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FEMS Microbiology Ecology 49 (2004) 71–82 www.fems-microbiology.org The role of microbial community composition and

FEMS Microbiology Ecology 49 (2004) 71–82

FEMS Microbiology Ecology 49 (2004) 71–82 www.fems-microbiology.org The role of microbial community composition and

www.fems-microbiology.org

The role of microbial community composition and groundwater chemistry in determining isoproturon degradation potential in UK aquifers

Andrew Johnson a, * , Neville Llewellyn a , Jennifer Smith a , Christopher van der Gast b , Andrew Lilley b , Andrew Singer b , Ian Thompson b

a Centre for Ecology and Hydrology Wallingford, Benson Lane, Wallingford, Oxfordshire OX10 8BB, UK b Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxfordshire OX1 3SR, UK

Received 28 November 2002; received in revised form 15 September 2003; accepted 23 March 2004

First published online 17 April 2004

Abstract

The community response of indigenous sandstone, chalk and limestone groundwater microorganisms to the addition of the commonly used herbicide isoproturon was examined. The addition of 100 lg l 1 isoproturon generally caused an increase in species diversity determined by chemotaxonomic analysis (fatty methyl ester analysis) of isolates resulting from incubation of cultures at 18 C for 4 days. Amongst the groundwater samples to which isoproturon was added, isoproturon degradation rates were correlated with increasing dominance of a few species. However, the changes in community profile associated with isoproturon degradation varied from site to site. Repeated sub-culturing with 100 lg l 1 isoproturon and sterile groundwater was carried out to examine whether this level of pesticide could exert a selection pressure, and hence stimulate more rapid degradation. Significantly increased degradation was observed in a groundwater sample from the chalk, but not in sandstone, or limestone samples. The addition of filter-sterilised sandstone groundwater to bacteria on filter paper from slow degrading limestone sites significantly improved their degrading performance. The addition of filter-sterilised limestone groundwater to the sandstone bacteria reduced their degradation rate only slightly. The data suggested that the nature of the indigenous community does influence pesticide degradation in groundwater, but that the groundwater chemistry may also play a role. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Groundwater; Isoproturon; Biodegradation; Microbial community response

1. Introduction

The Chalk, Permo-Triassic sandstone and Jurassic limestone aquifers supply approximately a third of the drinking water requirements of England and Wales in the UK [1]. Agriculture over these unconfined aquifers includes the production of winter cereal with which herbicide use is associated. Herbicides have penetrated to groundwater, albeit at low concentrations (sub

l g l 1 ), in the UK [2,3]. Despite its importance as a UK

* Corresponding

692424.

author.

Tel.:

+44-1491-838800;

fax:

+44-1491-

E-mail address: ajo@ceh.ac.uk (A. Johnson).

drinking water resource we know very little about the microbial ecology of the groundwater in the fractured rock aquifer environment. In particular, we do not know what factors influence the response of the indig- enous microorganisms of the groundwater environment to low levels of pesticide contamination. Isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea, is used frequently in Europe to suppress Blackgrass growth in winter cereals. It is the most widely used or- ganic pesticide used in the UK with approximately 3300 tonnes applied in 1997 [4]. It has been demonstrated that bacteria with the competence to degrade isoproturon can be found in chalk, limestone, sandstone and alluvial aquifers in the UK [5–8]. However, these studies have

0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.femsec.2004.03.015

72

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

shown that this potential is inconsistent, with wide variations in degradation rate occurring within the same field, and even boreholes ‘losing’ and regaining the ability to degrade isoproturon [6]. Also the degradation rates vary from negligible to fast between different aquifer types [7]. What are the limiting factors which cause this wide variation in degradation rates between different groundwater samples? A number of possibilities present themselves. Might the concentration of the pesticide, or previous exposure to high concentrations of pesticides in the groundwater influence subsequent degradation rates? Pahm and Alexander [9] observed reduced min- eralisation of p-nitrophenol at 1–5 l g l 1 compared to 100 l g l 1 . Similarly Torang et al. [10], observed differ- ences in the 2,4-D degradation rate depending on the spiking concentration in sandy aquifer samples from Denmark. Biodegradation rates achieved in microcosms from chalk groundwater samples in the UK were not correlated with previous exposure to isoproturon in the boreholes [6]. Thus, in groundwater subject to con- tamination by only very low and transient concentra- tions of pesticides, insufficient selection pressure probably exists to stimulate or sustain pesticide degrading communities. Might degradation be linked to the local ground- water chemistry? Unlike the UK, an isoproturon deg- radation potential has been absent in most Danish shallow sandy aquifers [11,12]. A possible explanation for its absence may be related to pH, since, many Danish groundwaters, appear to be more weakly acid (pH 5.3–6.4) than those mostly found in the fractured rock aquifers used for drinking water supply in the UK [13,14]. Studies into variations in isoproturon degrada- tion potential in a British soil found shorter half-lives were positively correlated with increasing pH [15]. However, as the majority of the major UK aquifers have a neutral to weakly alkaline pH, so pH alone cannot explain the inter-site differences observed in the UK [7]. If the degradation is cometabolic, due to a fortuitous similarity with a natural substrate, then concentration would be less important than the size of the existing degrading population and the presence of the cometabolite. For instance, secondary plant me- tabolites have been demonstrated to stimulate microbial degradation of xenobiotics a phenomenon referred to as analogue enrichment [16,17]. Another explanation is that differences in degradation rates are a function of the presence, or abundance of competent microbial consortia. Recent in situ studies in aquifers have revealed that continuous exposure to a mixture of herbicides (each at 40 l g l 1 ) caused accli- mation of exposed microbial communities, and the de- velopment of similar community structures [18]. Similarly, soil studies also suggest that isoproturon degradation may be dependent on subtle interactions

between different species in a microbial consortium [19]. Investigations into the impact of isoproturon on soil communities found that the composition and metabolic potential of the community can change with exposure [20,21]. For instance Variovorax, a genus that has pre- viously been implicated in the degradation of phenlyu- rea and other herbicides [22], has been reported to become more abundant. To better understand the microbial ecology and possible limiting factors associated with isoproturon degradation in groundwater the following hypotheses were tested:

(1) That the addition of isoproturon will influence the species diversity and community structure. (2) That rapid isoproturon degradation rates are associ- ated with particular changes in the microbial com- munity. (3) That rapid isoproturon degradation is related to and can be predicted by the presence of certain key species. (4) That competent degrading bacteria are universally present in groundwater samples, but that degrada- tion is stimulated by factors associated with the local groundwater chemistry. The principle method used was the incubation of fresh groundwater samples with 100 l g l 1 isoproturon. This technique has been used previously to test whether indigenous microorganisms have the competence to degrade isoproturon [5], and has revealed surprisingly variable responses in the microbial community to iso- proturon within the same field site [6]. Thus, whilst using isoproturon at the 100 l g l 1 level cannot tell us that degradation would actually occur in the natural situa- tion (usually no more than 0.3 l g l 1 [23]), it can reveal real differences in the microbial ecology between differ- ent groundwater sites.

2. Materials and methods

2.1. Sample collection from the field

In order to study the Triassic sandstone aquifer en- vironment, sites were selected on the outcrop near Mansfield in Nottinghamshire. The locations were at the Gleadthorpe farm (GT2), Welbeck, and Clumber Park, which are no more than 10 km from one another (Table 1). These sites are within the Eastern portion of the Sherwood Sandstone, where the groundwater chemistry is dominated by calcium-bicarbonate [24]. At present, around 10% of the drinking water requirement for En- gland and Wales comes from this type of aquifer. At Welbeck and Clumber Park groundwater samples were taken from a sample tap. To reduce the potential for contamination, the water samples were only collected after the sample taps had been opened for 30 min and over a 100 l discarded.

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

Table 1 Description of borehole sites used for groundwater sampling

73

Site

Site landuse

Soil type

Installation year

Borehole description

Isoproturon exposure/

 

presence?

GT2 (sandstone)

Experimental farm. Winter cereals used

Cuckney series, brown sand of slightly stony loamy sand to 0.9 m [35]

1995

Drilled to 17.6 mbs [7] with plastic casing extending to 12 mbs

Isoproturon applied in this, or adjoining field in 1996, 1998 and 2000

Clumber Park

In woodland with the nearest agriculture over 2 km away

As above

Pre-1909

7 mbs, in use to supply the estate with 67–135 m 3 drinking water per day

No measurements

(sandstone)

 

Welbeck

In a 0.25-ha wood surrounded by permanent pasture

As above

1988

90 mbs, cased out in mild steel, a sample tap in the pump house

No measurements

(sandstone)

 

Site WON (chalk)

In fields which undergo arable crop rotation

Andover series, slightly stony silty clay loam over fragmented chalk with brown soil to 0.5 mbs [36]

1991

10 mbs cased out with plastic tube

0.2–0.05 lg l 1 (1995–1998) [23]

Western Court

In fields which undergo arable crop rotation

As above

1992

15 mbs cased out with plastic tube

0.06–0.3 lg l 1 at the latter (D. Gooddy pers comm.)

(chalk)

 

Bridgets (chalk)

Permanent grassland

As above

1994

30 mbs cased out with plastic tube

No measurements

Coleby (limestone)

Beside road with sur- rounding fields being both arable and pasture

Elmton 1 series, brown, calcareous, slightly stony clay loam to san- dy clay loam to 0.3 mbs

1974

Monitoring borehole drilled to 15.5 m with a solid lining to 2 mbs

One measurement only carried out in 2000 revealed 0.04 lg l 1

 

[35]

Welbourn

Beside road, surrounded by arable farmland

As above

1976

Monitoring borehole drilled to 22.5 m with a solid lining to 3.5 mbs

No measurements

(limestone)

 

Chalk aquifers provide around 18% of the drinking water requirement for England and Wales [1]. For the chalk area, samples were taken from observation bore- holes previously installed by BGS at Site WON, Bridgets farm and Western Court which are 3 km distant from one another in Hampshire (Table 1). Limestone aquifers provide around 15% of the drinking water requirement for England and Wales [1]. For this area, samples were taken from Coleby and Welbourn (Table 1). These boreholes were within 14 km of each other. Ground- water was collected from the observation boreholes us- ing a small submersible electric pump. Five borehole volumes were pumped out and discarded before col- lecting samples in sterile bottles. Groundwater was stored (24 h maximum) at 4 C prior to use.

2.2. Site measurements

Determination of the unstable parameters pH, redox potential (Eh), dissolved oxygen (DO 2 ), alkalinity and temperature was carried out on site. Conductivity was also measured in the field using a portable conductivity bridge and meter (Mettler-Toledo, Schwerzenbach,

Switzerland). A flow through cell connected directly to the pumped supply was used for the measurement of pH, Eh (platinum electrode, Mettler-Toledo) and dis- solved oxygen (DO 2 membrane electrode and meter, Mettler-Toledo) in order to represent as closely as pos- sible the in situ conditions. Bicarbonate was measured in the field using a digital titrator (Hach, Loveland, USA). Following 0.45- l m filtration, dissolved organic carbon (DOC) was measured using a TOCsin II Aqueous Car- bon Analyser (Phase Separations Ltd, Queensferry, UK). The detection limit is 0.2 mg l 1 ( 0.1 mg l 1 ).

2.3. Degradation studies with microcosms

The microcosms were prepared by placing 12 g of matrix material previously extracted from the unsatu- rated zones 4–6 mbs of either site WON for incubation of chalk groundwater, Gleadthorpe for incubation of sandstone groundwater or Longwood (near Lincoln) for the limestone groundwater into 120 ml sterile screw-top plastic containers (Bibby-Sterilin, Stone, UK). These matrix samples had been recovered one year previously and stored in their original sealed 10 cm diameter, 50 cm

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A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

long core liners [6,7] using either dry percussion or ro- tary air flush techniques to avoid contamination with drilling fluids. The matrix samples were dried and ground to 1–4 mm diameter before use. The containers with their solid matrix components were then auto- claved at 121 C and 15 psi for 30 min. Triplicate 70 ml aliquots of the groundwater samples were added to the appropriate containers. Groundwater samples from site WON and Western Court were used to represent chalk groundwater, and GT2, Welbeck and Clumber Park to represent sandstone groundwater. Previous studies have demonstrated that in the absence of matrix material, groundwater degradation rates are minimal [5,25]. From a 150 mg l 1 isoproturon methanol stock, 3.2 ml were taken and made up to 60 ml in pure water to give an 8 mg l 1 working stock which was 0.45 l m filtered (PTFE Acrodisc, Gelman, Ann Arbor, USA). From this working stock 0.875 ml was used to spike the ground- water samples to give a final concentration of 100 l g l 1 . The controls including the relevant matrix material were autoclaved (30 min) as described above, but after the addition of groundwater and prior to spiking with the pesticide. In order to address concerns caused by trace quantities of methanol (0.06%) carried over into the microcosms and influencing the microbial community, 0.04 ml methanol was added to some 70 ml groundwater samples without isoproturon. To study the impact of isoproturon on the communities a third non-sterile treatment was left un-spiked by either isoproturon or methanol. Incubation was at 20 C and samples taken at approximately 50-day intervals over 250 days. The containers were sampled by drawing off 1.5 ml by sterile disposable pipette (polyethylene, Bibby-Sterilin, Stone, UK). Samples were then introduced to a 2-ml syringe and passed through a 0.45- lm PTFE filter (Gelman, Ann Arbor, USA) into a glass vial and stored at 4 C, prior to chemical analysis.

2.4. Microbial counts

On receipt at the laboratory and before use in the microcosms a simple viable count of the groundwater samples was carried out. This involved taking triplicate 1 ml sub-samples of groundwater which were added to 9 ml of quarter strength Ringer’s solution [26] and vor- texed for 1 min. Suspensions were serially diluted and 100 l l spread plated onto R2A (Difco, UK) amended with 50 mg l 1 cyclohexamide (Sigma, UK) to prevent fungal growth, to determine bacterial counts. The plates were incubated at 18 C for 4 days and those containing between 20 and 200 colonies counted. Counts were ex- pressed as colony forming units (cfu) per ml of groundwater. In addition samples were taken aseptically from microcosms; for the chalk groundwater samples 40 and 146 days and the sandstone samples 15 and 132 days after establishment, respectively. The microcosms were

vigorously shaken immediately before sampling to re- suspend the sediment, to assist counting of organisms both in the water and the sediment. Total cell counts were determined by microscopy using membrane filtra- tion and staining with 4 0 ,6-diamidino-2-phenylindole (DAPI) [27].

2.5. Bacterial isolate characterisation

In conjunction with the microbial count tests on the microcosms, 75 colonies were randomly selected from three replicate R2A plates (25 per plate) for each of the microcosm (3 aquifer types, 3 replicates, with and without the presence of isoproturon (850 in total). Plates with between 20 and 200 colonies after 10 days incubation and used to determine cfu, were selected for the isolation. Collected isolates were sub-cultured on TSBA to ensure purity. Isolates from the second sample (146 days for chalk and 132 days for sandstone) were characterised by analyses of the fatty acid methyl ester content (FAME) using gas chromatography [28]. In brief, cells were har- vested after 24 h incubation on TSBA at 28 C and whole cell fatty acids were saponified, methylated and extracted. FAME analysis was performed using a Hewlett–Packard HP6890 series gas chromatograph (Hewlett–Packard, Berkshire, UK) and using Microbial Identification Sys- tem (MIS) software (Microbial ID, Newark, DE). All isolates were identified using ‘‘Aerobe Library’’ version 4 (1999). In total 719 isolates were characterized by FAME, since a number of isolates were lost prior to FAME due to poor growth on TSBA.

2.6. Assessment of bacterial community structure

The diversity and structure of communities identified by FAME analysis was analysed further using three complementary indices. To do this, bacterial identifica- tions from FAME analysis were used to the species level [28]. The indices used were: (1) Species richness ðS Þ – this is simply the number of species identified in a sample. (2) Simpson’s index (L 0 ) – formally L 0 , is the probability that two isolates taken at random from a sample, will be the same species. Of the three measures this is the most sensitive to changes in the frequency of the more abundant species. (3) Shannon–Wiener index (H 0 ) – this is a combined figure that reflects the extent of diversity and the evenness of isolate distribution be- tween taxa. It is sensitive to changes in the frequency of common and less common (though not the rarer) spe- cies. These indices were calculated by:

s

Simpsons index of concentration ðL 0 Þ ¼ X

i¼1

s

n i ðn i 1Þ

NðN 1Þ

X p

i¼1

2

i

;

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

75

s

ShanonWiener index ðH 0 Þ ¼ X ðp i Þðlog e p i Þ

i¼1

(n i is the number of isolates in the ith species, s is the numbers of species in sample, p i is the proportion of species i in the sample, N is the number of individuals in the sample). The indices (species richness, Simpson’s index and Shannon–Wiener index) were adjusted using a resam- pling method [29] for a standard sample size of n ¼ 20.

2.7. Repeated enrichments in groundwater spiked with

isoproturon

An enrichment experiment with 100 l g l 1 isoprotu- ron and sterile groundwater was set up to examine whether this level of pesticide could exert a selection pressure on the microbial community, and hence stim- ulate more rapid degradation. Established microcosms used to study isoproturon degradation and between 200 and 280 days of age were used as the inoculum. For the chalk groundwater, 120-ml screw top containers were filled with 60 ml Western Court groundwater (stored at 4 C) and 12 g chalk and then autoclaved for 30 min. Isoproturon was then added to the autoclaved water to give a final concentration of 100 l g l 1 as described above, together with 10 ml from each of the replicates of the established microcosm degradation experiment (Bridgets). For the sandstone samples (GT2 and Clumber Park), this was repeated using GT2 ground- water, and for the Limestone samples Welbourn groundwater for Welbourn and Coleby were used (all groundwater previously stored at 4 C). When trans- ferred into the fresh sterile groundwater the chalk cul- tures were 170-day old, the sandstone cultures 153-day old, and limestone cultures 303-day old, respectively. Incubation and sampling followed the protocol de- scribed above. After 316 days of this new incubation, following vigorous mixing of the microcosms, 10 ml from each container was transferred to fresh sterile groundwater and sterile solid matrix as a second sub- culture. Sub-samples from the second sub-culture were transferred again to a third sub-culture after 240 days incubation. Degradation was monitored by sampling every 50 days for changes in isoproturon concentration and looking for the formation of metabolites.

2.8. Influence of groundwater type on isoproturon degra-

dation

To test the role of the groundwater chemistry in stimulating or retarding the potential of the indigenous microorganisms to degrade isoproturon, a filter switch technique was used. On the basis of previous contrasting performance the sandstone and limestone groundwaters

were compared. The sandstone samples were repre- sented by GT2 and Clumber Park, which had demon- strated previous rapid isoproturon degradation (Table 5). The limestone samples were represented by Welbo- urn and Coleby. The inoculum and groundwater used came from the second sub-culture experiment, after 240 days incubation with isoproturon. Each of these mi- crocosms were divided into two 20 ml aliquots. To separate the groundwater and indigenous microorgan- isms from one another, the samples were filtered through a sterile 0.2 l m PVDF filter (47 mm Durapore membrane filter, Millipore, Bedford, USA) held in a sterilized (autoclaved) magnetic 250 ml filter holder (Gelman, Ann Arbor, USA) with a Buchner flask. From previous tests with 14 C-labelled isoproturon, PVDF fil- ter discs had been selected as the best compromise be- tween practical filtration (not too hydrophobic) and not too sorptive of isoproturon (unlike cellulose-based fil- ters). Thus, for example a 20 ml sub-sample of one of the GT2 microcosms was 0.2 l m filtered. The moist filter paper with its retained bacteria was immediately placed aseptically into a 100 ml Sterilin pot with 7 g of sterile sandstone aquifer matrix (previously drilled from the GT borehole as described in 2.3) together with 40 ml of filter-sterilised GT2 groundwater, to act as a positive control (Fig. 1). The other 20 ml sub-sample of GT2 was similarly filtered, but in this case the filter paper con- taining the sandstone groundwater bacteria was trans- ferred to a 120 ml Sterilin pot containing 7 g sterile limestone aquifer matrix, and 40 ml filter-sterilised limestone groundwater from Welbourn. Thus, the sandstone bacteria were bathed in limestone ground- water in the presence of limestone aquifer material (from Longwood as described previously). The microcosms were then spiked as before to give a 100 l g l 1 final concentration. Similarly, the three replicates of the sandstone sample from Clumber Park second sub-cul- ture were divided and half incubated with limestone Welbourn groundwater and half placed back with its native groundwater. For the limestone bacteria of Welbourn and Coleby, these were divided and incubated either with their own groundwater or that of the sand- stone GT2. All the microcosms were then incubated at

20 C for 300 days and sampled approximately every 50

days to determine isoproturon degradation. To check the efficiency of the 0.2 l m PVDF filtration technique, 10 ml from GT2 and Clumber Park (sand- stone groundwater), and Welbourn (limestone ground- water) were passed through individual filter discs into sterile Buchner flasks. One millitres was taken from the filtrate and spread on 3% TSBA plates and incubated at

20 C for 3 days. Growth on the plates was compared

with 1 ml spread on TSBA plates taken from the original groundwater samples. In addition the effectiveness of the filtration was further examined by microscopy of DAPI- stained samples.

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A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

bacteria separated from native groundwater on filter paper Returned to native filtered groundwater Incubated with
bacteria separated
from native groundwater
on filter paper
Returned to native
filtered groundwater
Incubated with
foreign filtered
groundwater
Incubated with
foreign bacteria
(on filter paper)
0.2 µm filtration
of groundwater
sample
filtered
groundwater
Returned to native
bacteria (on filter paper)

Fig. 1. Schematic representation of experimental method associated with examining the influence of groundwater type on isoproturon degradation.

2.9. Analysis of isoproturon and metabolites

After filtration (0.2 l m) samples were stored in PTFE capped glass vials at 4 C prior to analysis. The samples were stored no longer than one week prior to analysis. All analytical standards were obtained from Sigma–Al- drich (Poole, UK). Isoproturon, isopropyl aniline, mono and didesmethyl-isoproturon were determined by high performance liquid chromatography (HPLC) with UV detection. A 150- l l sample was separated on a C18 column (Columbus from Phenomenex Ltd, UK; 250 21 mm, 5 l m) using an isocratic 35:65 acetoni- trile:water mobile phase at a flow rate of 200 l l/min. Detection was made at 240 and 220 nm, which also enabled the determination of peak identity and purity. Stock solutions were prepared in methanol with cali- bration standards and samples prepared in 50:50 meth- anol:water. The limit of detection and quantitation for these determinants was 1 and 5 lg l 1 , respectively. The appearance of any of the metabolites mentioned above was taken as confirmatory evidence of biodegra- dation having taken place [6,30,31].

3. Results

3.1. Groundwater sample characteristics

The chemical and microbiological characteristics of the groundwater samples used are shown in Table 2. All the groundwaters had dissolved oxygen at levels close to saturation, given a temperature of 9–13 C, whilst pH values were neutral to weakly alkaline (7.1–7.7) and DOC values between 0.7 and 2.8 mg l 1 . The lowest numbers of culturable bacteria were associated with the limestone groundwater samples and the highest with the sandstone. The water sampling method for all sites was

essentially the same, with borehole purging prior to sample collection. Clumber Park and Welbeck were different from the other sites in that these boreholes were equipped with their own pumps and the samples were taken from sample taps. The total amount of DOC in the groundwater and numbers of culturable organisms were not directly related to one another. Unfortunately when it came to measure the parameters at Welbourn the water level had fallen below the borehole depth, so further samples could not be taken.

3.2. The response of the indigenous microbial community to 100 l g l 1 isoproturon

Analysis of the microbial communities associated with the different treatments showed that the introduc- tion of isoproturon had no consistent impact on mi- crobial total or cultured counts either at the beginning or towards the end of the incubations. Total microbial counts decreased significantly (P < 0:05) in microcosms with chalk and sandstone from a mean 1.0 10 8 – 3.0 10 7 ml 1 after 117 d. incubation, in both spiked and un-spiked samples. The addition of 0.6% methanol alone (used as an isoproturon carrier solvent) had no detectable effect on the microbial communities (data not shown). All the groundwater samples collected from the chalk and sandstone sites degraded isoproturon and caused the formation of monodesmethyl-isoproturon (Figs. 2 and 3) that was not detected in sterile controls. The majority of the parent isoproturon was not converted to monodesmethyl-isoproturon, as has been previously observed in groundwater [6,7]. In fact with soil biodeg- radation studies the major metabolites are considered to be hydroxylated forms [30,31] which are not detectable with this eluent. To help compare the degradation rates for all the groundwater samples (Table 3), the data were

Viable count (R2A) and standard deviation from three replicates (cfu ml 1 10 3 )

Table 2 Chemical and microbial characterisation of groundwater samples investigated in this study (DO, dissolved oxygen; Eh, redox potential; SEC, electrical conductivity and DOC, dissolved organic carbon)

3.8 (0.09)

0.7 (0.3)

0.7 (0.4)

3.3 (0.2)

2.6 (0.5)

13 (0.1)

27 (7.8)

16 (1)

HCO 3 (mg l 1 )

114

176

187

328

298

273

281

301

DOC (mg l 1 )

ND

ND

1.2

1.2

0.7

2.8

2.3

1.1

SEC ( lS/cm)

1218

NC

502

590

716

517

618

951

Eh (mV)

ND

NC

444

364

147

309

415

418

* NA, information witheld; ND, carbonate prevented accurate measurement here and NC, data not collected.

DO (mg l 1 )

12.7

NC

9.4

7.0

7.6

9.8

8.8

9.1

NC

pH

7.2

7.2

7.7

7.7

7.5

7.3

7.1

Location

Pasture

Arable

Arable

Arable

Arable

Exptal

Park

Park

Watertable (1999–

15–19.5 mbs

8–12.0 mbs

8–10.0 mbs

24–26 mbs

4.5–6 mbs

5.5–7 mbs

6.8–9 mbs

7.5 mbs

2001)

Grid ref. Ordinance Survey 1:50,000

4594 3722

4518 1322

4522 1332

0153 1260

4604 3706

4622 3739

0079 6143

NA

Sandstone Welbeck Clumber Park Gleadthorpe (GT 2)

Chalk Bridgets farm no. 2 Western Court Site WON

Limestone

Welbourn

Borehole

Coleby

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

77

160 140 120 100 80 60 40 20 0 0 50 100 150 200 250
160
140
120
100
80
60
40
20
0
0
50
100
150
200
250
300
Isoproturon (µg/L)

Time (d)

Fig. 2. Degradation of isoproturon in chalk groundwater collected from Western Court (N), Western Court sterile treatment (M), Site WON (d) and Site WON sterile treatment (s) (mean of three obser- vations with standard deviations). Data are given for the metabolite monodesmethyl-isoproturon (– –) using the same site symbols (open) to represent the different treatments. Arrows indicate when micro- cosms sampled for viable counts, total counts and FAME character- ization.

120 100 80 60 40 20 0 0 30 60 90 120 150 180 210
120
100
80
60
40
20
0
0
30
60
90
120
150
180
210
240
270
Isoproturon (µg/L)

Time (d)

Fig. 3. Degradation of isoproturon in sandstone groundwater sampled from GT2 (N), Welbeck (d), Clumber Park (r) with sterile treatments (dashed lines, open symbols) (mean of three observations with stan- dard deviations). Data are given for the metabolite monodesmethyl- isoproturon (– –) using the same site symbols (open) to represent the different treatments. Arrows indicate when microcosms sampled for viable counts, total counts and FAME characterization.

entered into the Model Manager program (Cherwell Scientific, UK) to calculate the half-lives and give con- fidence levels for isoproturon in a simple first order model for the parent molecule. Whilst first order decay may not be entirely appropriate for these situations, it does provide a useful means of comparison:

M p ðtÞ ¼ M 0 expð k p tÞ;

where M p is the concentration of isoproturon, M 0 is the initial concentration of isoproturon and k p is the first order rate constant of the parent. As observed previously with studies on GT2 groundwater [7], its degradation of isoproturon was very consistent with good replication occurring (Table 3) and a half-life of around 100 d. Welbeck and Clumber Park showed greater variance between replicates with aggregate mean half-lives of 124 and 166 d. It should be noted that both these groundwater samples came from

78

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

Table 3 Isoproturon half-lives calculated as simple first order reactions for each individual replicate sample in microcosms used to determine commu- nity impact

Sample and

Half-life

95% confidence

R

2

code

(days)

range

Sandstone 24.11.99 GT2

 

SG1

106

69–143

0.95

SG2

96

89–103

0.99

SG3

97

88–106

0.99

Welbeck

SW1

181

72–287

0.87

SW2

91

64–118

0.97

SW3

102

67–138

0.96

Clumber Park

SC1

123

35–212

0.83

SC2

210

127–292

0.93

SC3

167

57–277

0.84

Chalk 10.11.99 WON4

 

CWO1

211

160–262

0.97

CWO2

239

199–277

0.99

CWO3

223

170–276

0.97

Western Court

CWC1

206

149–261

0.97

CWC2

89

25–154

0.88

CWC3

68

11–125

0.87

boreholes within parks which were a few km distant from agricultural farms. For the chalk, site WON had good replication with an aggregate half-life of 224 days (Table 3), but Western Court had some replicates showing rapid degradation of isoproturon (CWC2 and CWC3) and another slow (CWC1). Assessment by cul- ture based methods detected no consistent impact of the presence of 100 l g l 1 isoproturon on the taxa compo- sition of microbial communities isolated from chalk or sandstone groundwaters, after 146 and 132 days expo- sure, respectively. However, cluster analysis of the data grouped together all the isoproturon treated samples at 2 Euclids and likewise untreated at 2 Euclids, but sep- arated the treatments at 4.8 Euclids (data not shown), reflecting the distinctiveness of exposed communities. In most samples pseudomonad species, the most abundant group (Fig. 4), similarly showed no consistent difference between treatments in terms of the presence of specific species. However, when the data were analysed mathe- matically (Table 4) in terms of species richness (number of species) and community structure (diversity indices) differences were detected, irrespective of groundwater type. Overall, the presence of isoproturon increased species richness and diversity with SC (sandstone, Clumber Park) groundwater showing the most marked effect (H 0 increased from 1.75 to 2.07) followed by CWC (chalk, Western Court) and a more muted effect in SW (sandstone, Welbeck) groundwater (Table 4). In groundwater microcosms the isoproturon degradation

SW+

Met Pae Bre Sph 1% 3% 1% 1% Bac 1% Sta 1% 1%
Met Pae
Bre
Sph
1%
3%
1%
1%
Bac
1%
Sta
1%
1%

Pse

89%

Ace

6% Agr 7% SC+ Bac 12% Chr 1% Chy 1% Com Pse 1% 63% Fla
6%
Agr
7%
SC+
Bac
12%
Chr
1%
Chy
1%
Com
Pse
1%
63%
Fla
1%
Sph
NM
4%

1%

Aci Agr 1% 1% Azo Pse 16% 24% CWC+ NM 7% Str 1% Sta Bre
Aci
Agr
1%
1%
Azo
Pse
16%
24%
CWC+
NM
7%
Str
1%
Sta
Bre
1%
36%
Cel
Phe
Com
4%
1%
1%
Och
En
1%
1%
Cur Hyd Chr Stv 1% 1% 1% 1% NM 1% SW–
Cur Hyd
Chr
Stv
1%
1%
1%
1% NM
1%
SW–

Pse

93%

Azo Bac 3% 3% Noc 1% SC– Pse 43%
Azo
Bac
3%
3%
Noc
1%
SC–
Pse
43%

NM

19%

Aci

Sph

32%

13% Azo 1% Bre CWC– 3% Cel 1% Pse 49% Chr 18% Esc 1% Jan
13%
Azo
1%
Bre
CWC–
3%
Cel
1%
Pse
49%
Chr
18%
Esc
1%
Jan
Pho
6%
NM
Yer
3%
1%
1%

Fig. 4. Isolation frequencies of the culturable bacterial community detected in groundwaters in sandstone, Welbourn (SW) and Coleby (SC) microcosms sampled after 140 days and chalk Western Court (CWC) sampled after 130 days, with (+) or without ()) isoproturon present (mean of all three replicates). Ace, Acetobacter; Aci, Acidovo- rax; Agr, Agrobacterium; Azo, Azospirllium; Bac, Bacillus; Bre, Brev- undimonas; Cel, Cellulomonas; Chr, Chromobacterium; Chy, Chryseomonas; Com, Comamonas; Cur, Curtobacterium; En, Entero- coccus; Esc, Escherichia; Fla, Flavobacterium; Hyd, Hydrogenophaga; Jan, Janthinobacterium; Met, Methylobacterium; Noc, Nocardia; Och, Ochrobatrum; Pae, Paenibacillus; Phe, Phenylobacterium; Pho, Photo- bacterium; Pse, Pseudomonas; Sph, Sphingomonas; Sta, Staphylococcus; Str, Streptococcus; Stv, Streptoverticillium; Yer, Yersinia; and NM, No Match.

rates correlated (R 2 ¼ 0:56, P ¼ 0:009) with increased proportions of the three most numerous genera (Fig. 4). That is the presence of isoproturon increased the relative abundance of a few species that presumably were asso- ciated with increased rates of degradation. However, no consistent pattern could be detected in terms of the presence of key taxa and high rates of degradation. Thus, for example, the community profile associated with high degradation in the sandstone Welbourn site was different from the sandstone Coleby site (Fig. 5).

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

79

Table 4 Diversity measures – species richness (S ), Simpson’s index (L 0 ), and Shannon–Wiener index (H 0 ) – estimated for sandstone groundwater Welbourn (SW), and Coleby (SC) with chalk from Western Court (CWC) with (+) and without ()) isoproturon

Name

Total number

Estimated values

 

Ranking the estimated values of S , L and H 0a

of isolates

S

L 0

H 0

S

L 0

H 0

SW+

73

7

0.163

1.77

1–3

5

4

SW)

73

7

0.179

1.65

1–3

3

2

SC+

67

9

0.100

2.07

6

6

6

SC)

61

8

0.184

1.75

4–5

2

3

CWC+

65

8

0.174

1.79

4–5

4

5

CWC)

66

7

0.211

1.65

1–3

1

1

These values were standardised to a uniform sample size of 20 isolates by taking the median values from 1000 re-samplings with all replicates combined. The S and H 0 values rise with increasing diversity, while the L 0 values fall with increasing diversity. Also given are the rankings of these values. a Ranking of the estimated values of S , L 0 and H 0 is from least diverse (1) to most diverse (6).

3.3. Impact of enrichment on isoproturon degradation rates

To examine whether a more vigorous isoproturon degrading population would develop with repeated ad- ditions of the pesticide, an enrichment experiment was established. By comparing the degradation rates for the first microcosms and that after repeated sub-culturing

300 250 R 2 = 0.5507 200 150 100 50 0 30 40 50 60
300
250
R 2 = 0.5507
200
150
100
50
0
30
40
50
60
70
80
90
100
Half-Life (d)

% Dominant Genera

Fig. 5. Plot of the three most numerous genera against isoproturon degradation (half-life).

with fresh 100 lg l 1 isoproturon (Table 5), a variety of responses were seen. In some cases, degradation rates did not change, in others a transient improvement reverted to the original performance by the third sub- culture. In only one case, Bridgets, did the repeated sub- culturing lead to a putative increase in degradation rates. In this case, an original half-life began at 312 days, then dropped to 269 days on first sub-culture, 104 days on second sub-culture and finally 130 days on the final sub-culture (Table 5).

3.4. The influence of groundwater type on isoproturon degradation

In a test of the 0.2 l m filtration process, no colony growth whatsoever could be found when 1 ml of the filtrate was plated out on TSBA plates. However, tests with direct counting indicating some cell-like could pass through the filter. Using the filter technique the greatest change was detected when the normally slow degrada- tion of the limestone bacteria, from Welbourn and Co- leby (predicted half-lives 6.3 and 4.7 years, respectively), were incubated with groundwater (filter-sterilised) from the fast-degrading sandstone site GT2 (Fig. 6). In the

Table 5 Isoproturon half-lives (DT50) and standard deviation in parentheses calculated as simple first order reactions for microcosms established to de- termine the influence of repeated enrichment with 100 lg/L and sterile groundwater at 20 C

Material

Sandstone aquifer

 

Limestone aquifer

Chalk aquifer

Site

GT2

Clumber Park

Welbourn

Coleby

Bridgets

DT50 (days)

R 2

DT50 (days)

R 2

DT50 (days)

R 2

DT50 (days)

R 2

DT50 (days)

R 2

Original

71 (12)

0.98

240 (54)

0.75

759 (38)

0.89

247 (51)

0.95

312 (30)

0.99

1st sub-culture

78 (11)

0.99

112 (33)

0.96

765 (181)

0.73

247 (172)

0.87

269 (44)

0.94

(316 days)

2nd sub-culture

95 (25)

0.98

135 (77)

0.97

11,550 (350)

0.4

385 (86)

0.73

104 (10)

0.96

(240 days)

3rd sub-culture

73 (48)

0.97

266 (12)

0.92

577 (142)

0.77

552 (155)

0.70

130 (28)

0.97

(377 days)

The duration of the incubation period is given in the first column.

80

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

120 100 80 60 40 20 0 0 50 100 150 200 250 300 %
120
100
80
60
40
20
0
0
50
100
150
200
250
300
% Isoproturon remaining

Time (d)

Fig. 6. Degradation of 100 l g l 1 isoproturon by limestone Welbourn bacteria with its native groundwater (j), or filter sterilized sandstone GT groundwater ( ), similarly limestone Coleby bacteria incubated with its native groundwater (r), or filter sterilized sandstone GT groundwater (}) (mean of three observations with standard devia- tions). Data are given for the metabolite monodesmethyl-isoproturon (– –) using the same site symbols (open) to represent the different treatments.

120 100 80 60 40 20 0 0 50 100 150 200 250 300 %
120
100
80
60
40
20
0
0
50
100
150
200
250
300
% Isoproturon remaining

Time (d)

Fig. 7. Degradation of 100 l g l 1 isoproturon by sandstone GT bac- teria with its native groundwater (N), or filter sterilized limestone Welbourn groundwater (M), similarly sandstone Clumber park bac- teria incubated with its native groundwater (d), or filter sterilized limestone Welbourn groundwater (s) (mean of three observations with standard deviations). Data are given for the metabolite mo- nodesmethyl-isoproturon (– –) using the same site symbols (open) to represent the different treatments.

presence of the filter-sterilised sandstone groundwater the isoproturon degradation half-lives decreased to 161 days for Welbourn, and 187 days for Coleby, respec- tively. In contrast, when the fast degrading sandstone bacteria from GT2 and Clumber Park were incubated with filter-sterilised limestone groundwater from Wel- bourn, degradation was retarded only slightly (Fig. 7).

4. Discussion

In previous studies of the fate of isoproturon in groundwaters we demonstrated that its rate of degra- dation varied considerably, both spatially and tempo- rally [6,7]. An aim of this study was to determine the microbial basis of this observed variation and in par-

ticularly the possible correlation with microbial com- munity composition, abundance and groundwater chemistry. The results of this study demonstrated that the presence of 100 l g l 1 isoproturon increased the di- versity of the bacterial community. However, the extent to which isoproturon increased diversity at 100 l g l 1 , which is a much higher concentration than the trace levels (<1 l g l 1 ) detected in groundwaters in the UK, was small. The relatively weak selective effect of 100 l g l 1 of isoproturon suggests an even smaller likelihood that trace levels detected in groundwater in the field would select for specialist populations. The minor se- lective effect exerted by isoproturon was reflected in the response of the individual communities examined in this study, which were inconsistent in their response, re- vealing no trends in terms of preferential response of specific populations. In contrast to this general picture, repetitive sub- cultures of microbial communities from sandstone (Clumber Park) and chalk (Bridgets) led to decreased isoproturon half-lives, which in the latter case was maintained. The accelerated rates of degradation on repeated sub-culturing, observed in Bridget samples was the only exception. There is some evidence that accelerated degradation can occur in soil [32,33]. Some adaptation with another herbicide has been observed in groundwater microcosm studies containing sediment and groundwater exposed for 40 days to 30 l g l 1 phenoxy acid, the herbicide degraded more rapidly than in sediments not previously exposed [34]. This would suggest that some pesticides represent more valuable nutrient and energy sources than isoproturon, so preferentially selecting specific populations. Also it is probable that isoproturon concentrations of 0.1 l g l 1 , which more typically occur in the environment, are too low to have a significant lasting impact on exposed microbial communities, indeed in a previous study on chalk, the ability of the groundwater samples to degrade isoproturon was not correlated to previ- ous field exposure to isoproturon in their boreholes

[6,23].

The moderate and inconsistent effects of isoproturon, demonstrated in this study, suggest that the variable nature of isoproturon degradation in groundwaters are unlikely to be due solely to microbial processes, but may also be influenced by physico-chemical factors in the groundwater. The presence, or absence of key substrates might be playing a role, for instance, it has recently been demonstrated that the production of cometabolites by a bacterial strain in a consortia stimulated isoproturon breakdown by another member of the group [19]. The results of this and previous studies suggest that these controlling factors may be more complex than previ- ously suspected and certainly more subtle. This study contributes in several ways to pin-pointing the key controlling factors. We have demonstrated that:

A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82

81

The increased degradation rates, across the different groundwater microcosms, correlated with increasing dominance of the three most numerous genera. It is possible that these differences in community structure either correlated with, or generated, the classes of en- vironmental change that were important contributors to degradation rates. Despite this no consistent pat- terns in the terms of the presence or dominance of specific taxa could be correlated with degradation rates. This could be due to the fact that the popula- tions responsible for isoproturon degradation repre- sented only a small proportion of the total community, as well as the limits of the detection methods used.

Isoproturon exposure increased the extent of commu- nity diversity, but had no detectable effect on micro- bial counts.

Isoproturon degradation was detected in microbial communities that were very different in composition suggesting that the genes for degradation are widely distributed throughout the community.

Only in one case could isoproturon degradation rates be stimulated by enrichment using 100 lg l 1 isopro- turon. Given the absence of correlation of isoprotu- ron degrading potential related to previous exposure in groundwater noted in previous work on chalk [6], these data support the theory that fast de- grading microbial communities in groundwater are unlikely to occur as a result of continued exposure to isoproturon in the field.

Isoproturon degradation rates by the indigenous mi- crobial community could be improved or retarded ac- cording to the bathing groundwater, indicating a possible role of the groundwater chemistry in mediat- ing degradation rates.

Acknowledgements

This study was funded by the NERC CEH Integrat- ing fund. The authors thank Sarah Harman, Andy Dixon and Janice Trafford for technical support and Daren Gooddy for helpful information.

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