Microbial degradation and impact of Bracken toxin ptaquiloside on microbial communities in soil

Chemosphere 67 (2007) 202–209 www.elsevier.com/locate/chemosphere

Microbial degradation and impact of Bracken toxin ptaquiloside on microbial communities in soil Pernille Engel a, Kristian K. Brandt a,*, Lars H. Rasmussen b, Rikke G. Ovesen b, Jan Sørensen a b

a Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Department of Natural Sciences, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

Received 1 January 2006; received in revised form 31 July 2006; accepted 17 August 2006 Available online 2 November 2006

Abstract The carcinogenic and toxic ptaquiloside (PTA) is a major secondary metabolite in Bracken fern (Pteridium aquilinum (L.) Kuhn) and was hypothesized to influence microbial communities in soil below Bracken stands. Soil and Bracken tissue were sampled at field sites in Denmark (DK) and New Zealand (NZ). PTA contents of 2.1 ± 0.5 mg g1 and 37.0 ± 8.7 mg g1 tissue were measured in Bracken fronds from DK and NZ, respectively. In the two soils the PTA levels were similar (0–5 lg g1 soil); a decrease with depth could be discerned in the deeper B and C horizons of the DK soil (weak acid sandy Spodosol), but not in the NZ soil (weak acid loamy Entisol). In the DK soil PTA turnover was predominantly due to microbial degradation (biodegradation); chemical hydrolysis was occurring mainly in the uppermost A horizon where pH was very low (3.4). Microbial activity (basal respiration) and growth ([3H]leucine incorporation assay) increased after PTA exposure, indicating that the Bracken toxin served as a C substrate for the organotrophic microorganisms. On the other hand, there was no apparent impact of PTA on community size as measured by substrate-induced respiration or composition as indicated by community-level physiological profiles. Our results demonstrate that PTA stimulates microbial activity and that microorganisms play a predominant role for rapid PTA degradation in Bracken-impacted soils.  2006 Elsevier Ltd. All rights reserved. Keywords: Natural toxin; Soil contamination; Biodegradation; Soil respiration; Microbial activity; Community structure

1. Introduction The widespread Bracken fern (Pteridium aquilinum (L.) Kuhn) is known to cause intestinal and bladder carcinomas in cattle and sheep due to the toxic nor-sesquiterpene glycoside, ptaquiloside (PTA) (Hirono and Yamada, 1987). PTA is detected in all tissues of the Bracken and particularly high levels are found in the crosiers (Smith et al., 1994; Rasmussen and Hansen, 2004). A correlation between human cancer incidences and consumption of Bracken and/or PTA contaminated dairy milk has been

*

Corresponding author. Tel.: +45 35282612; fax: +45 35282606. E-mail address: [email protected] (K.K. Brandt).

0045-6535/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.08.025

observed (Alonso-Amelot et al., 2000; Marlie`re et al., 2000; Alonso-Amelot and Avendan˜o, 2002). The active carcinogenic agent is likely to be a highly unstable dienone intermediate formed spontaneously from PTA under weak alkaline conditions (Saito et al., 1989). In the Bracken field stands PTA is leached to the soil environment (Rasmussen et al., 2003a) where the compound is highly water-soluble and shows little sorption to soil particles (Rasmussen et al., 2005). Hence, the compound may leach to groundwater and/or surface aquatic environments and thus comprise a potentially carcinogenic contaminant of human drinking water (Galpin et al., 1990; Rasmussen et al., 2005). Little is known about the degradation of PTA in soils, although rapid chemical hydrolysis is known to occur under either strongly acidic (pH < 4) or

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alkaline (pH > 7) conditions (Agnew and Lauren, 1991). By contrast, PTA is chemically stable in weak acid soils (pH 4–7) where degradation by soil microorganisms (biodegradation) is expected to be predominant (Rasmussen et al., 2005). Many plant metabolites, e.g. terpenes (Amaral and Knowles, 1998) are known to be toxic to soil microorganisms, but only one study has so far reported a toxicity of PTA towards soil microorganisms (Schmidt et al., 2005). In this study a reduction of the actively respiring population of microorganisms was observed under increasing PTA exposure in a Danish agricultural soil (calcareous loamy Mollisol). In the present work, we undertook a comprehensive study of both degradation and toxicity effects of PTA in acidic/weak acidic Danish (DK) and New Zealand (NZ) soils under natural Bracken stands. The specific aims were to evaluate the importance of microbial activity for degradation of PTA and to investigate the toxicity of PTA to microbial communities in bracken-impacted soils. 2. Materials and methods 2.1. Field sites and sampling The two field sites supported dense Bracken stands and were chosen based on earlier measurements of high PTA levels in the fronds. The DK Bracken fronds (Pteridium aquilinum var. aquilinum) were fully grown, while some were still in the crosier stage in the NZ fronds (Pteridium aquilinum var. esculentum). The soils comprised a Typic Haplorthod at Præstø Fed in Denmark (DK, N: 6.115.795 E: 697.164) and a Typic Udorthent at Taumarunui in New Zealand (NZ, S: 342.475 E: 5.689.326) (Soil Survey Staff, 2003). Representative Bracken frond samples (n = 10; dried at 45 C and milled to a particle size less than 2 mm) were taken for determination of PTA content. Samples were also taken from representative soil horizons by combining approximately 1 kg of soil from each side of the soil profile into one composite sample for each soil horizon. The resulting composite samples were homogenized and split into three fractions which were either frozen at 18 C for determination of PTA content, air-dried and sieved through a 2-mm mesh screen for determination of soil characteristics, or kept at approx. 5 C for determination of PTA degradation and impacts on soil microorganisms. Separate samples were taken for determination of bulk density and water content in the soil horizons. 2.2. PTA contents in Bracken and soil PTA contents in aqueous extracts of Bracken were determined after cleaning the extracts with Polyamide 6 resin (Fluka, Steinheim, Switzerland), conversion of PTA to pterosin B (Agnew and Lauren, 1991) and quantification by HPLC (Rasmussen et al., 2005).

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2.3. Soil characteristics Soil profiles were classified according to Soil Survey Staff (2003). Size distribution of soil particles was measured using the hydrometer method (Bouyoucos, 1926). Soil pH was measured in a 1:2.5 suspension of soil and 0.01 M CaCl2 solution (Rowell, 1994). Organic carbon content was determined by dry combustion using an Eltra CS 500 Carbon Sulphur Determinator (Nelson and Somers, 1982). Total nitrogen content was determined by the Kjeldahl method (Rowell, 1994). All analyses were made in duplicate on large (>10 g), homogenized soil samples. 2.4. PTA preparations for degradation and impact studies in soil For studies of PTA degradation and impact in the DK soil, PTA was extracted and purified from the Bracken material harvested at the DK site using repeated aqueous extraction, low-pressure liquid chromatography (LPLC) and preparative HPLC (Rasmussen et al., 2005). The PTA preparations were either pure (100% purity) or crude (80% purity, omitting preparative HPLC step). 2.5. PTA degradation in soil Non-sterile and sterile DK soil from the A, E, B and C horizons were spiked with pure PTA at a level of 25 lg g1. The sterile soil samples were prepared by c-radiation (total dose of 18 kGy, applied twice) on a 10 MeV electron accelerator at Risø High Dose Reference Laboratory, Roskilde, Denmark. All incubations were performed in duplicate at 10 C with 2 g of moist, homogenized, sieved (2-mm mesh size) soil. PTA was extracted from soil following Rasmussen et al. (2005) except that the soil/water (wt/vol) ratio was changed from 1:1 to 1:4 to increase the extractable amount of PTA. Extractions were made at regular intervals and processed immediately. Rate constants for PTA disappearance were determined by data fitting using the software package TableCurveTM 2D 5.0 (Jandel Scientific, AISN Software inc., Mapleton, USA) and the fitting parameters reported by Rasmussen et al. (2005). PTA disappearance was well explained using either a one-component or a two-component first order kinetics model (see results for mathematical details). 2.6. PTA impact in soil (experimental setup) The impact of PTA on microbial communities was investigated in soil microcosms amended with either crude or pure PTA solutions (Experiments 1 and 2, respectively). Experiment 1 was performed with field-moist DK and NZ soils, respectively (A horizons only). The soils were homogenized and sieved (2-mm mesh) before being transferred (5 g field moist) to air-tight 150-ml serum flasks. The gravimetric water contents were initially

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6.1% and 38.5% in the DK and NZ soils, respectively. The water contents were adjusted to the theoretical field capacities based on their respective soil particle size distribution (Madsen et al., 1992). The soil microcosms were amended with 0, 29 and 105 lg PTA g1 dry wt from a crude PTA extract (80% PTA) at days 0, 7 and 15, respectively (3-weeks duration). Amendments were carried out immediately after each determination of PTA content by HPLC as described above. The repeated additions of PTA after one and two weeks were performed since PTA disappeared rapidly (see results). A total of 10 replicate microcosms for each PTA treatment were incubated at 15 C, and two microcosms per treatment were destructively sampled at regular time intervals for measurement of microbiological parameters (see Sections 2.7 and 2.8 below). Experiment 2 was performed with the following modifications: Microcosms were miniaturized (0.5 g field moist soil in 12-ml glass tubes, Venoject VT-100SU; Terumo, Leuven, Belgium) and soils were amended with 0, 22 and 107 g1 dry wt (pure PTA extract) at days 0 and 7, respectively (two weeks duration). 2.7. PTA impacts on soil microbial activity and growth Prior to and following PTA applications, basal respiration was determined as the rate of CO2 release from respiring microorganisms in the microcosms. This parameter could be measured non-destructively by gas chromatography (Brandt et al., 2003) as accumulating CO2 in the headspace of the soil incubations. Following each measurement (1–2 times per week) the headspace was outgassed and replaced with technical air (79% N2, 21% O2), ensuring that aerobic and low-CO2 conditions (<1%) were maintained in the headspace throughout the incubation period. In parallel, the impact of PTA on actual soil microbial growth (protein synthesis) was determined as the rate of [3H]leucine incorporation according to the microcentrifugation method developed by Ba˚a˚th et al. (2001). Briefly, these incubations were carried out in 1.5-ml soil suspensions prepared by centrifugation (1000 · g, 10 min, 22 C) of a 1:10 soil:water (wt/wt) slurry. Samples were added a mixture of L-[4,5-3H]leucine and unlabelled leucine (final concentration of 20 nM) and incubated for 2 h at 22 C, before the incubations were stopped by adding 160 ll icecold, 50% trichloroacetic acid (TCA). Corresponding blanks (negative controls) were prepared by adding TCA before addition of [3H]leucine. Subsequently, the samples were passed through a series of washing steps (5% TCA and 80% ethanol) to eliminate non-incorporated [3H]leucine label. Finally, the centrifugation pellet was re-solubilized in 1 M NaOH and the [3H]leucine incorporated in the microorganisms detected by scintillation counting. No attempts were made to estimate the degree of isotope dilution, and results thus only indicate relative differences in microbial growth rates.

2.8. PTA impact on microbial community size and composition A measure of PTA impacts on the size of the respiring microbial community was obtained by the substrateinduced respiration (SIR) assay. The SIR assay included detection of accumulating CO2 in 1-g soil samples supplemented with 12 mg glucose-talcum (ratio of 4 to 1) powder (Brandt et al., 2003); gas sampling and analysis by GC were performed at regular intervals between 0.5 and 4.5 h after glucose amendment as described previously (Brandt et al., 2003). Finally, a measure of the PTA impact on microbial community composition was obtained (only Experiment 1) by community-level physiological profiling (CLPP) using Biolog EcoPlatesTM (Biolog, Inc., Hayward, CA, USA). First, the soil suspension (which was also used for [3H]leucine incorporation assay, see above) was diluted 5 times with sterile distilled water and inoculated into the Biolog EcoPlates (150 ll per well). Subsequently, the plates were incubated on a flat bed shaker (50 rpm) and color formation (c = 590 nm) was measured twice a day for 4 days and again once after 7 days using an EL 808 Ultra Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). The number of cells in the soil suspensions used for inoculation of EcoPlates was estimated by epifluorescence microscopy after staining with SYBR-green I (chemical no. S-7567, Molecular Probes, Leiden, The Netherlands) and subsequent transfer of cells to black polycarbonate membrane filters (0.2 lm pore size, Poretics, Osmonics, Livermore, CA, USA) according to Weinbauer et al. (1998). The number of cells ranged from 0.9 · 106 to 1.9 · 106 cells per well (DK soil) and from 2.1 · 106 to 3.8 · 106 cells per well (NZ soil). Hence, bacterial communities were of similar size in all treatments and community size was not anticipated to affect the obtained community composition profiles following proper data normalization (Garland et al., 2001). Initially, the color development in each well was corrected by subtracting the absorbance of the corresponding control well. Normalization (scaling) was subsequently performed by dividing the absorbance value from each well with the average well color development (AWCD) as described previously (Garland et al., 2001). Analysis of normalized CLPP data was performed by principle-component analysis (PCA) as described previously (Brandt et al., 2004) using the Systat 10 software package (Systat Software, Point Richmond, CA). The number of positive wells were recorded after 7 days of incubation and defined as wells with an absorbance value (after subtracting control well background absorbance) of 0.5 or higher.

2.9. Statistical methods SAS software (edition 8.2, SAS Institute Inc., Cary, NC) was used for test of statistical significance. One- and two-

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way analysis of variance were used to determine impacts of PTA on basal respiration and [3H]leucine incorporation.

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to see if disappearance was due to chemical hydrolysis or microbial degradation. 3.2. Degradation of PTA

3. Results

The average frond height at the DK site was 2 m but only 0.6 m at the NZ site, while the average aboveground biomass was estimated to be about 470 and 50 g [dry wt] m2 at the DK and NZ sites, respectively. In contrast, the PTA content of the standing biomass was about twice as high at the NZ site (19.0 g m2) compared to the DK site (9.5 g m2), reflecting the much higher PTA content in NZ Bracken fronds (37.0 ± 8.7 mg g1) compared to DK Bracken fronds (2.1 ± 0.5 mg g1). High PTA contents in NZ Bracken fronds have been measured previously (Rasmussen et al., submitted for publication), but the extremely high PTA contents found in the present study are the highest ever observed. By comparison, the PTA content of the DK Brackens was comparable to earlier measurements (Rasmussen et al., 2003a,b). The texture of the DK and NZ soils were classified as sand and loam (United States Department of Agriculture soil classification system), respectively. The pH was approximately 4 in both soils, although the A horizon of the DK soil had a noteworthy low pH of 3.4. Further characteristics of the soils were the rapidly decreasing organic C and N levels with increasing depth as shown in Fig. 1. As also shown in Fig. 1, both soils contained PTA (2– 5 lg g1). A complete disappearance of PTA with increasing depth could only be observed in the DK soil. Since the DK soil showed the most complete profile including PTA disappearance with depth, this soil was selected for the detailed degradation experiments, including a first assay

Fig. 2a shows the disappearance at 10 C of PTA added in non-sterile and sterile soil samples from the DK site. In the non-sterile soil, PTA disappearance followed a firstorder reaction (Eq. (1); c(t) = amount disappeared at time t; a = initial amount; k1 = rate constant). cðtÞ ¼ 100  ðað1  ek1 t ÞÞ

DK soil 100

DK soil 5

60

40

20

0

A

0

5

10

100

200

600

700

500

600

700

80

50

Depth (cm)

2B

100

C 150 1

Fig. 1. % Organic carbon (m), % (·10 ) nitrogen (s) and PTA content (lg g1 [dry wt] soil) (·) as a function of depth (cm) in DK (A) and NZ (B) soils. Soils horizons are indicated.

% of PTA added

1B

50

10 0

500

A

E

B

300 400 Time (hours)

b 100 AB

Depth (cm)

0

NZ soil 10

0

15 0

A (0.998; 0.0184) E (0.998; 0.0081) B (0.996; 0.0063) C (0.942; 0.0061)

80

0

0

ð1Þ

Disappearance rate was clearly highest in the A horizon while comparable, but lower rates were observed in the underlying zones. Rate constants (k1) for the samples representing the different soil horizons are given in Fig. 2a. PTA disappeared completely within a week in the A horizon and within 2–4 weeks in lower soil horizons. In the sterile samples where microbial degradation was excluded, disappearance of PTA was generally much slower in all soil horizons (Fig. 2b). A rapid initial removal took place within the first day and most likely represented irreversible sorption to the soil particulate phase and dissolved organic matter. When fitting the PTA disappearance

% of PTA added

3.1. Characterization of field sites

60 40

As (0.950; 0.111; 0.0013) Es (0.906; 0.529; 0.0004) Bs (0.940; 0.380; 0.0002)

20

Cs (0.945; 0.037; 0.0001)

0

0

100

200

300 400 Time (hours)

Fig. 2. First-order degradation reactions in the DK soil horizons. Start PTA concentrations were about 25 lg g1 and incubations were at 10 C. (A) Non-sterile soil horizons A (h), E (), B (s) and C (·). R2 values and first-order rate constants (k1) are shown in brackets. (B) Sterile soil horizons As (h), Es (), Bs (s) and Cs (·). R2 values, first-order degradation (sorption) rate constants (k21) and forward rate equilibrium constants (k22) are shown in brackets.

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in the sterile samples with a two-component first-order kinetic model the rapid initial disappearance could be assigned one specific rate constant (k21), and the slow subsequent disappearance another one (k22) (Eq. (2); b = initial amount disappeared according to the second disappearance reaction). cðtÞ ¼ 100  ðað1  ek21 t Þ þ bð1  ek22 t ÞÞ

ð2Þ

Excluding the rapid initial sorption reaction, the disappearance reactions occurring in the sterile samples were assumed to represent chemical hydrolysis. Hydrolysis was clearly occurring in the A horizon, but was low in the underlying horizons. Excluding the rapid initial sorption reaction described by k21, a comparison of rate constants calculated for the non-sterile soils (k1) and sterile soils (k22) thus provided evidence that at least 90% of the PTA disappearance in the A horizon in the natural (non-sterile) DK soil was due to microbial biodegradation. By comparison chemical hydrolysis accounted for less than 10%. We suggest that the hydrolysis reaction could be due to the remarkably low pH (3.4) in this horizon. In the deeper horizons where pH was higher (4) chemical hydrolysis was even slower. Rapid PTA removal also occurred in soil microcosms used for studying impacts of PTA on soil microbial communities (see Sections 3.3 and 3.4). In the case of DK soil microcosms, only 2.8 ± 0.1 and 1.1 ± 0.02% of added PTA were recovered after one week in the low and high PTA treatments, respectively. Following 3 weeks of incubation with weekly additions of new PTA at days 0, 7 and 14 to the DK soil microcosms, PTA levels were below the detection limit for HPLC analysis. Corresponding figures for the NZ soil were 4.1 ± 5.9% (low PTA treatment, one week), 2.2 ± 2.4 (low PTA treatment, three weeks), below detection limit (high PTA treatment, one week), and 0.6 ± 0.7% (high PTA treatment, three weeks). 3.3. Impact of PTA on microbial growth Since microbial degradation (biodegradation) rather than chemical hydrolysis was shown to be predominant in the DK soil, it was further examined if the degradation DK soil Leu. incorporation rate 5 -1 -1 (DPM 10 g h )

5 4 3 2 1 0 0

5

10

15

Time (days)

Fig. 3. Incorporation of [3H]leucine (DPM · 105 g1 [dry wt] soil h1) in the DK soil spiked after 0 and 7 days with pure PTA at approx. 0 lg g1 (s), 22 lg g1 (·) and 107 lg g1 (m).

contributed to microbial growth. Addition of pure PTA significantly (P = 0.031, n = 6) increased microbial growth as detected by the [3H]leucine incorporation assay (Fig. 3). However, no effect was determined during the first 24 hours, showing that growth was not stimulated immediately after PTA amendment, but showed a lag phase comparable to what is expected for onset of growth (cell proliferation) in microbial populations after substrate addition. Hence, the experiment indicated that PTA or an associated degradation product served as a substrate for microbial growth. 3.4. Impact of PTA on soil microbial community size and composition In extended experiments with both DK and NZ soils, we subsequently tested the impact of PTA on a series of different microbiological parameters. Since pure PTA was difficult to retrieve we used crude PTA for these experiments, but included a repeat of the growth assays described above for comparison. Fig. 4a shows that stimulation of microbial growth in the DK soil using crude PTA (P = 0.001, n = 8) was similar to that observed using pure PTA (Fig. 3). Interestingly, however, we could not detect the stimulatory effect of PTA addition on the [3H]leucine incorporation in the NZ soil (Fig. 4b), perhaps because the background levels of protein synthesis were about 5 times higher in this soil than in the DK soil. Fig. 4c and d show that crude PTA addition also stimulated basal respiration rates significantly in both DK (P < 0.001, n = 10) and NZ (P < 0.001, n = 10) soils. The released CO2 accumulated linearly in all microcosms (R2 > 0.988) and we thus calculated one basal respiration rate for each microcosm (Fig. 4c and d). The basal respiration rates (without PTA addition) were about 10-fold lower in the DK than in the NZ soil, but in relative terms the stimulatory effect of PTA was higher in the DK soil (Fig. 4c and d). Since the measures of actual microbial activity (basal respiration) and growth ([3H]leucine incorporation) thus provided evidence for a role of PTA (or its degradation products) as a stimulatory substrate for the microorganisms, PTA application could further represent a stimulant affecting both the overall size and composition of the microbial communities. SIR rates of between 0.4–0.9 lg CO2-C g1 h1 and 8–11 lg CO2-C g1 h1 were obtained for the DK and NZ soil microcosms, respectively. However, no significant effects of PTA amendment were observed in any of the two soils (data not shown), indicating that the observed stimulatory effect of PTA amendments on actual microbial activity and growth described above (Figs. 3 and 4) was not immediately reflected in the overall size of the soil microbial biomass. Likewise, PTA addition did not immediately change the patterns of community composition in the DK and NZ soils, as indicated by the Biolog EcoPlate CLPP assay (Fig. 5). Both the number of substrates supporting color formation and

P. Engel et al. / Chemosphere 67 (2007) 202–209

12

5 -1

-1

(DPM 10 g h )

Leu. incorporation rate

NZ soil

DK soil

12

8

8

4

4

0

0 0

c

5

10 15 Time (days)

20

25

d

8 (µg CO2-C g-1 h-1)

Basal respiration rate

207

5

10 15 Time (days)

20

25

0

5

10

20

25

8

6

6

4

4

2

2

0

0

0

0

5

10

15

20

25

15

Time (days)

Time (days)

PC 2 (14. 1%)

Fig. 4. Incorporation of [3H]leucine (DPM · 105 g1 [dry wt] soil h1) in the DK (A) and NZ (B) soils and basal respiration (lg CO2-C g1 [dry wt] soil) in the DK (C) and NZ (D) soils. Crude PTA was added after 0, 7 and 15 days at approx. 0 lg g1(s), 29 lg g1 (·) and 105 lg g1 (m).

0. 40

4. Discussion

0. 20

4.1. Biological degradation and chemical hydrolysis of PTA in soil NZ soil

0. 00 -0 .20

DK soil -0 .40 -0 .60 -0 .80 0.60

0.70

0.80

0.90

1.00

PC1 (73.2%) Fig. 5. Principal component score plots of normalized Biolog-CLPP data. The percentage of the total variance explained by each principal component is indicated in brackets. PTA was added at approx. 0 lg g1 (•), 29 lg g1 (m) and 105 lg g1 () to the DK (filled symbols) and NZ (open symbols) soils. Error bars indicate standard deviations calculated for each principal component.

the patterns of average well color development (AWCD) were similar and unaffected by PTA addition. Principal component score plots were also unable to distinguish between PTA treatments, when using separate data input matrices for each soil site (data not shown). By contrast, the Biolog EcoPlate CLPP assay convincingly separated the bacterial communities present in DK and NZ soils (Fig. 5). Generally, bacterial communities in NZ soil supported faster color development (AWCD of 1 obtained within 2 days) and used more substrates (30 out of 31 substrates used following 7 days of incubation) than communities from the two DK soils (4 days and 24–26 substrates, respectively).

We report for the first time that microbial degradation (biodegradation) is the predominant process responsible for turnover of the Bracken toxin ptaquiloside (PTA) in Bracken-impacted soils. In DK soils incubated at 10 C at least 90% of the PTA removal was due to microbial activity, while the remainder could be explained by chemical hydrolysis. From other studies chemical hydrolysis has been shown to be important in acid soils (pH < 4) (Rasmussen et al., 2005). The low soil pH was therefore likely to affect PTA degradation in the most acidic A horizon of the DK soil (pH 3.4). Indeed, relatively high PTA removal rates were observed in the sterile samples of the A horizon, thus anticipated to represent chemical hydrolysis. By contrast, chemical hydrolysis was much slower in the lower less acidic (pH > 4) soil horizons in line with another recent study (Ayala-Luis et al., 2006). Disappearance of PTA followed first-order kinetics and rate constants were similar to those found by Rasmussen et al. (2005) for a number of sandy soils. Apart from kinetic constants defined for microbial degradation and chemical hydrolysis, data fitting to composite first-order kinetic models could explain a rapid initial disappearance of PTA due to irreversible sorption to soil particles and dissolved organic matter. In the present study, this was observed particularly in the sterile soil samples, where the initial and rapid disappearance of PTA was inferred as loss from the extractable soil pool. PTA is highly mobile in soil (Rasmussen et al., 2005) and some PTA may consequently escape both microbial degradation, chemical hydrolysis

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and irreversible sorption processes in the surface soil layers. However, the complete disappearance of PTA with increasing depth (even in the deepest C horizon) in the DK soil indicates that PTA is not lost to deeper soil layers. 4.2. Impact of PTA on soil microorganisms When PTA is degraded after amendment to the nonsterile soil (Fig. 2) the glucose moiety of PTA will be released and become available to the microorganisms. Following PTA amendments (up to about 105 lg PTA g1) the associated initial microbial degradation activity was approx. 2 lg PTA g1 h1 (assuming a rate constant of 0.0184 h1 for the DK A horizon, Fig. 2a). This activity corresponds to a conversion rate of 1.2 lg C g1 h1 for PTA, which is much higher than the basal respiration rate in the non-amended DK soil (less than 0.5 lg C g1 h1; Fig. 4c). Not all PTA degradation will undergo complete mineralization to CO2 by the respiring microorganisms, but the comparison illustrates that a stimulatory effect of PTA degradation on basal respiration may be expected. Indeed, we observed a 5-fold (maximum) stimulation of basal respiration (up to 2 lg C g1 h1) in the high-PTAamended DK soil (Fig. 4c). We interpret the increase of basal respiration rates in both the DK and NZ soils to result from a release of C substrate for further microbial metabolism associated with the PTA degradation. This is the first direct indication that PTA enhances microbial activity due to its likely role as a growth substrate for soil microorganisms. In contrast to the stimulation of actual microbial activity and growth (relatively high in DK soil, lower in the NZ soil), PTA amendment had no detectable impact on the size of the soil microbial community. Hence, the SIR assay (indicating active microbial biomass) was unaffected by PTA amendment. Several explanations for this apparent contradiction (increased microbial growth but unaffected active microbial biomass) may be proposed. Differential target populations for the leucine incorporation assay (primarily bacteria; Chin-Leo, 2002) and SIR assay (bacteria and fungi responding quickly to added glucose) is a likely explanation, but it should also be noted that we did not monitor processes responsible for removal of soil microorganisms such as predation and viral lysis. Even the very high amendment with PTA (about 120 lg g1 soil dry wt; i.e. more than 20-fold higher than the highest PTA concentration measured in the field soil samples) did not affect the microbial community size. Interestingly, these results are in contrast with a recent study, showing that the microbial community size (as measured by SIR) decreased progressively with increasing PTA exposure in a neutral (pH  7) agricultural soil, which had never before been exposed to PTA (Schmidt et al., 2005). One explanation for this observation could be that PTA or a degradation product such as the active dienone exerts a pH-dependent toxic effect on soil microorganisms (Ayala-Luis et al., 2006). Another explanation could be that the high and con-

tinuous field exposure to PTA could have promoted a community tolerance to the compound in the DK and NZ soils used for the present study. We finally concluded that also bacterial community structure (as measured by the Biolog community fingerprinting assay) was unaffected by PTA amendment in the DK and NZ soils. Given the significant stimulation of microbial activity and growth following PTA amendment, such a change might have been expected if some microorganisms were preferentially stimulated at the expense of others, but this was clearly not the case. This suggests that PTA-degrading bacteria responding by increased respiration and growth after PTA amendment were already abundant or representative of those expressing activity in the Biolog Ecoplate assay. However, the same results could actually be obtained if the PTA-degrading bacteria were unable to express activity in the Biolog Ecoplate assay. Hence, community-level physiological profiles do not necessarily reflect the full diversity of the inoculated microbial communities (Smalla et al., 1998). 4.3. Concluding remarks Our results indicate that even high doses of PTA are nontoxic to microorganisms in Bracken-impacted soils and that PTA may degrade rapidly in such soils. However, PTA is still highly mobile in soil (Rasmussen et al., 2005) and some PTA may escape microbial degradation, chemical hydrolysis or irreversible sorption in surface soil layers. Hence, there is a need for further studies in order to evaluate the risk of groundwater contamination under conditions facilitating PTA transport in soil such as preferential flow in soil pores during and after heavy rainfall. Acknowledgements The financial support of Carlsberg’s Mindelegat’s Scholarship, The Sigurd Tovborg Jensen’s Award and The Løvstrupga˚rd Foundation are gratefully acknowledged. We thank Peter Embling, Ruakura Research Center, New Zealand for his enthusiastic practical and technical assistance. References Agnew, M.P., Lauren, D.R., 1991. Determination of ptaquiloside in Bracken fern (Pteridium esculentum). J. Chromatogr. 538, 462–468. Alonso-Amelot, M.E., Avendan˜o, M., 2002. Human carcinogenesis and Bracken fern: a review of the evidence. Curr. Med. Chem. 9, 675–686. Alonso-Amelot, M.E., Castillo, U., Avendan˜o, M., Smith, B.L., Lauren, D.R., 2000. Milk as a vehicle for the transfer of ptaquiloside: a Bracken carcinogen. In: Taylor, J.A., Smith, R.T. (Eds.), Bracken Fern: Toxicity, Biology and Control. Proceedings of the International Bracken Group Conference, Manchester, 1999, Leeds. The International Bracken Group, United Kingdom, pp. 86–90. Amaral, J.A., Knowles, R., 1998. Inhibition of methane consumption in forest soils by monoterpenes. J. Chem. Ecol. 24, 723–734. Ayala-Luis, K.B., Hansen, P.B., Rasmussen, L.H., Hansen, H.C.B., 2006. Kinetics of ptaquiloside hydrolysis in aqueous solution. Environ. Toxicol. Chem. 25, 2623–2629.

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