 6.85 mg l1. Since Sphingomonas sp. SRS2 depends on an external source of certain amino acids (Sørensen et al., 2002), (NH4)2SO4 and KNO3 were replaced by 0.133 g casamino acids l1 (Difco) in the experiments with this bacterium. Malt extract (ME) broth (per liter): 10 g ME broth adjusted to pH 6.0. Potato dextrose agar (PDA) and R2A were purchased from Difco. R2B was prepared as described by Reasoner and Geldreich (1985). Specieller Na¨hrstoffarmer Agar (SNA) was prepared as described previously (Rønhede et al., 2005).

2.1. Chemicals and media

2.2. Soils

The analytical standards (purity 498%) for isoproturon (CAS no. 34123-59-6), MDIPU (CAS no. 34123-57-4), DDIPU (CAS no. 56046-17-4) and 4-isopropyl-aniline (CAS no. 99-88-7) were obtained from Dr. Ehrenstorfer

Four soils were used in the study. The soil designated ‘‘Jyndevad’’ was sampled from an agricultural field located near the village of Jyndevad in southern Jutland, Denmark. The field is used for organic farming, and no pesticides

Turnbull et al., 2001). In contrast to the above-mentioned soil bacteria, soil fungi metabolize isoproturon to hydroxylated metabolites. Thus 5 of 10 isolates from agricultural soil, including ascomycetes, basidiomycetes and zygomycetes, were found to hydroxylate isoproturon at the isopropyl side chain to generate 2-OH-IPU, 1-OH-IPU and 1-OH-MDIPU (Fig. 1) (Rønhede et al., 2005). Hydroxylated metabolites of isoproturon have also been detected in soil (e.g. Mudd et al., 1983; Schroll and Ku¨hn, 2004; Elkhattabi et al., 2004; Alletto et al., 2006), and 2-OH-IPU and 2-OH-MDIPU (Fig. 1) have been detected in run-off and in lysimeter leachate after heavy rainfall (Schuelein et al., 1996; Do¨rfler et al., 2006). The fate of hydroxylated isoproturon metabolites in soil has not been described, and whether or not they can be further degraded and mineralized by bacteria and other soil organisms or instead comprise dead-end metabolites of environmental concern is unknown. The aim of the present study is therefore to determine whether agricultural soils are able to mineralize hydroxylated isoproturon metabolites produced by fungi and whether previously isolated isoproturon-mineralizing and MDIPU-mineralizing bacteria are also able to degrade and mineralize hydroxylated metabolites of isoproturon.

ARTICLE IN PRESS S. Rønhede et al. / Soil Biology & Biochemistry 39 (2007) 1751–1758

have been applied since 1995. The pH was 6.9 and further characteristics of the soil are described in Bælum et al. (2006). The soil designated ‘‘Græse’’ was sampled from a field located near the village of Græse in Zeeland, Denmark. The field is used for conventional agriculture, and isoproturon has been applied on many occasions between 1990 and 1999 (Sørensen and Aamand, 2001). The soils designated ‘‘Deep Slade’’ and ‘‘Long Close’’ were from the Warwick HRI farm, University of Warwick, UK. Isoproturon had been applied regularly to the Deep Slade field over the 20-year period up to 2002, and is still being applied regularly to the Long Close field (Bending et al., 2001, 2006). All soil samples were collected from the plough layer, passed through a 4-mm sieve and stored at 5 1C until required. All soils had a pH between 6.7 and 6.9. 2.3. Fungi The isoproturon-hydroxylating fungi Phoma eupyrena Saccardo strain CBS 118522, Mucor hiemalis Wehmer strain CBS 118524 and Mortierella sp. strain CBS 118520 were isolated from the Græse soil by Rønhede et al. (2005), but were at that time designated Phoma cf. eupyrena Gr61, Mucor sp. Gr22 and Mortierella sp. Gr4. 2.4. Bacteria The isoproturon-mineralizing Sphingomonas sp. SRS2 was isolated from the Deep Slade field by Sørensen et al. (2001) and deposited in the Institut Pasteur Collection (CIP107349). A mixed bacterial culture able to mineralize MDIPU but not isoproturon was previously obtained by Sørensen and Aamand (2001) from a soil sample from the Græse field after successive liquid enrichment with MDIPU. 2.5. TLC of

14

C-labeled compounds

Isoproturon and its metabolites were measured using two TLC methods: RP-18 plates with a solvent of isopropanol–ethyl acetate–hexane–acetic acid (10 þ 40þ 50 þ 0:1 by volume) and silica gel 60 plates with a solvent of isopropanol–hexane–acetic acid ð30 þ 70 þ 0:1Þ (Rasmussen and Jacobsen, 2005). The compounds were spotted on the plates with 10-ml capillaries until 50 ml (equivalent to approx. 103 dpm) had been applied to the same spot. The plates were developed and placed on a Cyclone storage phosphor screen for 48 h for detection of b emissions. The screen was then scanned with a Cyclone storage phosphor system, and the resulting spectra were analyzed using OptiQuant image analysis software. The screens, instruments and software were obtained from Packard Instruments (Meriden, CT, USA). The retention factor (Rf) of isoproturon and MDIPU was identified by comparison with the reference standards. The Rf of 1-OH-IPU, 2-OHIPU and 1-OH-MDIPU was determined by comparison

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with samples shown to contain these compounds by highperformance liquid chromatography (HPLC) analysis with the method of Rønhede et al. (2005). 2.6. Bioconversion P. eupyrena was used to bioconvert isoproturon and MDIPU to 1-OH-IPU and 1-OH-MDIPU, respectively, as it is known not to N-demethylate the compounds and only produce traces of 2-OH-IPU (Rønhede et al., 2005). Clamydospores harvested from SNA-plates were used to prepare inocula. The spores were separated from the hyphae by vigorously shaking five 4-mm agar plugs with 1 g glass beads and 2 ml distilled water containing 1 g l1 Tween 80 and 8.5 g l1 NaCl in 5-ml vials for half an hour. Centrifuge tubes (50 ml; Nunc) containing 25 ml ME broth were each inoculated with 7.4  104 spores, loosely capped and incubated in darkness at 20 1C and 250 rev min1. After 65 h the mycelium had grown to a dry weight of 3176 mg (mean7standard deviation, n ¼ 5). The mycelium was drained through sterile gauze (28 threads inch1), washed with 25 ml fresh buffer 1 and drained again through fresh gauze. Each mycelium plus the remaining liquid was transferred from the centrifuge tube to a sterilized 100-ml screw cap bottle with a Teflon-lined lid (Schott-Duran). The bottles contained 20 ml buffer 1 and isoproturon or MDIPU at a final concentration of about 80 mmol l1. To produce 14C-labeled bioconverted metabolites, 14C-ring-labeled isoproturon or MDIPU were added to the buffer. Unlabeled and radiolabeled isoproturon or MDIPU were added from stock solutions in acetone or acetonitrile and the solvent evaporated before addition of buffer 1. The bottles were incubated in darkness at 20 1C and 150 rev min1. The culture was passed through a 0.5-mm hydrophilic polytetrafluoroethylene syringe filter (Advantec MFS) into sterile bottles and frozen until required. The purity of the isoproturon and MDIPU bioconversion products is shown for different incubation periods in Table 1. M. hiemalis was used to produce radiolabeled 2-OH-IPU as a standard for TLC analysis following the method described above for P. eupyrena except that the inocula consisted of 1:2  107 conidia from a PDA plate, the ME broth cultures were grown for 48 h at 25 1C (dry weight 3172 mg, n ¼ 12), and the mycelia were transferred to distilled water at a final concentration of 40 mmol isoproturon l1 and incubated for 16 days before harvesting of the metabolite-containing medium. 2.7. Mineralization in soil Wet soil equivalent to 10 g dry weight was added to sterilized 100-ml flasks. Solutions of isoproturon or MDIPU were prepared by adding isoproturon or MDIPU from stock solutions in acetone and 14C-labeled isoproturon or MDIPU from stock solutions in acetonitrile to sterilized screw-cap test tubes. The solvents were evaporated

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Table 1 Purity of the products resulting from the bioconversion of isoproturon and MDIPU by Phoma eupyrena as evaluated by TLC of 14C-labeled compounds (as % of total radioactivity) Product

Isoproturon MDIPU 1-OH-IPU 2-OH-IPU 1-OH-MDIPU Unknown 1 Unknown 2 Sumc

Rf on silica

0.47 0.60 0.24 0.24 0.33 0.33 0.33

Rf on RP-18

0.70 0.70 0.45 0.55 0.50 0.60 0.70

Substrate (incubation time) Isoproturon (5 days)a

MDIPU (5 days)a

Isoproturon (74 h)b

0 0 7477 974 0 0 0 8376

0 0 0 0 4970.04 1070.4 3473 9273

4.8–5.5 0 93.8–94.2 0 0 0 0 99

a

Mean of samples from three bioconversion experiments7standard deviation. The sample used for mineralization experiments. Values are the range of three measurements. c The remaining radioactivity up to 100% was accounted for by minor metabolites, or by test compound that did not migrate from the sample. b

and the compounds dissolved in fresh buffer 1. Solutions containing isoproturon or MDIPU were also prepared using buffer 1 that had been incubated with mycelium under identical conditions to those used for the bioconversion (see Section 2.6), but without the substrates (hereafter referred to as buffer 1+exudates). The aim of this was to test whether any fungal compounds that exuded during incubation of the mycelium in buffer 1 influenced mineralization of the isoproturon or MDIPU. All compounds were added to the soil in 0.5 ml buffer 1 at a final concentration of 0.8 mg g1 and about 104 dpm per flask. The compounds were evenly distributed on the surface of the soil. A 14CO2 trap consisting of a 5-ml test tube containing 2 ml 0.5 M NaOH was placed in each of the flasks, which were then closed with an airtight glass lid and incubated at 20 1C in darkness. The 0.5-M NaOH solution was collected and replaced twice a week. The sampled NaOH solution was mixed with 10 ml scintillation fluid (Wallac Optiphase HiSafe 3) and the 14 CO2 content determined using a Wallac 1409 scintillation counter. 2.8. Degradation and mineralization in bacterial cultures Inoculum of the mixed bacterial culture from the Græse soil was prepared by growing it in 100 mmol l1 MDIPU as the sole carbon source as described by Sørensen and Aamand (2001). After 10 days the cells were harvested by centrifugation for 10 min at 4260  g and washed once. Glycerol was added to a final concentration of 40% vol/vol and the cell suspension stored at 801C. Before use the cells were washed once and resuspended in medium A. Inoculum of Sphingomonas sp. SRS2 was prepared by growing it in R2B as described by Sørensen et al. (2001). The cells were harvested by centrifugation for 10 min at 5360  g and washed twice in medium A. Glycerol was added to a final concentration of 40% vol/vol and the cell suspension stored at 80 1C. Before use the cells were

washed once and resuspended in distilled water, which ensured the best dispersal of the cells. Degradation of isoproturon and MDIPU by bacterial cultures was performed by adding isoproturon or MDIPU from stock solutions in acetone to 100-ml sterilized flasks. The 14C-labeled isoproturon or MDIPU was then added from stock solutions in acetonitrile. The solvents were evaporated, after which 9.375 ml sterile medium A and 3.125 ml buffer 1 were added to yield a final concentration of 20 mmol l1 and 104 dpm per flask. Degradation of 1-OH-IPU and 1-OH-MDIPU by bacterial cultures was performed by mixing 3.125 ml buffer 1 with bioconverted and radiolabelled metabolites and 9.375 ml sterile medium A at a final metabolite concentration of about 20 mmol l1. Flasks with isoproturon or MDIPU were also prepared using 9.375 ml medium A and 3.125 ml buffer 1+exudates. The final pH of the various mixtures of medium A and buffer was 7.1–7.2. Sphingomonas sp. SRS2 was inoculated as 5  106 CFU (as counted on R2A) in 0.5 ml distilled water in each flask. The mixed bacterial culture from the Græse site was inoculated as 1  108 CFU (as counted on R2A) in 0.5 ml medium A in each flask. The 14CO2 trapped in the flasks was measured by scintillation counting as described in Section 2.7. In a parallel degradation experiment without 14C-labeled compounds the medium was sampled for HPLC. The experiment included abiotic controls and controls without isoproturon or metabolites. The hydroxylated, N-demethylated and aniline metabolites of isoproturon were assayed using the HPLC method of Rønhede et al. (2005) (qualitatively for 1-OH-MDIPU, 2-OH-IPU and 1-OHIPU and quantitatively for DDIPU, MDIPU, isoproturon and 4-isopropyl-aniline). The 1-OH-DDIPU (Fig. 1) that had been purified and characterized in our laboratory from degradation of MDIPU by the fungus Mortierella sp. strain CBS118520 was included as a qualitative standard (retention time 5 min).

ARTICLE IN PRESS S. Rønhede et al. / Soil Biology & Biochemistry 39 (2007) 1751–1758

Isoproturon

50 40 30 20

Cumulative 14CO2 production (in % of added 14C)

10 0 0

10

20

30

40

50

60

MDIPU

50

1755

and MDIPU mineralized in this soil (Fig. 2). As the amount of MDIPU that mineralized in the Jyndevad soil was less than the level of impurity in the 14C stock solution, however, it is unclear whether MDIPU mineralized at all. In the Deep Slade and Græse soils, 33% and 24% of the 1OH-IPU, respectively, mineralized in 30 days. This is comparable to isoproturon mineralization in the Deep Slade soil (26% in 30 days), whereas that in the Græse soil was slower (9% in 30 days) (Fig. 2). With MDIPU, mineralization was more similar in the Græse and Deep Slade soils (29% and 20% in 30 days, respectively) (Fig. 2). 3.2. Degradation and mineralization by bacterial cultures

40 30 20 10 0 0

10

20

30

40

50

60

20

30 Days

40

50

60

1-OH-IPU

50 40 30 20 10 0 0

10

Fig. 2. Mineralization of isoproturon, MDIPU and 1-OH-IPU in bottled samples of soil from the Jyndevad (crosses), Græse (open diamonds), Deep Slade (closed diamonds) and Long Close (triangles) sites. Broken lines indicate that the compound was added in buffer 1+exudates, while solid lines indicate that the compound was added in fresh buffer 1. Vertical bars indicate the standard deviation ðn ¼ 3Þ. The radiochemical purity of isoproturon, MDIPU and 1-OH-IPU was 99%, 90% and 94%, respectively.

3. Results 3.1. Mineralization in soil Mineralization of isoproturon and MDIPU was rapid in the Long Close soil, more than 40% being mineralized in 30 days (Fig. 2). The 1-OH-IPU also mineralized in the Long Close soil, but more slowly, with 29% being mineralized in 30 days. Mineralization was generally slow in the non-isoproturon exposed Jyndevad soil, although 25% of the 1-OH-IPU mineralized during the 63-day experiment. In contrast, less than 7% of the isoproturon

Isoproturon was degraded by Sphingomonas sp. SRS2 with transient accumulation of MDIPU as the only intermediate (Fig. 3a). MDIPU was also degraded by this bacterium, but without accumulation of metabolites (Fig. 3b). The mineralization of isoproturon and MDIPU by Sphingomonas sp. SRS2 occurred concomitantly with degradation. After 9 days of incubation, Sphingomonas sp. SRS2 had depleted all isoproturon and MDIPU from the medium, and mineralization peaked at approx. 45% (Fig. 3a, b and Fig. 4a, b). The 1-OH-IPU was also degraded by Sphingomonas sp. SRS2, but more slowly than isoproturon and MDIPU. About 10% of the added 1-OHIPU remained in the culture after 16 days (Fig. 3c). Degradation of 1-OH-IPU involved transient accumulation of 1-OH-MDIPU. The traces of 2-OH-IPU produced during bioconversion also degraded and were succeeded by traces of an unknown metabolite with a retention time of 6.25 min. The 1-OH-MDIPU degraded almost completely after 16 days, while the unknown metabolite remained unchanged (Fig. 3d). Degradation of 1-OH-IPU and 1-OH-MDIPU by Sphingomonas sp. SRS2 was not accompanied by mineralization to the same extent as with the non-hydroxylated compounds, since only 10% of the 14 C had mineralized in 42 days (Fig. 4c, d). While the 1-OH-MDIPU data are to some extent compromised by the fact that bioconversion of MDIPU resulted in a mixture of metabolites (Table 1), the low 14CO2 recovery nevertheless shows that Sphingomonas sp. SRS2 did not completely mineralize 1-OH-MDIPU (Fig. 4d). The mixed bacterial culture from the Græse site completely degraded MDIPU within 6 days (Fig. 3f) while concomitantly mineralizing the MDIPU (approx. 45% in 6 days; Fig. 4b). In contrast the mixed bacterial culture did not degrade isoproturon during 16 days of incubation (Fig. 3e), and less than 1% of the 14C mineralized within 42 days (Fig 4a). The mixed bacterial culture degraded both 1-OH-IPU and 1-OH-MDIPU, approx. 75% and 100%, respectively, of the metabolite disappearing from the medium in 16 days (Fig. 3g, h). Unlike with Sphingomonas sp. SRS2, the bioconversion product 2-OH-IPU and the unknown metabolite with a retention time of 6.25 min did not degrade in the presence of the mixed bacterial culture.

ARTICLE IN PRESS S. Rønhede et al. / Soil Biology & Biochemistry 39 (2007) 1751–1758

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15

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10 5

0 4

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0

Isoproturon degradation by mixed culture from Græse

8

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0

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0 12

200 100

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300 200 100 0

4

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1-OH-IPU degradation by mixed culture from Græse

Days

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300 200 100

16

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500

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400 300 200 100

0 0

4

1-OH-MDIPU degradation by mixed culture from Græse

500

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Area (arbitrary unit)

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15 µmol l-1

15 µmol l-1

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MDIPU degradation by mixed culture from Græse

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Area (arbitrary unit)

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1-OH-MDIPU degradation by Sphingomonas sp. SRS2

Area (arbitrary unit)

20

1-OH-IPU degradation by Sphingomonas sp. SRS2 Area (arbitrary unit)

MDIPU degradation by Sphingomonas sp. SRS2

µmol l-1

µmol l-1

Isoproturon degradation bySphingomonas sp. SRS2

0 0

4

8

Days

12

16

0

Days

4

8

12

16

Days

Fig. 3. HPLC analysis of degradation of isoproturon and its metabolites in liquid medium by Sphingomonas sp. SRS2 (a–d) and a mixed bacterial culture from the Græse site enriched on MDIPU (e–h). Closed squares: isoproturon, closed circles: MDIPU, open triangles: 1-OH-IPU, open squares: 2-OH-IPU, open circles: 1-OH-MDIPU, closed triangles: unknown compound with a retention time of 6.25 min. Vertical bars indicate the standard deviation ðn ¼ 3Þ. The purity of the 1-OH-IPU and 1-OH-MDIPU was 74% and 49%, respectively, as estimated from parallel bioconversion with 14C-labeled substrates (Table 1).

Cumulative14CO2 production (in % of added 14C)

Isoproturon

1-OH-IPU

70

70

60

60

50

50

40

40

30

30

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10

10 0

0 0

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1-OH-MDIPU and two unknown compounds

MDIPU 70

70

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60

50

50

40

40

30

30

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20

10

10 0

0 0

10

20 30 Days

40

0

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20 30 Days

40

Fig. 4. Mineralization of isoproturon and metabolites by bacterial cultures. Closed diamonds: Sphingomonas sp. SRS2; open diamonds: mixed bacterial culture from the Græse site. The experiments were carried out in medium A mixed with buffer 1+exudates (broken lines) or medium A with fresh buffer 1 (solid lines). Vertical bars indicate the standard deviation (n ¼ 3, but n ¼ 2 for isoproturon mineralization by Sphingomonas sp. SRS2). The radiochemical purity of the 1-OH-IPU and 1-OH-MDIPU was 94% and 49%, respectively, as evaluated by TLC (Table 1).

Moreover, degradation of the hydroxylated metabolites was not followed by mineralization (Fig 4c, d). There is no evidence for any abiotic degradation since the concentration of the compounds remained largely

unchanged in the bacteria-free controls between days 0 and 16 (paired t-tests, n ¼ 3). Mineralization of isoproturon and MDIPU was not suppressed by the presence of buffer 1+exudates as

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compared to fresh buffer (Figs. 2 and 4a, b). In fact it gave an earlier onset of mineralization in the bacterial cultures (Fig. 4a, b). 4. Discussion As authentic standards and radioisotopes are not commercially available for 1-OH-IPU and other hydroxylated metabolites of isoproturon, we attempted to produce these ourselves by fungal bioconversion of isoproturon and MDIPU and then used the resultant medium containing the hydroxylated and radiolabeled bioconversion products directly in the degradation experiments. This strategy proved successful for 1-OH-IPU, since the radiochemical purity achieved (94%) was sufficient to allow interpretation of the mineralization curves. Production of 1-OH-MDIPU was less successful, however, since the process also generated other major metabolites (Table 1). We were nevertheless able to follow the degradation of 1-OH-MDIPU in bacterial cultures using HPLC (Fig. 3). This study shows that agricultural soils can potentially mineralize the hydroxylated isoproturon metabolite 1-OHIPU in soil. This indicates that hydroxylated metabolites of isoproturon do not persist in the topsoil, thereby reducing the risk that they might leach to and contaminate groundwater and surface water. In the field, lower winter temperatures may limit mineralization and at these conditions leaching may occur. However, the central result from our laboratory incubations is that 1-OH-IPU is mineralized just as fast as isoproturon in soil. This implies that the persistence of 1-OH-IPU is comparable to that of isoproturon. In the soil from the non-isoproturon treated site at Jyndevad, 1-OH-IPU mineralized faster than isoproturon and MDIPU (Fig. 2). This could be attributable to the soil microbial community being better able to degrade 1-OHIPU or, alternatively, to the hydroxylated metabolite adsorbing less strongly to soil with a consequent greater bioavailability to degraders. Either way, the results indicate that fungal activity may benefit mineralization of isoproturon in the non-isoproturon exposed soil. In the isoproturon-exposed soils, mineralization of 1-OH-IPU was generally comparable to that of isoproturon and MDIPU. An exception is soil from the Græse site, which mineralizes isoproturon to a notably lesser extent than its metabolites, as has previously been reported for MDIPU (Sørensen and Aamand, 2001). The Deep Slade and Long Close soils are known to be adapted to fast degradation of isoproturon (Cox et al., 1996; Bending et al., 2006). The present study indicates that these soils are almost equally well adapted to the degradation of 1-OH-IPU. Sphingomonas sp. SRS2, which was isolated from Deep Slade soil by Sørensen et al. (2001), has been shown by denaturing gradient gel electrophoresis profiling to proliferate in the soil after treatment with isoproturon (Bending et al., 2003). The mixed bacterial culture from the Græse soil used here was enriched on MDIPU

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(Sørensen and Aamand, 2001). The isoproturon-mineralizing bacterium Sphingomonas sp. SRS2 and the MDIPUmineralizing mixed bacterial culture were unable to mineralize the ring carbon of the hydroxylated metabolites (Fig. 4), although they were capable of degrading the hydroxylated compounds (Fig. 3). Initial degradation of isoproturon by Sphingomonas sp. SRS2 takes place by consecutive N-demethylation. The strain was able to demethylate 1-OH-IPU to 1-OH-MDIPU, but the didemethylated degradation product 1-OH-DDIPU was not detected, possibly indicating that degradation beyond 1OH-MDIPU yields dead-end products undetectable by our HPLC method. Since 1-OH-IPU degraded but isoproturon did not, hydroxylation seems to have benefited degradation by the mixed bacterial culture (Fig. 3e and g). On the other hand, though, hydroxylation seems to have prevented subsequent mineralization since the mixed bacterial culture was able to mineralize MDIPU, but not 1-OH-MDIPU (Fig. 4c and d). Fungi might exude substances during bioconversion that bias degradation of bioconverted metabolites by stimulating or inhibiting the degrading organisms. This does not seem to have been the case in the present study, however, as mineralization of isoproturon and MDIPU did not differ markedly whether the incubation was performed using fresh buffer 1 or buffer 1 incubated with mycelium (Figs. 2 and 4), thus justifying the deduction that degradation of 1-OH-IPU was not biased by stimulatory or inhibitory fungal exudates. It seems unlikely that the potential of the soils to mineralize hydroxylated isoproturon metabolites is attributable to Sphingomonas sp. SRS2 or the organisms in mixed bacterial culture from the Græse site since these were unable to mineralize 1-OH-IPU or 1-OH-MDIPU in culture. This mineralization of 1-OH-IPU in soil is probably mediated by other bacteria that depend on preceding fungal hydroxylation of the isoproturon in the soil. In other words, the bacteria may exploit a niche created by fungi (de Boer et al., 2005). With polyaromatic hydrocarbons it has previously been proposed that fungal hydroxylation may stimulate their complete degradation by increasing the solubility of these otherwise hydrophobic compounds (Cerniglia, 1997). Thus initial transformation of benzo[a]pyrene and other PAHs by Penicillium janthinellum enabled bacterial cultures to mineralize these compounds (Boonchan et al., 2000). Similarly, a fungal metabolite of anthracene mineralized more rapidly than anthracene in soil and activated sludge (Meulenberg et al., 1997). In contrast, hydroxylation slowed the mineralization of phenanthrene (Meulenberg et al., 1997) and of pyrene in marine sediment (Giessing and Johnsen, 2005). With isoproturon, any stimulation of bacterial degraders by fungal hydroxylation is unlikely to be attributable to an increase in solubility since isoproturon is a polar pesticide. Apart from fungi-dependent degrader bacteria, mineralization of 1-OH-IPU in soil could also be attributable to isoproturon-mineralizing bacteria carrying out the first

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steps of the degradation of 1-OH-IPU, as Sphingomonas sp. SRS2 does (Fig. 3). In soil, other microorganisms could then mineralize the accumulating metabolites. Finally, isoproturon-exposed soils may contain bacteria with alternative degradation pathways for isoproturon that are able to accept 1-OH-IPU. We conclude that hydroxylation of isoproturon generally enhances mineralization of the ring carbon, but that the mechanism involved does not seem to entail stimulation of known isoproturon or MDIPU degraders as these are unable to mineralize hydroxylated isoproturon metabolites. The present finding that 1-OH-IPU degrades in soil could be of great relevance to environmental risk assessment as it implies a reduced risk that this isoproturon degradation product may leach from the topsoil and contaminate the aquatic environment. Acknowledgments The soil from Warwick HRI farm was a kind gift from Gary Bending. Ole Stig Jacobsen (GEUS) provided valuable assistance with TLC. S.R. was funded by the Danish Technical Research Council. References Alletto, L., Coquet, Y., Benoit, P., Bergheaud, V., 2006. Effects of temperature and water content on degradation of isoproturon in three soil profiles. Chemosphere 64, 1053–1061. Bælum, J., Henriksen, T., Hansen, H.C.B., Jacobsen, C.S., 2006. Degradation of 4-chloro-2-methylphenoxyacetic acid in top- and subsoil is quantitatively linked to the class III tfdA gene. Applied and Environmental Microbiology 72, 1476–1486. Bending, G.D., Shaw, E., Walker, A., 2001. Spatial heterogeneity in the metabolism and dynamics of isoproturon degrading microbial communities in soil. Biology and Fertility of Soils 33, 484–489. Bending, G.D., Lincoln, S.D., Sørensen, S.R., Morgan, J.A.W., Aamand, J., Walker, A., 2003. In-field spatial variability in the degradation of the phenyl-urea herbicide isoproturon is the result of interactions between degradative Sphingomonas spp. and soil pH. Applied and Environmental Microbiology 69, 827–834. Bending, G.D., Lincoln, S.D., Edmondson, R.N., 2006. Spatial variation in the degradation rate of the pesticides isoproturon, azoxystrobin and diflufenican in soil and its relationship with chemical and microbial properties. Environmental Pollution 139, 279–287. Boonchan, S., Britz, M.L., Stanley, G.A., 2000. Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal–bacterial cocultures. Applied and Environmental Microbiology 66, 1007–1019. Cerniglia, C.E., 1997. Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation. Journal of Industrial Microbiology and Biotechnology 19, 324–333. Cox, L., Walker, A., Welch, S.J., 1996. Evidence for the accelerated degradation of isoproturon in soils. Pesticide Science 48, 253–260. Cullington, J.E., Walker, A., 1999. Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biology & Biochemistry 31, 677–686.

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