FEMS Microbiology Ecology 49 (2004) 71–82

www.fems-microbiology.org

The role of microbial community composition and

groundwater chemistry in determining isoproturon
degradation potential in UK aquifers
a,*
Andrew Johnson , 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 drinking water resource we know very little about the

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
lg l
1 ), in the UK [2,3]. Despite its importance as a UK
*
Corresponding author. Tel.: +44-1491-838800; fax: +44-1491-
692424.
E-mail address: ajo@ceh.ac.uk (A. Johnson).
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 lg l
1 compared to
100 lg l
1 . Similarly Tor€
ang 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 lg 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 lg 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 lg l
1 level cannot tell us that
degradation would actually occur in the natural situa-
tion (usually no more than 0.3 lg 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 73

Table 1
Description of borehole sites used for groundwater sampling
Site Site landuse Soil type Installation year Borehole description Isoproturon exposure/
presence?
GT2 (sandstone) Experimental farm. Cuckney series, brown 1995 Drilled to 17.6 mbs [7] Isoproturon applied in
Winter cereals used sand of slightly stony with plastic casing this, or adjoining field in
loamy sand to 0.9 m [35] extending to 12 mbs 1996, 1998 and 2000

Clumber Park In woodland with the As above Pre-1909 7 mbs, in use to supply No measurements
(sandstone) nearest agriculture over the estate with 67–135
2 km away m3 drinking water per
day
Welbeck In a 0.25-ha wood As above 1988 90 mbs, cased out in No measurements
(sandstone) surrounded by mild steel, a sample tap
permanent pasture in the pump house

Site WON (chalk) In fields which undergo Andover series, slightly 1991 10 mbs cased out with 0.2–0.05 lg l
1
arable crop rotation stony silty clay loam plastic tube (1995–1998) [23]
over fragmented chalk
with brown soil to 0.5
mbs [36]

Western Court In fields which undergo As above 1992 15 mbs cased out with 0.06–0.3 lg l
1 at the
(chalk) arable crop rotation plastic tube latter (D. Gooddy pers
comm.)
Bridgets (chalk) Permanent grassland As above 1994 30 mbs cased out with No measurements
plastic tube
Coleby (limestone) Beside road with sur- Elmton 1 series, brown, 1974 Monitoring borehole One measurement only
rounding fields being calcareous, slightly drilled to 15.5 m with a carried out in 2000
both arable and pasture stony clay loam to san- solid lining to 2 mbs revealed 0.04 lg l
1
dy clay loam to 0.3 mbs
[35]
Welbourn Beside road, surrounded As above 1976 Monitoring borehole No measurements
(limestone) by arable farmland drilled to 22.5 m with a
solid lining to 3.5 mbs

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 (DO2 ), 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 (DO2 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-lm 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
74 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 lm 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 lg 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 ll 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 40 ,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 (L0 ) – formally L0 , 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:
Xs
ni ðni
1Þ
Simpson’s index of concentration ðL0 Þ ¼
i¼1
N ðN
1Þ
Xs
 pi2 ;
i¼1
 A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82
X
s
Shanon–Wiener index ðH 0 Þ ¼ ðpi Þðloge pi Þ
i¼1
(ni is the number of isolates in the ith species, s is the
numbers of species in sample, pi 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 lg 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 lg 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
75
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 lm 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 lm 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 lg 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 lm 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.
76 A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82
0.2 µm filtration
of groundwater
sample
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 lm) 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-ll sample was separated on a C18
column (Columbus from Phenomenex Ltd, UK;
250  21 mm, 5 lm) using an isocratic 35:65 acetoni-
trile:water mobile phase at a flow rate of 200 ll/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
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)
filtered
groundwater
Returned to native
bacteria (on filter paper)
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 lg 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  108 –
3.0  107 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
 A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82 77

Viable count (R2A) and

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

standard deviation from

160
140

three replicates (cfu

Isoproturon (µg/L)
120
100
ml
1  103 ) 80

3.8 (0.09)
2.6 (0.5)

3.3 (0.2)

0.7 (0.3)
0.7 (0.4)
13 (0.1)

27 (7.8)
60

16 (1)
40
20
0
(mg l
1 )
HCO3

0 50 100 150 200 250 300

301
298
281

176
114
187

328
273
Time (d)
DOC (mg l
1 )

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
ND
ND
1.2

0.7
1.2
2.3

1.1
2.8 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-
SEC (lS/cm)

ization.
1218

NC
618
590
716

502
517

951

120

100
Isoproturon (µg/L)
Eh (mV)

80
ND

NC
309
364

415
418
444

147
NA, information witheld; ND, carbonate prevented accurate measurement here and NC, data not collected.

60

40
DO (mg l
1 )

20

0
12.7

0 30 60 90 120 150 180 210 240 270

NC
7.6

9.1

7.0
8.8
9.4

9.8

Time (d)

Fig. 3. Degradation of isoproturon in sandstone groundwater sampled

NC
pH

7.2
7.1
7.2

7.7
7.7
7.5

7.3

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-
Location

Pasture
Arable
Arable

Arable
Arable
Exptal

isoproturon (––) using the same site symbols (open) to represent the
Park
Park

different treatments. Arrows indicate when microcosms sampled for

viable counts, total counts and FAME characterization.
Watertable (1999–

entered into the Model Manager program (Cherwell

15–19.5 mbs
8–12.0 mbs
8–10.0 mbs
24–26 mbs
6.8–9 mbs
4.5–6 mbs

5.5–7 mbs

Scientific, UK) to calculate the half-lives and give con-

7.5 mbs

fidence levels for isoproturon in a simple first order

2001)

model for the parent molecule. Whilst first order decay

may not be entirely appropriate for these situations, it
Grid ref. Ordinance

does provide a useful means of comparison:

Survey 1:50,000

Mp ðtÞ ¼ M0 expð
kp tÞ;

4522 1332
4518 1322

4594 3722
4622 3739
4604 3706

0153 1260
0079 6143

where Mp is the concentration of isoproturon, M0 is the

initial concentration of isoproturon and kp is the first
NA

order rate constant of the parent.

As observed previously with studies on GT2
Gleadthorpe (GT 2)
Bridgets farm no. 2

groundwater [7], its degradation of isoproturon was very

Western Court

Clumber Park

consistent with good replication occurring (Table 3) and

Site WON

a half-life of around 100 d. Welbeck and Clumber Park

Limestone
Welbourn
Sandstone
Borehole

Welbeck

Coleby

showed greater variance between replicates with

Chalk
carbon)
Table 2

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 Bre Met Pae Sph

1% 3%
Isoproturon half-lives calculated as simple first order reactions for each Bac 1% 1% Cur Hyd
Chr 1% 1% Stv
1% Sta
individual replicate sample in microcosms used to determine commu- 1%
1% 1% NM
nity impact 1%
1%

SW+ SW–
Sample and Half-life 95% confidence R2
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 Pse
Pse
89%
93%
Welbeck
Azo Bac
SW1 181 72–287 0.87 Ace
3% 3%
6% Agr Noc
SW2 91 64–118 0.97 7% 1%
SW3 102 67–138 0.96 SC+ SC–
Bac
12% Pse
Chr
Clumber Park 1% 43%
Sph
32%
SC1 123 35–212 0.83 Chy
1%
SC2 210 127–292 0.93 Com
Pse
SC3 167 57–277 0.84 63% Fla
1%

1%
Sph
Chalk 10.11.99 WON4 NM 4%
NM
CWO1 211 160–262 0.97 1%
19%
CWO2 239 199–277 0.99
CWO3 223 170–276 0.97 Aci
Agr
1% Aci
1%
Azo 13% Azo
Western Court Pse
16%
24% 1% Bre
CWC1 206 149–261 0.97
CWC+ CWC– 3%
CWC2 89 25–154 0.88 Cel
1%
CWC3 68 11–125 0.87 Pse
NM
49%
7% Chr
Str 18%
1%
Esc
Sta Bre 1%
boreholes within parks which were a few km distant 1% 36% Jan
Cel Pho 6%
from agricultural farms. For the chalk, site WON had Phe
1%
Com 4%
1%
NM
1%
Yer 3%
1%
good replication with an aggregate half-life of 224 days Och
1%
En
1%

(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 lg 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
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 (R2 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 (L0 ), 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 L0 H0 S L0 H0
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 L0 values fall with increasing diversity. Also given are the rankings of these
values.
a
Ranking of the estimated values of S  , L0 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
Half-Life (d)
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
R2 = 0.5507 degradation

200
In a test of the 0.2 lm filtration process, no colony
150
growth whatsoever could be found when 1 ml of the
100 filtrate was plated out on TSBA plates. However, tests
50 with direct counting indicating some cell-like could pass
through the filter. Using the filter technique the greatest
0
change was detected when the normally slow degrada-
30 40 50 60 70 80 90 100
tion of the limestone bacteria, from Welbourn and Co-
% Dominant Genera
leby (predicted half-lives 6.3 and 4.7 years, respectively),
Fig. 5. Plot of the three most numerous genera against isoproturon were incubated with groundwater (filter-sterilised) from
degradation (half-life). 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
2 2 2 2
DT50 (days) R DT50 (days) R DT50 (days) R DT50 (days) R DT50 (days) R2
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
120
% Isoproturon remaining
100
80
60
40
20
0 was small. The relatively weak selective effect of 100
0 50
Fig. 6. Degradation of 100 lg 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
% Isoproturon remaining
100
80
60
40
20
0
0 50
Fig. 7. Degradation of 100 lg 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-
A. Johnson et al. / FEMS Microbiology Ecology 49 (2004) 71–82
ticularly the possible correlation with microbial com-
munity composition, abundance and groundwater
chemistry. The results of this study demonstrated that
the presence of 100 lg l
1 isoproturon increased the di-
versity of the bacterial community. However, the extent
to which isoproturon increased diversity at 100 lg l
1 ,
which is a much higher concentration than the trace
levels (<1 lg l
1 ) detected in groundwaters in the UK,
100 150 200 250 300 lg l
1 of isoproturon suggests an even smaller likelihood
Time (d) 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 lg l
1
phenoxy acid, the herbicide degraded more rapidly
100 150 200 250 300 than in sediments not previously exposed [34]. This
Time (d) 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
lg 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
• 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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