Land Contamination & Reclamation, 9 (4), 2001 © 2001 EPP Publications

DOI 10.2462/09670513.600

In situ bioremediation of groundwater

contaminated with phenols, BTEX and
PAHs using nitrate as electron acceptor
Richard J.F. Bewley and Graham Webb

Abstract
This paper describes the implementation of an in situ bioremediation scheme to
address historical groundwater contamination at the site of a former oil works, now
occupied by an operating facility manufacturing bitumen-related products. Site investi-
gations had indicated that groundwater beneath the facility was contaminated with up to
20 600 µg/L phenols, 169 µg/L polyaromatic hydrocarbons (PAHs) and 440 µg/L ben-
zene, toluene, ethylbenzene and xylenes (BTEX). The site was mostly covered by
hardstanding so that the key risks posed by the contamination were to an adjacent river
located 50 m from the site boundary. The remedial scheme involved the installation of
four abstraction wells located towards the site boundary with the river from which
groundwater was abstracted at a typical rate of 40–50 m3/day and conveyed via reticu-
lation pipework to two sets of three reinjection wells located hydraulically upgradient of
the source area. Prior to reinjection, the groundwater was supplemented with a continu-
ous source of sodium nitrate as an oxygen source, together with nutrients, these being
supplied in the form of a commercial mixture of urea and diammonium phosphate. A
commercial mixture of phenol-degrading bacteria, PHENOBAC, was also applied peri-
odically in batch mode as point source injections to the most contaminated area, and to
the reinjected groundwater. Over the 2.5 years that the scheme was operating, mean
concentrations of phenols were reduced from 1100 µg/L at the start to 12 µg/L, PAHs
from 11 µg/L to 0.9 µg/L and BTEX from 58 µg/L to 19 µg/L. Following termination of
the scheme, ongoing monitoring of the groundwater indicated an initial increase in con-
centrations assumed to be due to ‘bounce-back’, followed by a further decline. Mean
concentrations of contaminants two years after termination were 93 µg/L phenols,
0.7 µg/L PAHs and 11 µg/L BTEX. Monitoring of groundwater indicated a general
increase in viable counts of bacteria, declining after termination, and a transient
increase in nitrite, indicative of biological dentifrication.

Key words: BTEX, in situ bioremediation, nitrate, PAHs, phenols

INTRODUCTION AND SITE DESCRIPTION
This paper presents the results of a groundwater reme-
diation programme undertaken at an operating facility
Received March 2001; accepted June 2001
Authors
Richard J.F. Bewley1 and Graham Webb2
1. Dames & Moore, 5th Floor, Blackfriars House, St. Mary’s
Parsonage, Manchester M3 2JA, UK (author for correspond-
ence).
2. Dames & Moore, Iveagh Court, 4th Floor, 6-8 Harcourt
Road, Dublin 2, Ireland
in the UK which currently produces a range of flooring,
damp proofing and roofing materials based on bitumen
and synthetic resins. The site is located on an industrial
estate in the north-west Midlands between a railway
line to the south and a major river approximately 50 m
to the north.
At the beginning of the 20th century, oil shale
extraction took place in the area. According to histori-
cal maps an ‘oil works’ formerly occupied the present
site and on a 1: 2600 Ordnance Survey map dated 1934,
the presence of a gasometer indicated potential coal
gasification processes. Prior to its current use part of the
site had also been occupied by a rail wagon repair yard.

335
 Land Contamination & Reclamation / Volume 9 / Number 4 / 2001

A series of investigations concerning soil and
groundwater quality within the site boundary were fol-
lowed by some localised remediation of soil contami-
nated by volatile organic compounds in an area
adjacent to a Low Flash products building. This
involved excavation and disposal of contaminated soil
to a licensed landfill, reinstatement with clean fill and
construction of protective measures for underlying soil.
An additional area of excavation and disposal involved
a former railway sleeper pit and adjacent contaminated
soil which was also removed to a licensed landfill, and
reinstated with clean granular fill.
Whilst these areas were both localised in nature and
accessible for soil remediation, much of the remainder
of the site was covered by hardstanding and buildings.
A series of boreholes were excavated over the site area,
to evaluate soil and groundwater quality, as shown in
Figure 1.
These indicated that the geological succession
beneath the site consisted of granular fill materials
overlying a thin natural clay, underlain by a succession
of fine to medium-grained sands. These were further
underlain by glacial clays as proved in borehole 401, on
the northern boundary.
In summary, the following sequence was inferred:
(1) Fill: predominantly granular but with some clay
and silt content up to 1.8 m thick;
(2) Clay: soft, firm and stiff, silty and occasionally
described as silt up to 1.2 m thick with a base at 2.0
– 2.8 m depth;
(3) Sand: loose to medium dense, black, very silty and
fine-grained becoming light grey, silty and fine –
medium and occasionally coarse-grained below
7 m and red/brown below 11 m, having a thickness
of approximately 10 m and a depth to base of
12.3 m (in BH401 on the northern boundary (Fig-
ure 1);
(4) Clay: stiff red brown, silty, slightly sandy and
gravelly, thickness or depth not proved but the lat-
ter is in excess of 13.5 m.

R I V E R

SEP 93 NOV 93 NOV 92 DEC 93 3.5-7.0m 9.2-12.0m

PHENOLS 815µg/l 7090µg/l PHENOLS NA PHENOLS 20600µg/l 1660µg/l DEC 92
PAHs 37µg/l 45µg/l PAHs NA PAHs 24µg/l 26µg/l PHENOLS 5µg/l
BTEX 440µg/l 137µg/l BTEX 4.5µg/l BTEX 305µg/l 60g/l PAHs 68µg/l
305
BTEX NA

105
401
304
SEP 93 NOV 93
202
PHENOLS 502µg/l 5930µg/l
PAHs 146µg/l 169µg/l
BTEX 88µg/l 55g/l
SEP 93
PHENOLS 2.3µg/l
PAHs 6.4µg/l
203 BTEX 0.3µg/l

DEC 92
PHENOLS 10300µg/l
PAHs 140µg/l 301
BTEX NA

SEP 93 NOV 93
PHENOLS 3.3µg/l 0.08µg/l
PAHs 1.8µg/l 4.7µg/l
302 BTEX 1.9µg/l 1.5µg/l
204
303
KEY
PHASE I SAMPLING LOCATIONS (GRAB SAMPLES)

250mm GROUNDWATER MONITORING WELL

DEC 92 150mm GROUNDWATER MONITORING WELL
DEC 92
PHENOLS 1357µg/l
PHENOLS 0.2µg/l
PAHs 3.9µg/l 75mm GROUNDWATER MONITORING WELL
PAHs 0.6µg/l
BTEX NA
BTEX 2.3µg/l 34mm GROUNDWATER MONITORING WELL

PAHs POLYAROMATIC HYDROCARBONS (16 USEPA LISTED)

BTEX BENZENE, TOLUENE, ETHYLBENZENE, XYLENES

USEPA TARGET COMPOUNDS

0 10 20 30 40 50m
NA NOT ANALYSED
APPROXIMATE SCALE

Figure 1. Site plan showing concentrations of key contaminants detected in groundwater

336
 In situ bioremediation of groundwater contaminated with phenols, BTEX and PAHs using nitrate as electron acceptor
Generally, the fill materials were found to be dry
and groundwater was encountered at shallow (2 – 3
metres) depth within the superficial sands. The sands
beneath the site formed a shallow aquifer, believed to
be in hydraulic continuity with the river, the bed level
of which was reportedly at 2 m AOD. The river was
tidal adjacent to the site with a tidal range of approxi-
mately 5 m. Groundwater levels at the site were mar-
ginally above mean water levels in the river and
inferred a net flow of groundwater towards the river. A
hydraulic gradient of 1:260 was estimated.
The soil and groundwater investigations identified
the occurrence of significant contamination in the
groundwater, specifically arising from benzene, tolu-
ene, ethylbenzene and o-, m-, and p-xylenes (BTEX),
phenols, particularly phenol itself, cresols (methylphe-
nols) and xylenols (dimethylphenols), and polyaro-
matic hydrocarbons (PAHs).
Concentrations of the above determinands identi-
fied in the groundwater during the various phases of
investigation are depicted on Figure 1.
On the basis that off-site migration of such contami-
nation could potentially impact the adjacent river, the
site owners retained Dames & Moore to design and
implement a volunteered remedial strategy with the
objective of minimising the potential risk to this recep-
tor.
SELECTION OF REMEDIAL APPROACH
Whereas potentially historic soil contamination existed
under the operational areas and other covered parts of
the site (as confirmed by the soil investigations), it
would not have been possible to have addressed this
without significant demolition of above ground struc-
tures and disruption of site activities. However,
because the majority of the site area was covered by
buildings and hardstanding then the amount of infiltra-
tion over such an area (with the potential to leach con-
tamination into the groundwater) was likely to be very
limited. The focus of the remedial scheme was there-
fore to address contamination in the saturated zone.
A review of potential remedial options was under-
taken and it was considered that the best practicable
approach involved in situ bioremediation. This was
based on the fact that the contamination was present as
dissolved rather than free phase. Also, given the poten-
tial for sorbed phase to be present it was considered that
an in situ approach offered significant advantages over
pump-and-treat, especially given the relatively
fine-grained nature of the aquifer. The fine-grained
nature of the aquifer also indicated that a passive in situ
approach would be of very limited effectiveness due to
the reduced radius of influence of dosing wells. A recir-
337
culatory approach was therefore selected as represent-
ing the most appropriate method of introducing
nutrients and an electron acceptor into the groundwater
to enhance in situ microbial activity. However, whereas
hydrogen peroxide had previously been used in treat-
ment of a sandy gravel aquifer (Bewley et al. 2001), the
prolonged travel time and assumed half-life of this rea-
gent were considered to represent serious limitations at
this site.
Nitrate, on the other hand, represented a soluble
electron acceptor (when present as the sodium or potas-
sium salt for example) that could be injected in poten-
tially high quantities into the aquifer. Under anaerobic
conditions, nitrate will undergo denitrification, with
reduction to gaseous oxides of nitrogen and nitrogen
gas. Additionally, it has been shown (primarily through
laboratory microcosms or studies of intrinsic biodegra-
dation) to be suitable as an electron acceptor during the
degradation of aromatics such as phenols (e.g. Bossert
and Young 1986; Lerner et al. 2000), naphthalene
(Al-Bashir et al. 1990) and BTEX compounds,
although in the case of the latter, the evidence for ben-
zene degradation has often been negative (Häner et al.
1995; Krumholz et al. 1996; Bewley 1996).
The nitrate reduction process for phenol can be sum-
marised thus:
28 28 + 14 29
C 6 H 6 O + ------ NO 3 + ------ H → 6CO 2 + ------ N 2 + ------ H 2 O
5 5 5 5
Reports of nitrate having been used in active in situ
bioremediation projects have largely been confined to
clean-up of BTEX and petroleum hydrocarbons (Bat-
termann et al. 1993; Hutchins et al. 1991; Werner
1985) although there is also a report of its use in treat-
ment of pentachlorophenol (Campbell et al. 1989).
The proposed scheme therefore involved abstract-
ing groundwater from an area hydraulically down-gra-
dient of the site, supplementing with nutrients and
nitrate as an oxygen source and re-injecting the
amended groundwater up-gradient of the contaminated
area. The nitrate as oxygenating agent and nutrients
would then accelerate the natural biodegradation proc-
esses taking place within the saturated zone (normally
limited by sub-optimal levels of these factors). Dia-
grammatic representation of the scheme is provided in
Figure 2.
The feasibility of using bioremediation was con-
firmed by a bench-scale laboratory test. This demon-
strated that in a sample of groundwater from the most
contaminated area, concentrations of the key contami-
nant groupings (BTEX, phenols and PAHs) could be
reduced to below 50 µg/L, albeit using an inoculum
and under aerobic conditions. A constant discharge
(pump) test was also undertaken to assess the hydraulic
 Land Contamination & Reclamation / Volume 9 / Number 4 / 2001

characteristics of the fine sand aquifer beneath the site.
Groundwater computer modelling was then carried out
to optimise the number and locations of abstraction and
re-injection wells, together with appropriate abstrac-
tion and re-injection rates.
The target concentrations for switch-off of the
scheme were:
(i) for phenols (as the sum of phenol, cresols and
xylenols on the US EPA Target List, i.e. phe-
nol, 2-methylphenol (o-cresol), 4-methylphe-
nol (p-cresol) and 2,4-dimethylphenol
(2,4-xylenol)) not to exceed 100 µg/L;
(ii) for BTEX, with benzenes, toluene, ethylben-
zene and each xylene isomer not to exceed
20, 50, 60 and 60 µg/L respectively and not
to exceed 100 µg/L;
(iii) for polyaromatic hydrocarbons (PAHs) as the
sum of 16 US EPA target list priority pollut-
ant compounds not to exceed 75 µg/L.
These targets were based on what was considered
practicably achievable from the bench-scale testing
and were broadly consistent with the Dutch guidelines
at the time, where applicable.

PLAN

R I V E R

NUTRIENT/NO3 RETICULATION
INJECTION POINT PIPEWORK

AW401 AW404
AW402
AW403

IW407
IW406
KEY
IW405
ABSTRACTION BOREHOLE (AW)
INJECTION BOREHOLE (IW)
DIRECTION OF INOCULUM INJECTION WELLS
GROUNDWATER BH302
BH303 FLOW MONITORING WELLS (BH)
IW410

IW408 0 20 40 60 80 100m
IW409 APPROXIMATE SCALE

SECTION 12m DEPTH OXYGEN

9m DEPTH
ABSTRACTION INOCULUM NUTRIENTS SOURCE: INJECTION
WELLS (AW) NITRATE WELL (IW)

NORTH SITE RETICULATION PIPEWORK

BOUNDARY
CONCRETE
RIVER CLAY
FILL
SAND
? CLAY CLAY
? ? SANDY CLAY
MEAN FINE WITH FILL
WATER SAND
LEVEL MEDIUM
SAND
DYNAMIC
GROUNDWATER
LEVEL
ZONE OF IN-SITU SAND BECOMING
BIOREMEDIATION COARSER

SUBMERSIBLE
PUMP

CLACIAL CLAY

Figure 2. Diagrammatic representation of remedial scheme

338
 In situ bioremediation of groundwater contaminated with phenols, BTEX and PAHs using nitrate as electron acceptor
Following termination of the active recirculatory
scheme, a monitoring period of up to two years was to
be carried out to take account of potential
‘bounce-back’ and intrinsic biodegradation.
METHODOLOGY
Installation of remediation system
A schematic of the remediation system is shown in Fig-
ure 2.
A series of four abstraction wells (AW401 –
AW404) were located along the down-gradient bound-
ary of the site. Each well was installed to the base of the
aquifer, a depth of approximately 12 m below ground
level and comprised 150 mm diameter well casing
screened from 2 m to 11 m below ground level,
installed within a 300 mm diameter borehole. The
annulus between the borehole wall and well casing was
filled with a sand filter pack across the screened sec-
tions of well casing, with a bentonite grout seal
installed above the filter pack. A submersible borehole
pump was installed within each of the four wells. For
each well location, a brickwork chamber was con-
structed to a depth of l m to house a flow control valve
and a sampling tap to enable collection of groundwater
samples directly from the rising main. A rising main
network comprising 75 mm diameter PVC pipe com-
bined the flows from the four abstraction wells into a
single discharge pipeline. Connected to the latter was a
nutrient dosing facility that enabled addition of nutri-
ents from a 500-litre mixing tank and pump (in batch
form), and continuous dosing of sodium nitrate and
Purisol 100 from 1000-litre IBCs.
The nutrient dosing facility also housed the electri-
cal supply and control panel for each of the abstraction
well pumps and dosing pumps. Discharge pipework led
from the facility to a series of six re-injection wells,
located as shown on Figure 2. These injection wells
were installed to depths varying from 9 to 11 m bgl.
Each hole was drilled at 460 mm nominal diameter and
single wall, screened PVC well casing of 250 mm nom-
inal diameter was installed from the base of each well
to 2 m bgl. Blank casing was installed above the
screened section, from ground level to 2 m bgl. A filter
pack was installed from the base of each well across the
screened section with a bentonite seal from 0.5 m to
2 m bgl.
The installation of three of the abstraction wells
(AW402, AW403 and AW404) and the six injection
wells (IW405 to IW410) was undertaken between 15
August 1994 and 16 September 1994. A fourth abstrac-
tion well, AW401, had been installed in November
1993 during a previous phase of work at the site. The
remaining components of the remediation system were
339
installed between 5 September 1994 and 30 September
1994.
Following initial pump testing of the abstraction
wells commencing during the week of 6 October 1994
(week 1) it was found that unacceptable amounts of silt
were migrating from the aquifer, through the well con-
struction and into the system pipework. To mitigate this
problem, each abstraction well was lined with a
100 mm nominal diameter well screen wrapped with an
80 micron nominal pore size geotextile. This work was
undertaken during week 3. Siltation problems were
also encountered within the injection wells, due to
materials in the aquifer being more fine-grained and
looser than expected. As a result of the siltation, it was
found during initial testing of the remediation system
that a number of the injection wells were unable to
accept the design flows from the abstraction wells. To
reduce this problem, and hence to increase the flow
capacity of the injection wells, each well was lined with
150 mm casing and the resulting annulus was filled
with a finer grained filter pack. This work was under-
taken during weeks 8 to 9.
In addition to the wells utilised in the abstrac-
tion/re-injection system, four inoculum injection wells
were installed in the vicinity of well AW401, using
‘window sampling’ equipment. The wells were drilled
at 75 mm nominal diameter to a depth of 4 m bgl. A
piezometer comprising 38 mm PVC screen, a sur-
rounding filter pack and a bentonite seal was installed
at each location. Stop-cock covers were installed at
each location to protect the installations. Monitoring
boreholes (BH302 and BH303) had previously been
installed on site in September 1993 as part of the inves-
tigation works. The boreholes had been advanced to
between 3 and 4 m depth with the installation of
150 mm and 75 mm ID piezometers in BH302 and
BH303 respectively.
Commissioning and operation
Commissioning trials of the remediation system
included stepped discharge pump tests and a constant
discharge pump test which were performed during
weeks 9 to 10. The pumping trials indicated the follow-
ing operational characteristics of the system:
(i) the maximum potential abstraction rate from
any one abstraction well was of the order of 4
– 5 m3/hr;
(ii) the maximum potential injection rate into any
one of the injection wells was of the order of
0.6 m3/hr (wells 408, 409, 410) to 0.8 m3/hr
(wells 405, 406, 407);
(iii) the results of the test indicated a steady-state
draw-down of the water table at the abstrac-
tion wells of the order of 1.0 – 1.5 m at the
 Land Contamination & Reclamation / Volume 9 / Number 4 / 2001

Table 1. Nutrient and nitrate addition to groundwater prior to re-injection

Date Week Methoda Urea Dipotassium Magnesium Sodium Purisol

(kg) phosphate sulphate nitrate 100e (T)
(kg) (kg) (36%) (T)
b
19/12/94 11 B 100 25 10 – –
21/12/94 11 B 200 50 20 – –
04/01/95 13 B 200 50 20 – –
d
06/01/95 13 B 200 50 20 – –
c
19/12/94 11 C – – – 24 –
09/01/95 13 C – – – – 5

Key
(a) Method of addition as indicated.
(b) Batch addition made on date shown.
(c) Continuous flow commencing on date shown (Purisol 100 added on alternate weeks from April 1995).
(d) On this occasion constituents added to inoculum injection wells, not reinjected groundwater.
(e) Commercial mixture consisting of urea and diammonium phosphate supplied by ICI Chance & Hunt.

final flow rate, and an increase in water levels
at the injection wells of approximately 0.5 m
for the southern three injection wells 408,
409 and 410. The increase in water table lev-
els in the vicinity of the northern three wells
405, 406 and 407 was estimated to be of the
order of 0.8 m;
(iv) hydraulic conductivity of the aquifer was of
the order of 1 m/day and total abstraction
(and re-injection) rate was of the order of 100
m3/day.
Initial testing of the groundwater re-circulation sys-
tem took place in October 1994. Groundwater abstrac-
tion and re-injection commenced in December 1994
and continued to be operated until August 1997 (week
148). Following completion in August 1997, periodic
monitoring of the groundwater continued through
1999.
The groundwater re-circulation system was mostly
run for 24 hours per day with abstraction taking place
from up to four wells at a time, although this was later
reduced to three wells from early 1995. Groundwater
abstraction was subsequently concentrated at wells
AW403 and AW401, with groundwater abstraction
from well AW403 only, during 1997. Fouling of the
re-injection wells significantly reduced their efficiency
so that the typical groundwater abstraction rate was
approximately 40–50m3/day over the course of the
treatment, significantly below that during the pumping
trials.
Due to this build-up of slime within the screen of the
re-injection wells, it was necessary to shut down the
system temporarily for a period of three weeks during
March 1995 (weeks 22 to 25) whilst the wells were
treated with concentrated hydrogen peroxide. The
slime was considered to have originated from rapid
build-up of microbial cells within the screen owing to
the favourable conditions for microbial activity (i.e.
presence of nutrients in a relatively aerobic environ-
ment).
The process was repeated during July 1995 (weeks
41 to 42) and subsequently at two to three month inter-
vals.
Nutrient and inoculum application
Application of nutrients (as batch additions of urea,
dipotassium phosphate and magnesium sulphate)
together with sodium nitrate (as oxygen source) com-
menced on 19 December 1994 following completion of
the pump tests.
The purpose of the initial nutrient addition was to
provide a readily available source of nitrogen, phos-
phorus and potassium at the same time as the inoculum
was added. This would primarily provide the inocu-
lated bacteria with a source of nutrients as they were
transported through the zone of contamination by the
re-circulated groundwater. To avoid too rapid micro-
bial growth (which would produce a high oxygen
demand and exacerbate pore blockage from slime for-
mation) the nutrients were added in four batches during
weeks 11 to 13, as shown in Table 1.
For ongoing supply of nitrogen and phosphorus,
Purisol 100, a commercial mixture of urea and ammo-
nium phosphates, supplied by ICI Chance & Hunt, was
added following completion of the batch additions. The
Purisol 100 and the 36% sodium nitrate solution were
each injected as a continuous feed into the discharge
pipeline using peristaltic pumps. Both these supple-
ments were stored in separate 1 m3 IBC containers. As
from April 1995, the Purisol 100 was added every alter-
nate week, rather than continuously, to reduce accumu-

340
 In situ bioremediation of groundwater contaminated with phenols, BTEX and PAHs using nitrate as electron acceptor
lation of slime. Later, the supply of Purisol 100 was
terminated, once contaminant concentrations appeared
to have diminished substantially, and it was judged that
the groundwater was sufficiently well-charged with
nutrients. The nitrate application was continued how-
ever, through 1995 and 1997 as it was considered that
oxygen would continue to be a limiting factor.
The inoculum used was PHENOBAC, a proprietary
mixture of naturally occurring bacteria and growth
stimulants supplied by Microbac Ltd., Durham. The
product had received previous US EPA authorisation
for release into navigable waters on a case-by-case
basis. Following rehydration and filtering, a slurry of
the inoculum was added in batch form to the ground-
water re-injection and inoculum injection wells. This
was performed initially during week 10 and was
repeated three times during weeks 13 and 14. Addi-
tional inoculation was performed during weeks 44 and
81.
Monitoring
Samples of groundwater from the abstraction and mon-
itoring wells were taken periodically for chemical anal-
ysis. The initial sampling (week 0) took place at the
time of the initial pump tests in October 1994 (other
samplings being reported to the nearest week). The
final sampling during the re-circulation of the ground-
water took place in May 1997 (week 137). Following
switch-off in August 1997 (week 148), sampling was
carried out through May 1999 (week 240). Sampling of
the abstraction wells was carried out from taps allow-
ing water to be withdrawn during the course of the
treatment. The remaining boreholes were sampled
using either a Teflon bailer or an MPI pump, having
purged each one of at least three borehole volumes to
ensure representative samples. Each groundwater sam-
ple was temporarily stored in a cool box refrigerated at
3 to 4°C prior to despatch to the laboratory. All chemi-
cal and microbiological analysis was performed by
AES, Wallsend and included benzene, toluene, ethyl-
benzene and xylenes by purge and trap, GC/MS, pol-
yaromatic hydrocarbons by GC/MS following Soxhlet
extraction, phenols by HPLC/electrochemical detec-
tion (British Gas Method) and other determinands by
standard ‘MEWAM’ methods or similar. Colony
counts were carried out at 22°C using HMSO pub-
lished methods (Anon 1983). More detailed speciation
of phenols was carried out using GC/MS on the final
sampling occasion in May 1999 and undertaken by
Chemex, Cambridge.
341
RESULTS
Microbial populations
The total viable counts of microbial populations
(expressed as colony forming units, cfu/mL) following
incubation at 22°C are shown in Figure 3. Within the
abstraction wells there was a significant increase from
before the pump test to the start of the remediation pro-
gramme. The effect of the pump test in drawing
groundwater through the aquifer may have resulted in a
stimulation of microbial activity through disturbance
and localised oxygenation of the groundwater.
Microbial population levels then fluctuated consid-
erably. The numbers following switch-off in week 169
were particularly elevated in all wells, but especially in
monitoring well BH303 (2.9 x 107 cfu/mL) and
abstraction well AW404 (1.8 x 107 cfu/mL). By week
199 microbial counts had also increased to >107
cfu/mL in all wells apart from AW403 and AW404
where there was a decline to 105 cfu/mL since January.
However, by the final sampling during week 240 there
had been a significant decline in all wells sampled.
The reasons for such fluctuations cannot always be
readily explained but are probably due to variations in
the chemical (particularly indigenous nutrient) compo-
sition of the abstracted groundwater, or in other cases
localised enhancement of microbial populations
through inoculated microorganisms, either into the
inoculum or groundwater, injection wells. It should be
stressed that the population levels in the groundwater
do not in themselves necessarily indicate the overall
level of microbial activity. Microorganisms responsible
for contaminant degradation will also be present sorbed
onto the solid phase, and it is these, which may be
responsible for the bulk of the contaminant degrada-
tion. Also, the plate count method is selective for bacte-
ria, which can be readily cultured under laboratory
conditions and is likely to underestimate total popula-
tions present. The main value of the viable count data
was to provide a relatively rapid index to assess if there
was any gross inhibition of microbial activity which
appears not to be the case, even on the occasions where
high concentrations of contaminants had been identi-
fied.
Concentrations of ammonia and oxidised nitrogen
Variations in concentrations of ammoniacal and oxi-
dised nitrogen are shown in Figure 3 and summarised
in Table 2. Relatively low levels of ammoniacal, nitrite
and nitrate nitrogen were present prior to the pump
tests in all wells. The high level of ammoniacal nitro-
gen in the abstraction wells (particularly AW403) at the
56-week sampling may represent some localised
breakthrough of nutrients added to the inoculum injec-
tion wells during August. Ammoniacal nitrogen con-
 Land Contamination & Reclamation / Volume 9 / Number 4 / 2001

Table 2. Concentrations of target compounds, ammoniacal and oxidised nitrogen at the start of the remediation
programme, on termination of the recirculation system and two years afterwards

Mean concentration ± S.E.M.(a)

Prior to
Determinand Unit Two years
At start of remediation(b) termination of following
active
remediation termination

Week 0 Week 11 Week 137 Week 240

PAHs(c) µg/L 11 ± 6.5 4.0 ± 2.1 0.90 ± 0.34 0.67 ± 0.67
Target phenols(d) µg/L 88 ± 65 1100 ± 1100 12 ± 4.6 93 ± 87(e)
Phenol µg/L 6.8 ± 3.2 75 ± 75 3.8 ± 1.2 <1
Cresols µg/L <1 450 ± 450 0.83 ± 0.54 32 ± 31
Benzene µg/L 13 ± 6.1 <10 <10 0.67 ± 0.49
Toluene µg/L 14 ± 8.4 12 ± 3.8 <10 2.5 ± 2.3
Ethylbenzene µg/L 3.8 ± 3.8 <10 <10 0.83 ± 0.4
Total xylenes µg/L 27 ± 15 16 ± 16 19 ± 15 6.7 ± 5.3
BTEX µg/L 58 ± 31 28 ± 18 19 ± 15 11 ± 7.4
NH4-N mg/L 0.97 ± 0.37 – (f) 2.4 ± 0.7 0.98 ± 0.26
N02-N mg/L 0.01 ± 0.005 – 0.04 ± 0.03 0.09 ± 0.08
N03-N mg/L 0.02 ± 0.022 – 10 ± 9.0 0.97 ± 0.61

Key
(a) Mean of four abstraction wells and two monitoring wells.
(b) The week 0 data follow pump testing of abstraction wells which may have resulted in localised transient charges in ground-
water quality. Week 11 data were taken at the start of the groundwater recirculation.
(c) As sum of the 16 US EPA ‘priority pollutant’ polyaromatic hydrocarbons.
(d) As sum of phenol, cresols, dimethyl and ethyl phenols except where stated.
(e) As sum of phenol, cresols and 2,4-dimethylphenol (target compounds).
(f) – not sampled for nitrogen.

centrations in the monitoring wells however, reflect the
input of Purisol 100 until this was discontinued.
Apart from the 56-week result for ammoniacal
nitrogen, consistently elevated concentrations of
ammonia, nitrite and nitrate in the monitoring wells
were observed over the course of the groundwater
recirculation, both compared to previous concentra-
tions and to concentrations in the abstraction wells.
Nitrite nitrogen, an intermediate in biological nitrate
reduction, was detected in groundwater within the
monitoring wells throughout the treatment period and
two peaks were observed at 29 and 92 weeks. Much
smaller peaks were observed in the abstraction wells at
47 and 121 weeks, potentially representing some
breakthrough. In the case of nitrate, peaks were
observed in the monitoring wells at 39 and 92 weeks. In
the abstraction wells however, only a single peak was
observed at 56 weeks, following which nitrate concen-
trations remained at mostly non-detectable levels for
the remainder of the treatment and post-treatment peri-
ods. Following termination of the recirculation system
concentrations of ammoniacal nitrogen appears to have
stabilised in both the abstraction and monitoring wells.
Concentrations of both nitrite and nitrate, whilst con-
siderably lower than during the early phase of the
re-injection, remained elevated in the monitoring wells
compared to the abstraction wells following termina-
tion.
The continued presence of nitrite provided evidence
of continuing microbial denitrification of the added
nitrate.
Polyaromatic hydrocarbons (PAHs)
The clean-up criteria proposed for PAHs for the termi-
nation of the active remedial scheme were that the sum
of the 16 US EPA Target List priority pollutant PAHs
should not exceed 75 µg/L. Mean and maximum con-
centrations of the sum of the 16 US EPA Target List pri-
ority pollutants over the course of treatment are shown
in Figure 4 and the concentrations before and after
treatment are summarised in Table 1.
Concentrations of PAHs were somewhat lower at
the commencement of remediation than in 1993 in

342
 In situ bioremediation of groundwater contaminated with phenols, BTEX and PAHs using nitrate as electron acceptor

CFU's
NH4-N
Switch off
9
10 Switch off
8 20
10 AW Wells
7 BH Wells
CFU's/ml (Geometric mean)

10
6 15
10

NH4-N (m g /l )
5
10
10
4
10
3
10 5
2 AW Wells
10 BH Wells
1 In excess of
10 these values
0
0 0 50 100 150 200 250
10 Weeks
0 50 100 150 200 250
Weeks

NO2-N NO3-N
Switch off Switch off
1.8 16
AW Wells AW Wells
1.6 BH Wells BH Wells
14

1.4
12
NO3-N (mg/l)
NO2-N (mg/l)

1.2
10
1
8
0.8
6
0.6
4
0.4

0.2 2

0 0
0 50 100 150 200 250 0 50 100 150 200 250
Weeks Weeks

Figure 3. Viable counts of microorganisms in groundwater samples (geometric means) of colony forming units (CFUs) expressed
logarithmically and concentrations of ammoniacal nitrogen, nitrite nitrogen and nitrate nitrogen (arithmetic means) in abstraction
and monitoring wells during treatment. Asterisked results are approximate and indicate CFUs in excess of indicated value.

AW401 and in AW402 (compared with the adjacent centrations were below the limits of detection. The
BH304) as illustrated in Figure 1. maximum detectable concentration of PAHs in AW403
Whilst concentrations were below the clean-up cri- had then fallen to 1.2 µg/L. However, when this well
teria, both in the abstraction and monitoring wells, the was re-sampled three months later, in week 212, none
results indicate a general decrease over the treatment of the 16 PAHs were present above their detection lim-
period, with the mean concentration falling to 0.9 µg/L its.
(maximum 2.1 µg/L) at week 92. Because of the con- At the final sampling during week 240, concentra-
centration being significantly below the criteria on tions of PAHs were below detectable concentrations in
each sampling occasion, and the consistent decrease all wells except for AW404, where a total concentration
observed, no further analyses were undertaken during of 4 µg/L of naphthalene was identified. None of the
the operation of the active scheme from week 92 to other PAHs (including the more toxic 4, 5 or 6-ringed
switch-off at week 148. compounds) were present above detection limits.
On re-sampling during week 169, an increase in
PAH concentrations was observed compared with the Phenols
previous monitoring in July 1996, with a maximum The clean-up criteria proposed for phenols for the
concentration of 5.3 µg/L. Total concentrations of the active remedial scheme were that the sum of phenol,
16 priority pollutant PAHs then subsequently cresols and xylenols on the US EPA Target List should
decreased in all wells (both abstraction and monitoring not exceed 100 µg/L; these compounds being phenol,
wells) and by week 199 were the lowest since monitor- 2-methylphenol, 4-methylphenol and 2,4-dimethylphe-
ing began. In both of the monitoring wells, PAH con- nol.

343
 Land Contamination & Reclamation / Volume 9 / Number 4 / 2001

Mean Total (USEPA) PAHs Maximum (USEPA16) PAHs

START OF START OF
REMEDIATION SWITCH OFF REMEDIATION SWITCH OFF
12

10
100

µg/l (LOG SCALE)

8
µg/l

10
4

1
0
-93 0 50 100 150 200 250
0 50 100 150 200 250 300
WEEKS WEEKS

Mean Target Phenols Maximum Target Phenols

START OF START OF
REMEDIATION SWITCH OFF REMEDIATION SWITCH OFF
1200 100,000

1000
10,000
µg/l (LOG SCALE)

800
µg/l

1000

600

100
400

10
200

0 1
0 50 100 150 200 250 300 -93 0 50 100 150 200 250

WEEKS WEEKS

Mean Total BTEX Maximum Total BTEX

START OF SWITCH OFF START OF
REMEDIATION REMEDIATION SWITCH OFF
100 1,000

80
µg/l (LOG SCALE)

100
60
µg/l

40
10

20

0 1
0 50 100 150 200 250 300 -93 0 50 100 150 200 250
WEEKS WEEKS

Figure 4. Mean and maximum concentrations of key contaminants detected in abstraction and monitoring wells before and after
remediation. Maximum concentrations detected are expressed logarithmically and also show concentrations two years prior to
remediation. (Target phenols may also include ethylphenols apart from final sampling indicated by *.)

For monitoring purposes during the active remedial programme are provided in Table 1 and are depicted
scheme however, phenols were determined by the graphically in Figure 4.
‘British Gas’ (HPLC) method (HPLC being the method In summary, both the mean and maximum concen-
recommended for analysis of phenols arising from gas- trations of phenols demonstrated a substantial decrease
works sites (Department of the Environment 1987)). over the treatment period, albeit with some fluctua-
Because this method provides a quantification of phe- tions. Following switch-off there was an initial increase
nol, total cresols (methyl phenols) and dimethyl plus followed by a decrease in concentration.
ethyl phenols (as reported in these categories), other As with PAHs and BTEX, total target phenols were
phenols may have been included besides the specific also generally lower than comparative samplings in
target compounds listed above. The concentrations of 1993 (Figures 1 and 4). The mean total concentration of
total ‘target phenols’ (reported as the sum of phenol, target phenols in the groundwater being abstracted
cresols, dimethyl plus ethyl phenols) over the remedial from AW403 where the highest concentrations were

344
 In situ bioremediation of groundwater contaminated with phenols, BTEX and PAHs using nitrate as electron acceptor
detected, increased to a relatively high level (6550
µg/L) between the start of the pump tests and the start
of the groundwater recirculation in week 11. However
this subsequently fell by almost an order of magnitude
during weeks 26 and 27. Conversely, concentrations in
groundwater from AW402 increased from 243 µg/L in
week 11 to 1430 µg/L in week 26 before declining.
Two further increases (though to a lesser degree) were
observed in the abstraction wells, one in week 47, the
second in week 92 (the latter occurring mostly in
AW403). Both of these were followed by decreases in
concentration and are probably indicative of heteroge-
neities in contaminant distribution beneath the site, i.e.
concentrations fluctuated as localised pockets of con-
tamination were drawn towards the abstraction wells.
In the case of the week 47 sample this was taken after
the system had been temporarily shut down for five
weeks, so that the increase here could have been due to
desorption.
Loss of phenols was most pronounced in the moni-
toring wells. In BH303 concentrations reached a maxi-
mum of 206 µg/L in week 29 but then decreased to
below 10 µg/L. In BH302, located further down-gradi-
ent from the nearest injection well than BH303, con-
centrations were at a maximum in week 47 (64 µg/L),
then decreased to 21 µg/L. Since week 78, concentra-
tions of target phenols in this well remained below 10
µg/L.
In all wells, total concentrations of target phenols
were below the clean-up criterion of 100 µg/L for the
final two samplings in 1997 prior to switch-off (weeks
121 and 137). Following switch-off, an increase in phe-
nols was observed in most of the abstraction wells
when re-sampled in week 170. In two of these, AW402
and AW403, concentrations exceeded 100 µg/L (133
µg/L and 320 µg/L respectively). In monitoring well
BH302, 15 µg/L total phenols was detected whilst in
BH303 concentrations had decreased to below the limit
of detection.
By week 199, concentrations of target phenols were
in excess of 100 µg/L only in well AW403, where a
concentration of 764 µg/L was obtained, in the remain-
der, concentrations were of a similar order to week 170,
some slightly higher, others lower. An additional sam-
ple from this well only (not shown on Figure 4) was
then taken for more detailed speciation of phenols by
GC/MS in week 212 to examine the four specific target
phenols, these being phenol, 2-methylphenol, 4-meth-
ylphenol and 2,4-dimethylphenol. None of these target
phenols, nor any of the other semi-volatile organics on
the US EPA Target List were present above their detec-
tion limits.
At the final sampling in week 240, GC/MS was also
used to speciate the listed phenols. Concentrations
were again below detection limits in AW403 as was
345
also the case in wells BH302 and BH303. Apart from a
single value of 529 µg/L in AW402, concentrations in
the remaining wells were well below 100 µg/L, namely
24 µg/L in AW401 and 4 µg/L in AW404. As noted in
Table 1, the ‘target phenols’ results for this final sam-
pling are not entirely comparable with the previous
dataset given the fact that these were performed using
GC/MS rather than the HPLC method (which includes
the two other xylenol isomers plus any ethylphenols
which may be present). However, phenol itself was
below its detection limits (<1 µg/L) in all wells (Table
1) with cresols present only in well AW402 at 189 µg/L
(Table 1). In all other wells, cresols were below
10 µg/L.
Concentrations of BTEX
The clean-up criteria for BTEX compounds, for the
active remedial scheme were for benzene not to exceed
20 µg/L, toluene not to exceed 50 µg/L, ethylbenzene
not to exceed 60 µg/L, xylene (each isomer) not to
exceed 60 µg/L, sum of above (BTEX) not to exceed
100 µg/L. The reductions in concentrations of each of
the individual BTEX compounds are summarised in
Table 1, whilst total BTEX concentrations are shown in
Figure 4.
As with the other contaminants, concentrations of
benzene, toluene, ethylbenzene and xylenes at the start
of the remediation were generally lower than in
December 1993, in AW401 and also in AW402, com-
pared with the concentrations detected in adjacent
BH304 in September and November 1993 (Figure 1).
The groundwater abstracted from AW403 and AW404
was most contaminated with respect to BTEX com-
pounds. Concentrations in these, as with the other
abstraction wells, fluctuated considerably with time, as
the groundwater was abstracted (Figure 4). By week
137 concentrations of all individual BTEX compounds
had fallen below their target concentrations in all
abstraction wells, with total BTEX below 100 µg/L
(Table 1, Figure 4). On resampling during week 170
following switch-off, concentrations of total BTEX in
AW404 were still below 100 µg/L, with only xylenes
being detectable (56 µg/L total isomers). BTEX con-
centrations in AW403 had increased slightly above 100
µg/L (105 µg/L), with benzene at 40 µg/L. Other indi-
vidual BTEX compounds remained below their respec-
tive targets. By the final sampling however, there had
been subsequent decreases in both individual and total
BTEX compounds (Table 1, Figure 4), none of the indi-
vidual BTEX compounds being present above 20 µg/L
in any abstraction well or total BTEX above 50 µg/L.
Concentrations of each of the individual BTEX
compounds in the two monitoring wells BH302 and
BH303 remained consistently less than 10 µg/L from
week 21 onwards, including all of the monitoring
 Land Contamination & Reclamation / Volume 9 / Number 4 / 2001
rounds, following switch-off, through week 240 in
1999.
GENERAL DISCUSSION
Comparison of the concentrations of the target contam-
inants one to two years before the start of the remedia-
tion (Figures 1 and 4) indicates that there had already
been a degree of reduction in contamination levels
prior to active remediation. Some of this may have
been attributable to localised source removal, and
improvements in surface integrity including extending
concrete hardstanding over previously unprotected
areas and thereby minimising leaching from the
unsaturated zone. Natural attenuation may also have
been a contributory factor. In studies at a former coal
tar distillery, Lerner et al. (2000) demonstrated that
whilst concentrations of (mostly) phenol contaminants
(in excess of 1000 mg/L TOC), were inhibitory within
the central core of a plume within the Sherwood Sand-
stone, natural attenuation within the more dilute areas
at the fringe of the plume readily took place.
Bench-scale testing had established that microbial
activity was not significantly inhibited by the elevated
phenol concentrations, which was also supported by
the plate counts (notwithstanding the limitations of this
technique). The addition of an inoculum to this area of
contamination may only have had a marginal effect on
degradation and was considered appropriate in this
example as a precautionary measure. Addition of inoc-
ula in bioremediation (bioaugmentation) has often
been the subject of controversy (Bewley 1996),
although there have been instances of controlled condi-
tions (i.e. with or without bioaugmentation) where a
significant difference was observed, especially with the
more recalcitrant contaminants (e.g. Bewley et al.
1989).
Although definitive verification of bioremediation
full-scale is not possible, the results of the monitoring
post switch-off have indicated that the treatment car-
ried out has achieved its remedial objectives and that
the degree of microbial activity within the aquifer has
been sufficient to address desorption of any residual
contamination present. The mean concentrations of tar-
get contaminants were all below the target concentra-
tions both at termination of the recirculation scheme
and after two years of switch-off. This was also the case
for concentrations within the individual wells except
for one single concentration of total phenols within
well AW402 (529 µg/L). Where there had been some
evidence of ‘bounce-back’ in concentrations of con-
taminations following completion of the active recircu-
lation system (Figure 4), this appeared to be transient in
nature as concentrations of all determinands subse-
346
quently declined over the following two years. Where
the bounce-back occurred, this took place in the most
contaminated area: in well AW403 for example where
concentrations of phenols were 34 µg/L at termination
rising to 320 µg/L 33 weeks later, and 764 µg/L after a
further 29 weeks. Thirteen weeks later however, con-
centrations had fallen below detectable levels, which
was the case after a further 28 weeks at the final sam-
pling. The phenol concentrations in AW402 were
therefore assumed to follow a similar cycle. Concentra-
tions of PAHs and BTEX also showed similar trends as
the phenols; in each case however, all such determi-
nands were below their respective target concentrations
on both the penultimate and final sampling occasions.
Supporting indicators of biological degradation
were the occurrence of nitrite as an intermediate in den-
itrification during the nitrate reduction process, the
depletion of nitrate and the occurrence of a high bacte-
rial count in the aqueous phase. As discussed, given the
selectivity of isolation methods and the likelihood of
microbial activity being concentrated on the solid
phase the results of the microbial counts in the free
phase need to be interpreted with caution. However, it
is of interest that there is a general decline at the end of
the monitoring phase, potentially indicative of an over-
all decline in degradable carbonaceous material.
This case study therefore illustrates that in situ
bioremediation using nitrate as an electron acceptor
represents a viable remedial solution for groundwater
contaminated with low molecular weight monoaromat-
ics, PAHs and phenols. It also demonstrates that
enhanced passive remediation may be of significance
following completion of active remediation in main-
taining residual concentrations at acceptable levels.
ACKNOWLEDGEMENTS
We wish to thank the owners of the site for permission
to publish this paper and acknowledge the support of
our colleagues, Mr John Alexander during detailed
design, Mr Richard Andrews for hydrogeological mod-
elling and Mr Mark Jones for field engineering support.
The support of the Environment Agency during the
implementation of this project is also appreciated. The
views expressed in this publication are those of the
authors and do not necessarily reflect the views of URS
Dames & Moore.
REFERENCES
Al-Bashir, B., Cseh, T., Leduc, R. and Samson, R. (1990)
Effect of soil/contaminant interactions on the biodegradation
 In situ bioremediation of groundwater contaminated with phenols, BTEX and PAHs using nitrate as electron acceptor

of naphthalene in flooded soils under denitrifying condi-
tions. Appl. Microbiol. Biotechnol., 34, 414-419.
Anon (1983) The bacteriological examination of drinking
water supplies. Reports on Public Health and Medical Sub-
jects, 71. HMSO, London.
Battermann, G., Fried, R., Meier-Lohr, M. and Werner, P.
(1993) Pilot and large-scale experiences in the in-situ biore-
mediation of a refinery site polluted with hydrocarbons, pp.
1135-1144. In F. Arendt, G. J. Annokkee, R. Bosman and W.
J. van den Brink (ed.) Contaminated Soil '93. Kluwer Aca-
demic Publishers, Dordrecht.
Micro-organisms in the Treatment of Hazardous Materials
and Hazardous Wastes. Hazardous Materials Control
Research Institute, Silver Spring, MD.
Department of the Environment (1987) Problems Arising
from the Redevelopment of Gasworks and Similar Sites (2nd
ed.), prepared by Environmental Resources Limited. HMSO,
London.
Häner, A., Höhener, P. and Zeyer, J. (1995) Degradation of
p-xylene by a denitrifying enrichment culture. Appl. Envi-
ron. Microbiol., 61, 3185-3188.

Bewley, R.J.F. (1996) Field implementation of in-situ biore-
mediation: key physicochemical and biological factors. In
Soil Biochemistry, Volume 9, G. Stotzky and J.M. Bollag
(eds.), pp. 473-541. Marcel Dekker Inc., New York.
Bewley, R., Ellis, B., Theile, P., Viney, I., Rees, J. (1989)
Microbial clean-up of contaminated soil. Chemistry and
Industry, 23, 778-783.
Bewley, R.J.F., Alexander, J.G. and Webb, G.H. (2001) Ex
situ and in situ bioremediation of former oil distribution ter-
minals. Land Contamination and Reclamation, 9 (3),
269-277.
Bossert, I.D. and Young, L.Y. (1986) Anaerobic oxidation of
p-cresol by a denitrifying bacterium. Appl. Environ. Micro-
biol., 52, 1117-1122.
Campbell, J.R., Fu, J.K. and O’Toole, R. (1989) Biodegrada-
tion of PCP-contaminated soils using in-situ subsurface
bioreclamation, pp. 17-27. In Biotreatment – The Use of
Hutchins, S.R., Sewell, G.W., Kovacs, D.A. and Smith, G.A.
(1991) Biodegradation of aromatic hydrocarbons by aquifer
microorganisms under denitrifying conditions. Environ. Sci.
Technol., 25, 68-76.
Krumholz, L.R., Caldwell, M.E. and Suflita, J.M. (1996)
Biodegradation of ‘BTEX’ hydrocarbons under anaerobic
conditions. In Bioremediation: Principles and Applications,
ed. R.L. Crawford and D.L. Crawford, pp. 61-99. Cambridge
University Press, Cambridge, UK.
Lerner, D.N., Thornton, S.F., Spence, M.J., Banwart, S.A.,
Bottrell, S.H., Higgo, J.J., Mallinson, H.E.H., Pickup, R.W.
and Williams, G.M. (2000) Ineffective natural attenuation of
degradable organic compounds in a phenol-contaminated
aquifer. Groundwater, 38, 922-928.
Werner, P. (1985) A new way for the decontamination of
polluted aquifers by biodegradation. Wat. Supply, 3, 41-47.

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