Environmental Pollution 81 (1993) 229-249

BIOREMEDIATION OF SOIL CONTAMINATED WITH

POLYNUCLEAR AROMATIC HYDROCARBONS (PAHs):
A REVIEW

Susan C. Wilson & Kevin C. Jones

Institute of Environmental and Biological Sciences, Lancaster University, Lancaster, LA1 4 YQ, UK

(Received 20 February 1992; accepted 3 June 1992)

Abstract compounds. It has advantages over thermal and some

physico-chemical techniques in terms of cost and because
Polynuclear aromatic hydrocarbons ( PAHs) constitute a
the soil as a living system suitable for plant growth is
group of priority pollutants which are present at high
not destroyed. Costs associated with excavation and
concentrations in the soils of many industrially contami-
disposal of contaminated material, and void in-filling
nated sites. Criteria established for the removal or treat-
with clean material, are not incurred. Furthermore, the
ment or both of soils contaminated with PAHs vary
contamination is not transferred elsewhere, as with
widely within and between nations. The bioremediation of
landfilling. However, despite the widespread interest of
contaminated soils with in-situ, on-site, and bioreactor
Government and private industry in bioremediation,
techniques is reviewed, together with the factors affecting
uncertainties remain concerning its effectiveness for
P A H degradation. Current in-situ remediation techniques
certain compounds, including the PAHs, and also its
are considered ineffective for the removal of most PAHs
applicability in certain environments. Detailed research
from contaminated soil. On-site 'landfarming' methods
is clearly needed to establish optimum conditions for
have been used successfully (and within a reasonable
cost-effectiveness.
period of time) to degrade only those PAHs with three or
PAHs are ubiquitous in the environment (Blumer,
fewer aromatic rings. Bioreactors have proved most effec-
1976) and are found in high concentrations on many
tive for soil remediation, since conditions for enhanced
industrial sites, particularly those associated with the
degradation can be achieved most readily. However, bio-
petroleum, gas-production and wood-preserving indus-
reactors are still at the development stage, and further
tries. They cause concern as environmental pollutants
research is required to optimise their efficiency and econ-
because some are carcinogens and mutagens (World
omy for routine use. Degradation of the more recalcitrant
Health Organisation, 1983). PAHs are relatively stable
high-molecular-weight PAils in contaminated soil has not
and recalcitrant in soils and less easy to degrade than
been particularly successful to date. Further research
many other organic compounds. Consequently, they
needs are identified to help develop bioremediation into a
are difficult to remove from contaminated soil by using
more cost-effective technology. The importance of full site
the treatments that have been used successfully to clean
assessments and treatability studies for successful applica-
soils contaminated with more degradable or volatile
tion in the field is emphasised.
organic compounds.

INTRODUCTION
PAHs IN CONTAMINATED LAND
The remediation and reclamation of land contaminated
Sources of PAHs
with hazardous materials has received increasing
PAHs consist of two or more fused benzene rings in
attention internationally in recent years, with enhanced
linear, angular, or cluster arrangements. By definition,
awareness of the potential adverse effects on human
they contain only C and H atoms, although N, S, and
health and the environment. Biological remediation
O atoms may readily substitute in the benzene ring
techniques have been used at a number of sites con-
to form heterocyclic aromatic compounds, commonly
taminated with organic compounds, including the
grouped with the PAHs. Some of the more common
well-publicised clean-up of oil spilt from the Exxon
PAHs are shown in Fig. 1. They are formed whenever
Valdez in Prince William Sound, Alaska, in 1989
organic materials are burnt, with temperature influenc-
(Claxton et al., 1991).
ing the specific mixture of PAHs formed. PAHs are
Bioremediation is the use of micro-organisms to
formed naturally during thermal geologic reactions
break down hazardous organic materials to harmless
associated with fossil-fuel and mineral production, dur-
Environ. Pollut. 0269-7491/93/$06.00 © 1993 Elsevier Science ing the burning of vegetation in forest and bush fires,
Publishers Ltd, England. Printed in Great Britain and also by some plant and bacterial reactions (Blumer,
229
230 S. C. Wilson, K. C. Jones

Naphthalene Fluorene
Acenaphthene Acenaphihylene
(CloHs) * (C131~h0) *
(C12Hlo) * (C12Hs) *

Phenanthrene Anthracene (C14H10) * F l u o r a n t h e n e (C16H10) *

(C1.H1o) *

C h r y s e n e (Clsth2) *
Pyrene Benz[alanlhracene
(C1~HIo) * (C1alh2) *

B e n z o l k l f l u o r a n t h e n e (C20H12) *
B e n z o l b l f l u o r a n t h e n e (C20H12) *

B e n z o [ a l p y r e n e (C20th2) * D i b e n z [ a h l a n t h r a c e n e (C221hO *

l n d e n o 11,2,3,cdlpyrene
Benzo[ghilperylene (C221 h2) * (C221112) *
C o r o n e n e (C24H12)

• E P A Priority
Pollutants
Fig. 1. Chemical structures of some common polynuclear aromatic hydrocarbons.

1976). However, anthropogenic sources, particularly
the burning of fossil fuels (Freeman & Cattell, 1990;
Benner et al., 1990), are significant sources of PAHs to
the environment (Sims & Overcash, 1983). Anthropo-
genic combustion activities are a principal source of
PAHs to soils in industrialised countries via atmospheric
deposition. As a result, soil concentrations have tended
to increase over the last 100-150 years, particularly in
urban areas (Jones et al., 1989a, 1989b).
The presence of PAHs on grossly contaminated sites
is associated with industrial activities, usually involving
the processing, combustion, and disposal of fossil fuels,
or fossil-fuel-derived products, such as coal tar and
carbon black (Nishioka et al., 1986). Fractionation
products derived from crude oil, such as diesel,
petroleum, fuel oil, lubricating oil, etc., contain PAHs.
The crude-oil source (Grimmer et aL, 1983) and frac-
tionation process used have an effect on the PAH
content of the final fuel products, and higher concen-
trations of PAHs are associated with the higher-
boiling-point distillation products (Nyer & Skladany,
1989). Sites where refining and distillation have occurred
 Bioremediation o f soil contaminated with P A H s : A review 231

Table 1. Industrial activities associated with the production,
processing, use, and disposal of PAH-containing materials
Gasification/liquefaction of fossil fuels
Heat and power generation by using fossil fuels
Coke production
Catalytic cracking
Carbon-black production and use
Asphalt production and use
Coal-tar/coal-tar-pitch production and use
Refining/distillation of crude oil and crude-oil-derived products
Wood-treatment processes
Wood-preservative (e.g. creosote/anthracene-oil) production
Fuel/oil storage, transportation, processing, use, and disposal
Landfill/waste dumps
Open burning (tyres/refuse/coal etc.)
Incineration
Industrial activities commonly associated with the
production, use, and disposal o f significant quantities
of PAH-containing materials are summarised in Table 1.
There have been relatively few reports of specific PAH
concentrations on contaminated sites. However, those
available are presented in Table 2. It must be appre-
ciated that sampling methods and locations vary at
each site, and the values in Table 2 have therefore been
presented to give only an indication of the concen-
trations of specific PAHs that have been detected on
different sites and are not intended as an inter-site
comparison. Bioremediation would have obvious
application if it could be applied to such grossly
contaminated sites.

are frequently contaminated with PAHs and other
aromatic and aliphatic hydrocarbons. P A H contamina-
tion on industrial sites is commonly associated with
spills and leaks from storage tanks (under or above
ground) and with the conveyance, processing, use, and
disposal of these fuel/oil products. PAH contamination
is also commonly associated with wood-treatment
activities, since PAHs are major constituents of
creosote (approximately 85% PAHs by weight) and
anthracene oil, which are commonly used wood-
treatment pesticides (Bos et al., 1984; Bumpus, 1989;
Mueller et al., 1989; Walter et al., 1990).
A S S E S S M E N T OF P A H S O I L C O N T A M I N A T I O N
An important part of any site remediation scheme is to
evaluate the hazard and risk associated with the
contamination and determine the degree of remediation
required, whatever clean-up method is to be adopted.
Approaches vary between and within nations. Some
countries undertake site assessment on a case-by-
case basis with reference to any available local or
national guidelines and land end-use. Others undertake
a uniform site assessment according to standard
national clean-up criteria. Siegrist (1990) reviewed
approaches adopted to establish clean-up criteria in
the USA, Canada, Western Europe, and Scandinavia.

Table 2. Concentrations of PAHs at contaminated sites.

PAH Wood-preserving~ Creosote Wood Coking Cokin~ Gasworks e

productionb treatmenff plant ¢ plant a

Surface-soil Subsoil mean range mean range

Naphthalene 1 3925 1313 <1-5769 91.8 56 59

1-Methyl naphthalene 1 1452 901 <1-1617 87
2-Methyl naphthalene 1 623 482 6-2926 112
2,6-Dimethyl naphthalene 2 296
2,3-Dimethyl naphthalene 1 168
Acenaphthalene 5 49 33 6-77 187
Acenaphthene 7 1368 29 2 0-11
Fluorene 3 1792 650 49-1294 620 7 245 225 113-233
Phenanthrene 11 4434 1595 76-3402 1440 27 277 379 150-716
Anthracene 10 3037 334 15~93 766 6 130 156 57-295
2-Methyl anthracene 14 516
Fluoranthene 35 1629 682 21-1464 1350 34 2174 614-3664
Pyrene 49 1303 642 19-1303 983 28 285 491 170-833
2,3-Benzo(b)fluorene 8 288
Chrysene 38 481 614 8-1586 321 11 135 345 183-597
Benzo(a)pyrene 28 82 93.7 14 92 45-159
Benz(a)anthracene 12 171 356 16 200 317 155-397
Benzo(b)fluoranthene & 38 140 260 108-552
Benzo(k)fluoranthene 238 152-446
Dibenz(ah)anthracene 10-1 2 2451 950-3836
Indeno(123cd)pyrene 10 23 207 121-316

All concentrations in mg k g 1 dry matter

a Mueller et al. (1991b, 199It)--composite samples.
b Ellis et al. (1991)--samples are 1.5 m or 3.5 m.
c Weissenfels et al. (1990a)--no range or sampling details provided.
a Werner et al. (1988)--no range or sampling details provided.
e Bewley et al. (1989)--samples taken from prototype treatment bed.
232 S. C. Wilson, K. C. Jones
Explicit national guidance is available in only a few
cases and clean up criteria vary widely. Furthermore,
the legislative system is currently in a state of flux in
many countries. However, established criteria and
priority-pollutant lists that include PAHs in soil or
associated groundwater have been reviewed here.
USA
Under the Comprehensive Environmental Response,
Compensation, and Liability Act (CERCLA), or
'Superfund', the National Priority List (NPL) of sites
was established in the United States. These sites were
eligible for remediation funding through the Superfund.
Under CERCLA, the US Environmental Protection
Agency (EPA) was granted authority to clean up the
sites on the NPL. The NPL sites are assessed on a
case-by-case basis by using appropriate Federal and
state requirements, where available. The Superfund
Amendments and Reauthorisation Act 1986 (SARA),
Section 121, Clean Up Standards, provides rules and
specific approaches for remediation. However, because
of the variety of releases and contamination encountered
at hazardous-waste sites, specific remedial actions and
clean-up levels are determined on a site-by-site basis
(Balba, 1991). Remediation-assessment procedures for
sites not on the NPL vary widely between states.
Clean-up standards have not been determined by the
EPA on a Federal level, and State agencies are charged
with development of their own management strategies
for contaminated land based on suggested approaches.
Many states have established their own uniform standards
or guidance levels. For example, under the EPA's
Underground Storage Tank Program, some states have
developed soil clean-up levels for petroleum-con-
taminated soils. Groundwater protection is a major
consideration in setting standards, and most states
specify criteria for either total petroleum hydrocarbon,
or benzene, toluene, ethyl benzene, and xylene. PAHs
are seldom used as a basis for determining the extent
of remediation. The clean-up levels and compounds
specified vary between states, even within the same
region, and may be used as either guidance or formal,
uniformly enforced standards (Anon, 1991).
However, PAHs have been included on a number of
lists of hazardous materials which are regulated at a
Federal level with regard to their release or disposal
to the environment. These are included in parts of
CERCLA, SARA, and the Resource, Conservation,
and Recovery Act (RCRA) (McCoy & Associates, Inc.,
1988). The list most frequently used with regard to
water pollution was established by the EPA in a 1978
'Consent Decree'. This identified the 'priority pollu-
tants', recognised water pollutants, for which technol-
ogy-based effluent limitations and guidelines would be
determined to limit or prevent their introduction to
water (Bedding et al., 1983; Keith & Telliard, 1979).
The sixteen PAH compounds included in this list are
illustrated in Fig. 1. The list is frequently used by
regulatory authorities to identify site contamination
and specify monitoring parameters.
Canada
The Canadian Council of Ministers of the Environ-
ment has recently initiated the National Contaminated
Sites Remediation Programme for high-priority sites in
Canada. To promote consistency in the assessment and
remediation of sites under this programme, interim
environmental-quality criteria for contaminated sites
were published in September 1991. These were estab-
lished from existing criteria and guidelines in use in
various Canadian provinces, such as Quebec, Alberta,
and British Columbia (CCME, 1991). Canadian Water
Quality Guidelines and Guidelines for Canadian Drink-
ing Quality are also included. The criteria consist of:
(i) assessment criteria, which are approximate back-
ground concentrations or approximate analytical-
detection limits for contaminants in soil and
water and refer to a representative ambient level
of the contaminant; and
(ii) remediation criteria, which are considered gener-
ally protective of human and environmental health
for specified uses of soil and water and may be
used to evaluate the need for further investiga-
tion. The three land uses for which remediation
criteria have been set are agricultural, residential/
parkland, and commercial/industrial.
The assessment and remediation criteria established
for PAHs are presented in Table 3 and are intended as
guidance rather than national uniform standards. Each
site is assessed on a case-by-case basis. The criteria are
interim and will be modified as appropriate scientific
data become available.
The European Community (EC)
Soil contamination has not been a priority of EC
environmental policy until very recently, although to a
certain extent the articles of certain directives regarding
waste disposal may be applied for the benefit of soil
protection (Murillo Mantilla, 1990). To date, no direc-
tive specifically defines guidelines or standards for clean
up of soil contaminated with organic substances. How-
ever, Council Directive 80/68/EEC on the protection of
groundwater against pollution by certain dangerous
substances (OJ L20 26.1.80) contains a List I of sub-
stances that must be prevented from introduction to
groundwater and a List II of those that must be limited
in their introduction to groundwater (Haigh, 1990).
Although PAHs are not specifically identified in any
category (this has been left to the decision of the
Member State authorities), in view of their properties
and composition, most PAHs would be included in the
following List I categories:
Category 4 substances that possess carcinogenic,
mutagenic, or teratogenic properties in
or via the aquatic environment.
Category 7--mineral oils and hydrocarbons.
In view of the interaction of the soil-groundwater
system, this directive is directly related to the preven-
tion of soil pollution.
 Bioremediation of soil contaminated with PAHs: A review 233

Table 3. Clean up criteria for PAHs on contaminated land"

EPA Priority Dutch standards UK Canadian standards

pollutants
Assessment Remediation

Soil Groundwater Threshold Action Soil Water Soil Water

mg kg 1 dry matter /~g litre-t mg kg I mg kg l /zg litre i mg kg I dry matter ttg litre I
* B C * B C air-driedsoil dry matter A* B* C* A* B* C*

Naphthalene 0.015 5 50 0.1 7 30 0.1 0.2 0.1 5 50

Acenaphthene
Acenaphthylene
Fluorene
Phenanthrene 0.045 10 100 0-02 2 10 0.1 0.2 0-1 5 50
Anthracene 0.05 10 100 0-02 2 10
Fluoranthene 0-015 10 100 0.005 1 5
Pyrene 0.1 0.2 0.1 10 100
Chrysene 0.02 5 50 0-002 0-5 2
Benzla)anthracene 0.02 5 50 0-002 0.5 2 0.1 0.01 0.1 1 10
Benzo(a)pyrene 0.025 1 10 0-001 0.2 1 0.1 0.01 0-1 1 10 0.01-
Benzo(b)fluoranthene 0.1 0.01 0-1 1 10
Benzo(k)fluoranthene 0.025 5 50 0-001 0.5 2 0.1 0.01 0.1 1 10
Benzo(ghi)perylene 0.02 10 100 0-0002 1 5
Dibenz(ah)anthracene 0.1 0.01 0.1 1 10
Indeno(123cd)pyrene 0.025 5 50 0-0004 0.5 2 0.1 0.1 0.1 1 10

Total PAH 20 200 -- 10 40 50/` 500"

1000+ 10000+

a Denneman (1991) (1992), ICRCL (1987); CCME (1991) + -- Landscaped areas, buildings, hard cover
* -- Target value at 10% organic matter and 25% clay on a dry-matter basis A* -- Agricultural (no PAH criteria)
(concentration of dissolved PAH for groundwater) B* = Residential, parkland (no PAH criteria)
B -- (Indicative) value for elaborate investigation C* -- Commercial, industrial
C = (Indicative) value for clean up investigation and clean-up operation ~ = Drinking water
^ -- Domestic gardens, allotments, play areas

EC Directive 76/464/EEC (1976) on pollution caused
by certain dangerous substances discharged into the
aquatic environment of the C o m m u n i t y (OJ L129
8.5.76) sets a framework for the elimination or reduc-
tion of pollution of inland, coastal, or territorial waters
by discharges of particularly dangerous substances and
in theory relates to all discharges to surface waters and
groundwater. This Directive also contains a List I
(Black List) and List II (Grey List) of substances that
must be eliminated and reduced, respectively, from
water, (these lists are similar but not identical to the
Lists I and II in Directive 80/68/EEC). Daughter Direc-
tives have been set to fix standards for Black List
substances, although only seven have been agreed to
date, and these only specify environmental quality
standards for specific substances in surface waters,
none of which refers to PAHs.
However, the following P A H s are included in a list
of 129 Grey List substances to be considered as Black
List substances (European Commission, 1982):
P A H (in particular 3, 4-benzopyrene (benzo(a)pyrene);
benzofluoranthene (benzo(b)fluoranthene));
anthracene;
naphthalene.
In practice, this list is often used to indicate sub-
stances that m a y be controlled with respect to ground-
water, particularly in view of the interaction between
surface and groundwater.
The Fourth Environmental Action Programme (1987-
1992) has changed the strategy of the C o m m u n i t y to
multi-media pollution control, and soil protection is
now considered of primary importance, with increased
allocation of funding for research.
Individual European and Scandinavian N a t i o n s
The majority of nations in Europe and Scandinavia
generally assess each contaminated site on a case-by-
case basis, and m a n y adopt the Dutch List of soil
clean-up criteria for general guidance. However, some
are currently developing their own national standards,
e.g. G e r m a n y (Siegrist, 1990).
The Netherlands has a national policy of maintain-
ing soil multi-functionality and has established a list of
generic criteria, target values, B and C levels (the C
levels are currently under review), for evaluating the
significance of soil and groundwater contamination.
Contaminated land must usually be cleaned up to
multi-functional quality (usually the target value). The
criteria currently established for P A H s are listed in
Table 3 (Denneman, 1991, 1992).
By contrast, the U K has a policy of cleaning up
contaminated sites on a case-by-case basis with regard
to the use to which the land is to be put. A series
of guidance notes on contamination associated with
particular industrial activities, e.g. gas works, has been
published, with 'trigger concentrations' for site clean-
up. Action and threshold trigger concentrations have
234 S. C Wilson, K. C Jones
been specified for total PAHs related to land end-use
(ICRCL, 1987) and are listed in Table 3. In addition,
implementation of EC Directive 80/68/EEC on ground-
water protection is progressing, with steps to make the
National livers Authority legally responsible for classi-
fying substances as List I or List II in accordance with
criteria agreed by central Government.
BIOREMEDIATION
Bioremediation involves the use of micro-organisms to
degrade hazardous organic constituents to harmless sub-
stances, such as carbon dioxide and water. The degra-
dation process may be enhanced by changing chemical
or physical conditions in the soil, such as soil pH,
moisture, and aeration, and also by nutrient addition.
The addition of specifically adapted micro-organisms
may also enhance the process. Bioremediation methods
used to treat soil contaminated with organic compounds,
including PAHs, have been reviewed in detail by a
number of researchers (Bradford & Krishnamoorthy,
1991; Golueke & Diaz, 1989; Morgan & Watkinson,
1989; Ryan et al., 1991; Soczo & Staps, 1988), and
consist of one or a combination of the three systems
described below.
(1) In situ
The contaminated soil is treated in place and essentially
remains undisturbed during in-situ treatment (Hilberts
et al., 1985; Wilson & Brown, 1989). The most common
form of in-situ treatment is the biodegradation of
contaminants within the saturated zone of the soil. This
involves the addition of nutrients, an oxygen source
(usually hydrogen peroxide), and sometimes specifically
adapted micro-organisms to enhance degradation and
may be achieved by drilling a series of wells through-
out the contaminated area and directly injecting the
appropriate solutions. Groundwater is often recovered
(usually down-gradient) and recirculated after some
form of surface treatment. The soil amendments may
be added to the treated groundwater prior to recircula-
tion. Groundwater treatment is usually achieved by
using one of the following systems: activated sludge,
aerated lagoon, trickling filter or rotating biological
contactor. The success of in-situ treatment is highly
dependent on the soil permeability owing to the necessity
for oxygen transfer.
In-situ treatment can be used for unsaturated soils
but tends to be less easy to control. Treatment gener-
ally relies on the application of nutrient-rich water by
percolation or through pressure-injection with multiple
injection points. Forced-air/vacuum-extraction systems
may be used to enhance the air exchange within the
soil.
(2) Prepared bed/On-site
Landfarming of contaminated material was one of the
first forms of on-site treatment and has been widely
used in the oil industry for the disposal of oily sludges
(Morgan & Watkinson, 1989). The waste material is
applied to the soil as a slurry and the area managed by
fertilisation, irrigation, and lime addition to maintain
optimum conditions of nutrient content, moisture
content, and pH. The area is also tilled to improve
aeration and to ensure that degradation and immobili-
sation of contaminants within the upper and under-
lying soil layers occur. Micro-organisms used in the
degradation process are most often the indigenous
soil population. However, specifically adapted micro-
organisms may be added to the soil to enhance the
process. The major disadvantage with landfarming is
the possibility of contaminant movement from the
treatment area.
More recently, prepared beds, which involve a
greater degree of engineering and containment, have
been used. The contaminated soil is removed to a
specifically prepared area, which is usually lined with
low, permeability material, such as high-density
polyethylene or clay. The bed is managed to optimise
degradation with fertilisation, irrigation, pH control,
and sometimes microbial and surfactant additions. The
beds are designed to enhance treatment and minimise
contaminant movement off-site. They often encompass
leachate-collection and sometimes emission-control
systems and are usually situated elsewhere on-site or
on the area from which the contaminated material is
removed.
Composting is another form of prepared-bed type
of treatment that has been used to treat highly con-
taminated material. This is a specific process involving
a succession of mesophilic and thermophilic micro-
organisms and consists in piling the soil and mixing
with an organic bulking agent, such as straw or wood
chips. The pile is aerated by either forced aeration or
pile turning, and the moisture content, pH, and nutrient
content, etc., are controlled. Composting has not
been widely applied for treatment of hydrocarbon-
contaminated soil.
(3) Bioreactors
The contaminated soil is excavated and removed to a
specific reactor for treatment. The bioreactor may have
been established on-site or elsewhere in a dedicated
treatment area. Usually, the soil is slurried with water
and then treated in the reactor, where conditions for
bioremediation are enhanced. There is considerable
control over the operating conditions, which often
results in relatively quick and effective treatment.
The bioreactors are usually of the horizontal-drum
and airlift type and may be batch or continuous, but
are usually batch-mode. An acclimatised microbial
population from a previously treated soil batch is
usually introduced to each new batch to enhance the
degradation rate. After treatment, the material is passed
through a water-separation system and the water
recycled. Contaminated groundwater/effluent may also
be treated in bioreactors that are either the fixed-film or
the stirred-tank type.
Bioreactors have not yet been used extensively to
treat PAH contaminated soil.
 Bioremediation o f soil contaminated with P A H s : A review
Bioremediation has only recently been considered as
a viable treatment method for contaminated soils.
However, it is now being used or is under consideration
by the EPA for clean-up in over 135 Superfund and
UST sites, as well as many other contaminated sites
(Carraway & Doyle, 1991; Sims, 1990; US EPA, 1989).
FACTORS AFFECTING BIOREMEDIATION OF
PAHs
A number of different factors affect the likelihood of
successful bioremediation. In the first instance, this is
dependent on inherent properties of the PAH com-
pounds and the soil. Soil properties that alter the rate
of degradation, such as organic matter, structure, and
particle size, have been reviewed previously (Manilal &
Alexander, 1991; Sims et al., 1990; Steiber et al., 1990).
Environmental factors, including temperature, pH, and
soil aeration, also affect the rate of degradation and
may be modified to enhance degradation. The presence
of contaminants that are toxic to micro-organisms,
such as metals and cyanides, may hinder degradation.
(1) Properties of PAHs
Although PAHs behave in certain general ways in the
environment, each PAH compound obviously has a
unique set of physical and chemical properties. The
stability of PAHs is indicated by the ring arrangement,
linear being the most unstable and angular (rings in
step) the most stable (Blumer, 1976). PAHs are rela-
tively insoluble in water and are therefore associated
primarily with the particulate phase, in particular the
organic matter (Means et aL, 1980). A wide range of
solubilities is displayed with solubility generally
decreasing with an increasing number of fused rings
(Sims & Overcash, 1983). Volatility also decreases with
an increasing number of fused rings.
(2) Fate of PAHs in soil
The soil-removal and decomposition processes that
determine the fate of PAHs are volatilisation, abiotic
losses (e.g. leaching, hydrolysis), and biodegradation
(Sims et al., 1990).
Biodegradation by natural surface and subsurface
soil micro-organisms appears to be the process primarily
responsible for the removal of PAHs in a multiphase
soil system (Sims & Overcash, 1983; Sims et al., 1988).
Song and Bartha (1990) illustrated the importance of
biodegradation in removing jet fuel from soil. In a
study of fourteen PAHs in two soils under unsaturated
conditions, Park et al. (1990) found that volatilisation
was negligible, except for naphthalene and substituted
naphthalenes. Mechanisms of abiotic loss appeared to
be potentially important for two- and three-ring PAH
compounds, although neither volatilisation nor abiotic
losses played a significant role in determining the fate
of PAHs with more than three rings.
Wild and Jones (1992) studied the loss of PAHs from
sewage-sludge-amended soils in glass microcosms, and
235
found that abiotic processes had a significant affect
only on PAHs with fewer than four aromatic rings.
Bossert and Bartha (1986) studied structure-biodegrad-
ability relationships for ten PAHs in the presence of
1-phenyldecane as a primary substrate. Primarily volatil-
isation, but also abiotic-loss processes, had a significant
effect on the loss of the three-ring PAHs, but not those
with more than three rings. An inverse correlation
between the number of PAH rings and their loss from
soil was determined. Biodegradation correlated posi-
tively with water solubility rather than with the degree
of condensation (cluster against linear arrangement of
the same number of rings).
The biodegradability of two- and three-ring PAHs
has been shown to be extensive, whereas that of four-,
five-, and six-ring PAHs is considerably less significant
(Tabak et al., 1981; Herbes & Schwall, 1978). In
laboratory studies, Sims et al. (1988) showed that the
degradation of two-ring PAHs in sandy soils was
extensive, with half-lives of approximately two days.
Comparative half-lives for the three-ring PAHs,
anthracene and phenanthrene, were 16 and 134 days,
respectively. Four-, five-, and six-ring PAHs generally
exhibited half-lives of >200 days. Heitkamp and
Cerniglia (1987) found a similar pattern of PAH degra-
dation in sediment:water microcosms. McGinnis et
al. (1988) performed laboratory-treatability studies on
creosote-waste constituents in soil from wood-treat-
ment sites. They found that PAHs with two rings
generally exhibited half-lives of <10 days and those
with three rings half-lives of <100 days. However, most
four- and five-ring PAHs generally exhibited half-lives
of >100 days.
Studies on the persistence of PAHs in sewage-sludge-
amended soil have shown that half-lives estimated for
soils under artificial laboratory conditions may be
considerably shorter than those actually observed in
the field (Wild et al., 1990, 1991). It is important to
realise the limitations of laboratory-based studies,
although the information they provide is useful in
predicting trends in biodegradation in the field.
Biodegradation has been shown to be a major
removal mechanism for many PAHs from soil. Its
augmentation to accelerate PAH decomposition may
therefore become an effective means of PAH removal
from soil. Clearly, however, knowledge of the mechan-
ism of degradation and the factors controlling it is
necessary to achieve effective bioremediation.
(3) Microbial degradation of PAHs
( i) Acclimatisation and adaptation
Studies have shown that prior exposure and acclimatisa-
tion of subsurface and surface microbes to PAHs or
other hydrocarbons in soil or sediment enhance degra-
dation rates (Thomas et al., 1989; Bauer & Capone,
1985, 1988; Heitkamp & Cerniglia, 1987; Heitkamp
et al., 1987). Herbes and Schwall (1978) found that
absolute rates of PAH transformation were 3000-725 000
times as great in contaminated soil as in unacclimatised
soil. Although the factors limiting acclimatisation are
236 S. C. Wilson, K. C. Jones
not well understood, it is apparent that a threshold
PAH concentration or specific time is necessary.
In many cases, micro-organisms may adapt to
specific site conditions. Hence, in order to reduce the
acclimatisation period and increase the rate of PAH
biodegradation, microbes used in remediation projects
are frequently isolated from the contaminated site itself.
Those that show the fastest rates of PAH degradation
are cultured and reapplied to the soil for bioremedia-
tion. Grosser et al. (1991) reported enhanced minerali-
sation of pyrene (55% enhancement in two days) after
bacteria isolated and cultured from the contaminated
soil were reintroduced at 106-108 cells per gram of soil.
In some studies, specifically adapted strains from one
site are cultured in the laboratory and applied to a second
site contaminated by similar compounds. Vecchioli et
al. (1990) reported a 22% increase in hydrocarbon
removal when this was carried out in petroleum-
contaminated soil. Problems could be expected with
this approach, however, since micro-organisms intro-
duced from one soil to another may have difficulty in
adap.ting to the specific ecological conditions of the soil
requiring remediation.
There has also been some work on the use of geneti-
cally engineered micro-organisms for the degradation
of PAHs (Grund & Gunsalus, 1983; Blackburn et al.,
1987). However, again problems arise with survival of
laboratory cultures in the natural environment during
bioremediation. The fate and effects of bioengineered
organisms in the environment are also of concern, and
regulation of their use is stringent.
(ii) Microbial degradation in the field
The ultimate usefulness of any micro-organism to degrade
PAHs in a clean-up of contaminated soil depends on
its survival and performance in diverse, contaminated
ecosystems. Limitations to growth will include competi-
tion from other micro-organisms, micro-organism die-off,
the presence of substrates that are degraded in prefer-
ence to PAHs, and site conditions. Heitkamp and
Cerniglia (1989) demonstrated that Mycobacterium sp.
survived in microcosms for six weeks and mineralised
multiple doses of pyrene. Competition with indigenous
micro-organisms did not adversely affect survival of the
bacteria or pyrene degradation. However, few studies
have demonstrated the survival in the natural environ-
ment of laboratory cultured microbes isolated from
contaminated soil that are known to have the potential
to degrade PAHs, particularly those with more than
three rings. Demonstration of effectiveness in diverse
OH
~ OH
OH
COOH
OH
Catechol Gentisic Acid
Fig. 2. Common intermediate metabolites.
environments is essential for successful application in
the field.
( ifi) Biochemical pathways
The ultimate aim of bioremediation is to degrade
organic contaminants completely to harmless consti-
tuents, such as carbon dioxide and water. Intermediate
metabolites include dihydrodiols, phenols, and arene
oxides, many of which have been identified as carcino-
genic, mutagenic, and teratogenic. As well as being
toxic (Park et al., 1988), the intermediate compounds
are often more soluble than the parent compounds.
Microbial degradation of PAHs has been reviewed
by a number of researchers (Cerniglia, 1984a, 1984b;
Cerniglia & Heitkamp, 1989; Sims & Overcash, 1983)
and has therefore not been discussed in detail here.
Degradation of the unsubstituted, saturated-aromatic-
ring structure of PAHs is dependent on the ability of
enzymes produced by micro-organisms, such as bacteria,
fungi, and algae, to incorporate oxygen into the ring
structure. The enzymes are generally compound-
specific, and many different microbes may be required
to degrade all the PAH compounds on a contaminated
site. Aerobic biodegradation is currently the most
common form of bioremediation practised for soil
contaminated with PAHs.
The initial activation and oxidation of PAHs involve
enzymes called oxygenases produced by the micro-
organisms that catalyse oxygen-fixing reactions. The
two groups of oxygenases are monooxygenases and
dioxygenases. Fungi (and mammals) generally produce
monooxygenases, which incorporate one oxygen atom
into the substrate to form arene oxides (electrophilic
arene oxides bind to nucleophilic sites in DNA to
produce tumor inhibition). This is followed by the
enzymatic addition of water to yield trans-dihydrodiols
and phenols.
Bacteria characteristically produce dioxygenases,
which incorporate two oxygen atoms into the substrate
to form dioxethanes. These are further oxidised to
cis-dihydrodiols and then to dihydroxy products.
The initial ring oxidation is the rate-limiting step in
the biodegradation reaction of PAHs (Cerniglia &
Heitkamp, 1989), after which degradation proceeds
relatively quickly with little or no accumulation of
intermediates (Herbes & Schwall, 1978).
Although initial reactions of specific PAH degrada-
tion differ to yield different initial-oxidation products,
only a few common intermediate metabolites, namely,
catechol, protocatechuic, and gentisic acids (see Fig. 2),
Protocatechuic Acid
 Bioremediation of soil contaminated with PAHs: A review
are produced. Catechol is the most frequently formed.
The specific compound produced depends on whether
the hydroxyl groups in the dihydrodiols are ortho or
para to each other. These metabolites are degraded by
five similar pathways, which include ring cleavage, to
produce succinic, fumaric, pyruvic, and acetic acids and
acetaldehyde, all of which are utilised for cell-protein
synthesis and energy by micro-organisms with the
production of carbon dioxide and water (Sims &
Overcash, 1983). The degradation pathways of PAHs
are summarised in Fig. 3.
Substrate M~......ganism~__Cell Protein + CO2 + H20
Oxygen
There has been much interest recently in the ability
of the white-rot fungus, Phanerochaete chrysosporium,
to degrade a number of xenobiotic compounds, includ-
ing PAHs (Bumpus et al., 1985; McFarland et al., 1989;
Lin et al., 1991). The white-rot fungus produces an
extracellular lignin-degrading enzyme (ligninase), which
has the potential to degrade high-molecular-weight
(HMW) and/or insoluble compounds that will not pass
through bacteria cell walls (Hammel et al., 1986). The
ligninases rely on a supplemental enzyme system to
supply the necessary hydrogen peroxide to start the
oxidation of lignin. Lignin has a random composition
and a high-polymer structure. The ligninases therefore,
have a wide substrate range and low specificity and
thus have the potential to degrade many different
organic pollutants. This contrasts with the degradation
strategies discussed above.
(iv) Micro-organisms
PAHs are microbially degraded in one of two ways,
either as the sole source of carbon and energy to the
Arene Oxide
~ Phenol
PA~,,~ E~
Oo t e s :
Q~ Fungi,Mammals
trans-Dihydrodiol
V Pmca ryotes:~
Bacteria ~ i
V ~H
Dioxethane
ds-Dihydrodiol
Fig. 3. Microbial degradation of PAHs (adapted from Cerniglia 1984a & b).
237
micro-organisms or by co-metabolism (or co-oxidation
if oxidation occurs). In the latter, an enzyme produced
for degradation of a certain growth-supporting compound
also degrades a PAH that is not used nor essential for
growth. Little is known about the mechanism and
factors controlling co-metabolism. Co-oxidation has
been observed to enhance the degradation of high-
molecular-weight (HMW) PAHs in particular, i.e.
those with four or more aromatic rings (Keck et al.,
1989; Sims & Overcash, 1983). Sims et al. (1988)
observed that H M W PAHs were more resistant to
degradation when present as pure compounds in soil
than in the same soil in complex waste mixtures.
During soil bioremediation, use of the PAH as the
sole carbon and energy source is considered favourable,
because complete oxidation to carbon dioxide and
water can occur. Complete mineralisation of low-
molecular-weight (LMW) PAHs (two- and three-ring)
in contaminated soils by a variety of naturally occur-
ring micro-organisms that use these PAHs as sole
carbon and energy sources has been demonstrated by
a number of researchers (Cerniglia, 1984b; Cerniglia
& Heitkamp, 1989; Efroymson & Alexander, 1991;
Goldsmith & Balderson, 1989; Mueller et al., 1989;
Sorkhoh et al., 1990; Walter et al., 1990; Weissenfels
et al., 1990b. Bacteria genera are the most commonly
reported and include: Aeromonas, Alicaligenes, Bacillus,
Beijerinckia, Corynebacteria, Cyanobacter, Flavobacterium,
Micrococcus, Mycobacterium, Nocardia, Pseudomonas,
Rhodococcus, and Vibrio.
PAHs with four or more condensed rings are less
soluble, more stable, and therefore more recalcitrant.
Only recently, in laboratory studies, have a number of
researchers isolated micro-organisms from contaminated
OH
RingCleavage Acids/
~" Aldehydes
H / Catechol
OH
238 S. C. Wilson, K. C. Jones
soil that have demonstrated the ability to mineralise
four-ring PAHs as their sole carbon and energy total
source. Bacterial mineralisation of PAHs with four or
more rings has generally been reported to occur via
co-metabolism (Heitkamp & Cerniglia, 1988; Heitkamp
et al., 1988a, 1988b; Heitkamp & Cerniglia, 1989;
Mahaffey et al., 1988; Mueller, et al., 1990). However,
those bacterial species that have been reported to use
four-ring PAHs as their sole carbon and energy source
are: Rhodococcus sp., which degraded pyrene and
chrysene (Walter et al., 1991), Pseudomonas paucimobilis,
which degraded fluoranthene (Mueller et al., 1990), and
Alicalienes denitrificans, which degraded fluoranthene
(Weissenfels et al., 1990b).
Fungal metabolism of PAHs with more than three
aromatic rings is less well studied but has been
reviewed by a number of researchers (Cerniglia, 1984a,
1984b; Cerniglia & Heitkamp, 1989; McMillan et al.,
1987). Most mechanisms reported are co-metabolic.
However, at least 22 two-, three-, and four-ring PAHs,
including phenanthrene, fluorene, fluoranthene, anthra-
cene and pyrene, underwent 70-100% removal in the
laboratory, some to CO2, by the white-rot fungus,
Phanerochaete chrysosporium, during 27 days of incu-
bation with nutrient-limited cultures (Bumpus, 1989).
Degradation of benzo(a)pyrene to carbon dioxide and
water has also been reported (Sanglard et aL, 1986).
Although the potential of this fungus has yet to be
demonstrated effectively in the field, it may be used in
the future to degrade HMW substrates, with bacteria
aiding degradation of the resulting lower-molecular-
weight (LMW) metabolites.
(4) Environmental factors affecting hiodegradation
The effectiveness of PAH biodegradation can be im-
proved by controlling environmental conditions within
the soil (Lapinskas, 1989; Morgan & Watkinson, 1989;
Sims et al., 1990; Sims, 1990; Song et al., 1990). The
ranges reported for microbial activity have been sum-
marised in Table 4 (Sims et al., 1990) and conditions
reported for optimum PAH degradation have also been
incorporated, where available. The attainment of opti-
mum conditions for degradation is a major part of any
treatment project. The soil pH and C : N : P ratio may
be maintained by the addition of appropriate materials,
such as lime, nutrients, and fertilisers. Moisture content
is controlled and soil texture improved by sorting and
homogenisation (Sims et al., 1990).
On-site land-treatment beds are tilled periodically
to improve aeration, although many bioremediation
schemes use forced aeration. However, provision of an
adequate supply of oxygen to enable aerobic microbes
to mineralise the organic contaminants completely is
difficult to achieve, particularly for in-situ projects,
owing to the low solubility of oxygen. It is also expen-
sive. Hydrogen peroxide (H202) has therefore been
used to provide oxygen at higher levels than can be
achieved by aeration (Barenschee et al., 1990). Hydrogen
peroxide, however, is highly reactive and may be toxic
at high concentrations. Its uncontrolled decomposition
Table 4. Environmental conditions affecting degradation of
organic contaminants in soil
Parameter Conditions required Optimum values
for microbialactivity for PAH degradation
Soil moisture 25-85%of water-holding 30-90%(1)
capacity
Soil pH 5.5-8.5 7.5-7.8 (1), 7.0 (3)
Redox potential Aerobesand facultative
anaerobes >50 mV,
Anaerobes <50 mV
Oxygencontent Aerobic,minimumair- 10-40%02 (2)
filledpore space of 10%,
Anaerobic < 1%by volume
Nutrient content N and P for microbial P (5), C:N 60:1 (1),
growth, C:P 800:1 (1),
C :N :P 120: 10: 1 Salt concentration
(optimumvalue <4% (6)
approximately)
Temperature (°C) 1545 30 (2,3,7) 20 (1),
27 (4), 24--30(6)
(Adapted from Simset al. (1990)
(1) Dibble and Bartha (1979) (5) Manitaland Alexander(1991)
(2) Bauerand Capone(1985) (6) Heitkampet al. (1988a)
(3) Weissenfelset al. (1990b) (7) Walteret al. (1991)
(4) Songet al. (1991).
N -- nitrogen,C-- carbon, P=phosphorus.
can result in wasteful off-gassing (Morgan & Watkinson,
1992). This decomposition has been attributed to
inorganic catalysts in the soil, such as iron, and also to
the bacterial production of the enzyme catalase (Spain
et al., 1989). Further research is required to understand
fully and stabilise decomposition at each specific site.
Maintenance of an appropriate temperature for
microbial degradation is also difficult to achieve, partic-
ularly for in-situ projects, and is most easy to maintain
by using bioreactors. Tent-like, heated tunnels have
been erected over treatment beds during on-site studies
to elevate temperatures, particularly in winter (Ellis et
al., 1991). Volatile emissions can also be contained and
treated in these tents.
(5) Surfactant addition
Many PAHs sorb strongly onto soil, which limits their
availability for microbial degradation. Indeed, low
aqueous solubility may be the rate-limiting factor
controlling degradation rates of the HMW PAHs in
soils. Surfactants have been shown to enhance desorp-
tion and solubilisation of PAHs (Edwards et al., 1991;
Jafvert, 1991) with appreciable desorption in excess of
the critical micelle concentration of surfactant. Non-
ionic octyl and nonyl phenylethoxylates with from nine
to twelve ethoxylate units have been shown to be most
effective for enhancement of the desorption of the
PAHs anthracene, phenanthrene, and pyrene in soil-
water suspensions (Liu et aL, 1991). For soil:water
ratios of 1:7-1:2, more than 0.1% by volume of
surfactant was required to initiate solubilisation, and
1% by volume resulted in 70-90% solubilisation.
 Bioremediation o f soil contaminated with P A H s : A review
The concentration of surfactant used is important.
High concentrations may inhibit microbial activity, or
the surfactant may be preferentially used as the parent
substrate. Furthermore, provision of high surfactant
concentrations is expensive. Application concentrations
in excess of the critical micelle concentration have
usually been reported as most successful. However,
enhanced biodegradation of phenanthrene without
appreciable increase in PAH desorption has been
observed after the addition of non-ionic alcohol
ethoxylate surfactants at low concentrations (10/xg gl
soil) (Aronstein et aL, 1991). Further research is
required to understand fully the mechanism responsible
before accurate prediction of the required surfactant
concentrations in the field is possible.
Degradability of the surfactants used is also impor-
tant to limit further contamination. Knaebel et aL
(1990) showed that linear alcohol ethoxylate and linear
alkyl benzene sulphonate surfactants were degraded by
natural soil micro-organisms and suggested that this
occurred at very low concentrations (<50 ng g-1 soil).
Micro-organisms themselves produce biosurfactants in
the form of glycolipids (Oberbremer & Muller-Hurtig,
1989). There has been some success with the degrada-
tion of hydrocarbons by using biosurfactants in model
systems after an initial microbial-adaptation period
(Goclik et al., 1990; Muller-Hurtig et al., 1990).
Because of the high costs involved with surfactant use,
micro-organisms that produce biosurfactants under
conditions of PAH degradation could be used to
reduce costs of treatment. However, microbes that
produce biosurfactants and degrade PAHs have yet to
be identified and their use in the field has to be proven.
Many problems still exist with surfactant use in the
form of clogging, toxicity, and degradability, and
research to overcome these problems is continuing
(Bryniok et al., 1990; Werner & Brauch, 1988).
TREATMENT OF PAH CONTAMINATED SOIL
Conditions and contaminants vary for each site. A full-
scale site survey is necessary to evaluate site conditions
and contaminants prior to the commencement of any
bioremediation (Sims et aL, 1990). Treatability studies
in the laboratory and on-site are also essential to evalu-
ate optimum biodegradation conditions. It is important
that treatability studies incorporate some assessment of
detoxification of groundwater and soil in order to eval-
uate the effectiveness of degradation. Jones and Peace
(1989) observed a correlation between mutagenicity in
soils and elevated total PAH concentration by using
the Ames test, and Ames response is therefore recom-
mended to give a measure of detoxification of soil con-
taminated with PAHs (Jones & Page, 1989). The results
from the site survey and treatability tests are used to
determine the most appropriate remedial action for
a particular contaminated site. This should result in
effective and economic site remediation. This section
describes treatability studies and case studies for soil
contaminated from different sources by using data to
239
assess the fate of individual PAHs where available.
Remediation studies on oil-contaminated soil have also
been included where relevant because of the presence of
PAHs, particularly in the heavier fractions of oil.
(1) In-situ treatment
In-situ treatment of contaminated soil has been reviewed
by a number of other researchers (Madsen, 1991;
Wilson & Brown, 1989; Hilberts et aL, 1985). Details of
all in-situ treatability and case studies reviewed here are
presented in Table 5.
Aprill et al. (1990) evaluated in-situ bioremedia-
tion processes for two types of wood-preserving
and petroleum-refining wastes in unacclimatised soils
(~PAH ranged from 490 to 6646 mg kg -l soil). Con-
ditions within the soil were not altered to enhance
degradation. Soil and water detoxification was assessed.
The range of percentage decrease in mass of PAH
added after 354 days' incubation for the four waste
types is shown in Table 6. Degradation of LMW PAHs
was generally greater than H M W PAHs. There was no
detectable degradation of five-ring PAHs. Decrease in
the H M W PAHs was correlated with the oil and grease
contents of the wastes and therefore attributed primarily
to co-oxidation processes. There was no correlation of
oil and grease content with decrease in LMW PAHs.
Weissenfels et al. (1990a) set up trickling columns for
two soils, one from a wood-impregnation plant with
high concentrations of LMW PAHs (Soil A), and a
second from a former coking plant with high concen-
trations of H M W PAHs (Soil B). Results are illustrated
in Table 6 and show that, after eight weeks' incubation,
the percentage degradation of LMW PAHs was gener-
ally greater than that of H M W PAHs. There was no
significant degradation of any PAHs in soil from the
coking plant, which contained a higher proportion of
H M W PAHs.
Morgan and Watkinson (1990) also assessed the
potential for in-situ clean-up of sandy soil contaminated
with crude oil, lubricating oil, and gasoline. These
materials all contain some concentration of PAHs (crude
oil has the higher concentrations of H M W PAHs),
which contribute to the total hydrocarbon content.
The authors concluded that bioremediation by in-situ
micro-organisms would appear to be most effective
for low- and medium-distillate hydrocarbons, such as
lubricating oil, which consists of lower ring PAHs,
volatile aromatics, and aliphatic compounds, although
provision of an adequate oxygen supply and nutrients
is important to enhance degradation.
Full scale in-situ bioremediation has generally con-
sisted in recirculation and treatment of contaminated
groundwater. At a former asphalt-production plant in
the Netherlands, where soil was contaminated with
volatile organics, phenols, and PAHs (XPAH (16 EPA
priority pollutants) = 24 000 mg kg -1 soil, naphthalene
-- 8400 mg kg -1 soil), groundwater was extracted and
treated in three biological reactors, both on-site and in
the laboratory (Van der Hock et aL, 1989). Greatest
PAH removal (95%) from the groundwater on-site was
240 S. C. W i l s o n , K . C. J o n e s

Table 5. In-situ treatability studies and full-scale projects

Author Treatability studies Case studies

Aprill et al. Weissenfels Morgan and Van der Hock Ellis et al.
(1990) et al. (1990a) Watkinson (1990) et aL (1989) (1991)

Location Deventer Blekholmstorget,

The Netherlands Stockholm
Soil type Sandy, loam, -- Sandy, Fill material underlain
Unacclimatised contaminated by clay (2-15 m)
Contamination Protoleum refinery Contaminated soil Gasoline, Former asphalt Creosote: from
and wood-preserving from wood-impregnation lubricating oil, plant--volatile <10 to >32000 mg kg-j
wastes added to soil and coking plants, crude oil organics, phenols, soil, 0.9-4.5-m depth
PAHs present PAHs up to 10-m depth
PAHs 24000 mg kg -l soil
Description Batch reactors, Trickling soil columns Batch cultures, Groundwater Groundwater
Soil columns (soil slurry) recirculation and recirculation and
treatment treatment, 15 000 m 3,
sheet-pile contaminant
Conditions No treatment 100 g Raschig rings Nutrient addition- MSM,
used 150 g soil, 100 ml fertilisers, KNO 3 DO 8.5 ml/l, + 35% H202-
circulating mineral NH4NO 3 NaH2PO 4 (100 mg litre t), nutrients
salts medium (MSM), urea, 20°C, No pH --K2HPO4, bacterial
ambient temp, pH 7.2 control inoculation, ambient
temperature, surfactants--
Ethylan BCP & CD916,
Lankro (0.5% w : w)
Duration 354 days 8 weeks 2-5 years
of treatment

achieved by using an upflow aerated column (UAC). In
the laboratory, high PAH-removal efficiencies were
achieved (99-100% EPAH after 146 days) when a
rotating biological contactor (RBC) was run in parallel
with a UAC. Only 8.1% of the PAHs partitioned
into the RBC sludge, primarily those with three or
more rings. However, testing of the soil revealed that,
after prolonged flushing (146 days/up to 675 times
flushed), 46% of the initial PAH soil concentration
remained.

Table 6. Percentage PAH loss in treatability studies

Duration In situ On-site Bioreactor

(1) (2) Soil A (2) Soil B (3) (2) Soil A (2) Soil B
354 days 8weeks 8weeks 2weeks 12weeks 4 weeks *4 weeks

Naphthalene 100 (69-273) 97 (8) 100 84 (92) 0 (56)

Methyl naphthalene 99 (100) 100
Dimethyl naphthalene 99 (241) 100
Trimethyl naphthalene 100 (210) 100
Fluorene 59-97 (21-177) 70 (670) 0 (7) 95 (670) 45 (7)
Phenanthrene 40-97 (120-833) 70 (1440) 0 (27) 71 (89) 98.8 97 (1440) 30 (27)
Methyl phenanthrene 61 (208) 100
Dimethyl phenanthrene 27 (191) 100
Anthracene 46-97 (20-243) 33 (766) 0 (6) 68 (766) 32 (6)
Fluoranthene 11-38 (55-2600) 70 (1350) 3 (34) 95 (1350) 30 (34)
Pyrene 35-74 (143-3000) 71 (983) 2 (28) 29 (27) 97 94 (98) 19 (28)
Methyl pyrene 46 (16) 90
Benz(a)anthracene 0-31 (14--320) 62 (356) 4 (16) 87 (356) 17 (16)
Chrysene 30-62 (21-173) 53 (321) 9 (11) 78 (321) 6 (11)
Benzo(a)pyrene 29 (94) 3 (14) 74 (94) 11 (14)
Dibenz(ah)anthracene 17 (10) 0 (2) 73 (10) 0 (2)

1) Aprill et aL (1990).
2) Weissenfels et al. (1990a).
3) Wang et al. (1990).
* Not inoculated.
Figures in parentheses indicate initial PAH concentration in mg kg 5.
 Bioremediation o f soil contaminated with P A H s : A review 241

Table 7. Results from treatment of contaminated creosote- Song et al. (1990) assessed the potential for bio-
production sitea remediation of soil contaminated with five fuels, ranging
from gasoline to heavy fuel oil. Biodegradation played
Treatment Mean percentage removal
a relatively minor role in reduction of the lower-
In situ On-site distillate fuels compared with vaporisation and was
ineffective in removal of the heavier fuels. However,
Treatment duration 4 months 88 days bioremediation of soil contaminated with medium-
distillate fuels was effective.
Naphthalene 100 (1313) 100 (35)
2-Methyl naphthalene 99 (901) 100 (18) Outdoor lysimeter treatability tests on spills of the
1-Methyl naphthalene 99 (483) 100 (20) medium-distillate fuels (i.e. jet fuel, heating oil, and
Acenaphthalene 3 (33) 88 (36) diesel, all of which contain PAHs, although diesel fuel
Dibenzofuran 83 (652) 96 (43) contains the higher concentrations of HMW PAHs)
Fluorene 60 (650) 90 (72) showed that bioremediation, consisting in pH control,
Phenanthrene 8 (1595) 75 (197)
Anthracene 21 (334) 69 (94) fertilisation, and weekly tilling and involving the use of
Fluoranthene 53 (682) 58 (202) indigenous micro-organisms, was effective in increasing
Pyrene 61 (646) 54 (177) the rate of biodegradation (Wang & Bartha, 1990).
Chrysene 11 (614) 50 (130) After 20 weeks, the hydrocarbon content in soil for all
fuels was reduced from 50-70 mg gt soil to <5 mg g ~.
a Ellis et al. (1991)
Figures in parentheses represent initial mean PAH concentra- Soil contaminated with jet fuel was rapidly detoxified
tions in mg kg 1. ('MICROTOX '® test) within two weeks. The toxicity
of soil contaminated with the heating and diesel oils
initially increased but then decreased to background
Ellis et aL (1991) used in-situ treatment at a disused toxicity concentrations in 20 weeks. The toxicity of the
creosote-production site in central Stockholm, which soil contaminated with diesel oil decreased the most
was contaminated with high concentrations of both slowly. Phytotoxicity became insignificant after hydro-
LMW and HMW PAHs. Total creosote ranged from carbon residues were reduced to <15 mg g-i soil, which
<10 to >32 000 mg kg -l soil, with a mean of 4329 mg was attained after between four and six weeks at
kg -~ soil, and extended in depth from 0-9 to 4.5 m. summer temperatures. In further experiments, Wang et
Phenols, oil hydrocarbons, and metals were also detected. al. (1990) showed that bioremediation of diesel-oil-
Site conditions and treatment details are presented in contaminated soil increased the rate of PAH degrada-
Table 5. The in-situ treatment could be used only on an tion, all PAHs being reduced to low levels within twelve
area where clay layers were absent. Mean percentage weeks (greater than or equal to 90% degradation).
reduction of PAHs in soil samples from the site after Results for the degradation of specific PAHs are
the first four months of treatment (June-September presented in Table 6.
1990) is presented in Table 7. Over-all degradation of However, soil from gas works and wood-preserving
PAHs was significant. However, after four months, sites is frequently contaminated with high concen-
reduction in concentrations of PAHs with three or trations of both LMW and HMW PAHs, as well as
more rings was generally considerably <60%. Owing to phenols and other toxic materials (see Table 2).
the temperature-sensitivity of the process, it could be Successful biological reclamation of these sites has been
operated only for seven months when ambient tempera- limited (Sherman et al., 1990) and much research is
tures were >8°C. ongoing (Srivastava et al., 1990).
The data provided by these treatability studies and Mueller et al. (1991a, 1991b), conducted treatability
full-scale projects indicate that in,situ treatment is studies on soil and groundwater taken from the American
inefficient for removal of many PAHs from con- Creosote Works, Pensacola, FL, USA, and analysed
taminated soil within a reasonable time period, owing for 21 PAHs. Initial PAH concentrations are presented
to strong adsorption of PAHs to soil and low PAH in Table 2. The groundwater produced a positive toxicity
aqueous solubility. Furthermore, limitations in terms of and teratogenic response, even after bioremediation for
temperature and soil type also exist. fourteen days, as a result of the presence of HMW
PAHs. Tillage and fertilisation of the subsurface and
(2) On-site treatment surface soil at the site was undertaken for twelve
Details of all on-site treatability and case studies weeks. Results are summarised in Table 9. The general
reviewed are presented in Table 8. rate of biodegradation for the contaminated material
On-site bioremediation has frequently been used to was: phenolics > heterocyclics > LMW PAHs > HMW
treat oily wastes (Bossert et aL, 1984). A number of PAHs > pentachlorophenol.
environmental factors may enhance the effectiveness of Bioremediation enhanced the degradation of LMW
landfarming oily sludge (Dibble & Bartha, 1979). How- PAHs in surface soil, and the average degradation was
ever, the degradation of specific crude-oil distillates and >50% after twelve weeks. However, the percentage loss
associated individual PAHs has been studied in only a of HMW PAHs was considerably lower (see Table 9).
few cases, and often only the decrease in total hydro- Initial PAH concentrations in subsurface soils were
carbon or XPAH content has been assessed. high and after twelve weeks of bioremediation remained
 t~

Table 8. On-site treatability studies and full-scale projects

Authors Treatability studies Case studies

Song et aL Wang and Bartha, (1990) Mueller et al. Bewley et al. Bewley et al. Ellis et al.
(1990) Wang et al. (1990) (1991b) (1989) (1990) (1991)
Location Blackburn, U K Hanover, F R G Blekholmstorget,
Stockholm
Soil Type Loam sand, 68% sand, 16% silt, Sandy 30 500 m 3
clay 22% org. matter, subsurface and fill and clayey
16% clay surface soil soil
Contamination Fuel products, Jet fuel, Creosote Gasworks, Disused refinery, Creosote
low-high distillate heating oil, coal tar, phenols crude and mineral oil 3500 m 3
fuels added to soil diesel oil (pilot study) 10 000-30 000 mg kg -l
2-3 ml cm e
applied to soil

Description Soil columns Outdoor lysimeters Landfarming Layered, Homogenised soil bed Treatment bed,
chamber homogenised, on gravel base, lined concrete based, bunded
treatment bed with HDPE, leachate gravel underlayer, leachate
collection, collection
3 x 0.2-m layers, 8 0 m × 6 0 m x 0-75m
45mx 7mx0.7m
heated polytunnel
Conditions Weekly tilling, Watering, Nutrient soln. Moisture added, Moisture 15% w/w, Moisture 20% w/w,
moisture 50% soil- weekly tilling to 15 cm, 50 mg/3 kg soil inoculated in layers 20-25°C, surfactants, rotovation (x 2 weekly),
holding capacity, pH 7.4 (lime) tiling, pH 7.1, with microbes from microbial inoculations, MSM, nutrients,
pH 7.5-7.6 (lime), fertilizer-urea 10 mg cm 2 moisture 8-12% site, nutrients, pH 6.6-7-4 (Ca(OH)2) microbial inoculations
fertilisers-NH2NO3, superphosphate 4-3 mg cm 2 23°C, surfactants nutrients (MSM, 106 cell/g soil each
K2HPO4, 17-37°C C : N 200: 1, C : P 1000:1 no inoculation (spray appln.) fertilizer) treatment season,
16-25°C (ambient) bed rotovation surfactants 0.5% w : w
ambient temp. Ethylan BCP & CD916,
Lankro.
Time 4 weeks 30 days 12 weeks 12 months
HDPE -- High-density polyethylene.
 Bioremediation o f soil contaminated with P A H s : A review 243

Table 9. Comparison of on-site and bioreaetor treatments on Successful on-site biological treatment of soil
creosote-contaminated soil in terms of percentage PAH loss" contaminated with high concentrations of HMW
PAHs, i.e. those on a gasworks site, has been less
Duration On-site Bioreactor
common (Stroh et al., 1990). Soil excavated from a
12 weeks 30 days creosote-production site in central Stockholm was treated
in lined beds with leachate collection and recirculation
Surface Sediment Surface Sediment (Ellis et al., 1991). Data are presented in Table 7. Total
soil soil PAH was reduced from 1024.4 to 324.1 mg kg-~ soil.
Although a significant reduction in EPAH concentra-
Naphthalene 60 96.8. 100 100
2-Methyl naphthalene 100 73-2 100 99-9 tion is apparent, the HMW PAHs were again degraded
l-Methyl naphthalene 100 75-1 100 100 much less than the LMW PAHs. A considerable increase
Biphenyl 100 68.5 100 100 in the degradation of PAHs with three or more rings
2,6-Dimethyl naphthalene 100 67.3 100 100 was apparent (except for fluoranthene and pyrene) by
2,3-Dimethyl naphthalene 100 64-4 100 93.6 using on-site treatment when compared with in-situ
Acenaphthylene 46.2 32.7 100 100
degradation on the same site (Table 7).
Acenaphthene 83.1 29.6 100 99.8
Fluorene 100 26.7 72 99.9
The results of these studies indicate that the rate of
Phenanthrene 57.1 24.4 42-2 99.6 degradation of PAHs in contaminated soil is increased
Anthracene 88.5 26.4 86-6 99-5 during on-site treatment compared with in-situ treat-
2-Methyl anthracene 76.3 23.8 72.2 98.9 ment of the same soil, because conditions for degrada-
Anthraquinone 77.2 2.2 59.6 96 tion are more easily enhanced. Degradation of LMW
Fluoranthene 56.2 21.6 51-3 99-3 PAHs during on-site treatment is generally significant,
Pyrene 62.8 24 52.4 77-1
and treatment of soil contaminated with PAHs con-
Benzo(b)fluorene 51.9 28.1 67-4 94.8
Chrysene 59.5 31.3 57.3 94.7
taining predominantly fewer than four aromatic rings,
Benzo(a)pyrene 43.4 27.5 48.4 70.7 such as medium-distillate fuels, is relatively successful.
Benz(a)anthracene 10.9 45.8 15-5 85-7 However, the removal of HMW PAHs is limited within
Benzo(b)fluoranthene and a reasonable period of time. Careful soil preparation is
Benzo(k)fluoranthene 27-9 16 34-3 24-5 necessary for successful on-site treatment, and there are
Indeno(123cd)pyrene 18.2 30.1 23-8 40 a number of limitations, including the presence of other
materials toxic to micro-organisms. However, on-site
Mueller et al. (1991b, 1991c).
Data are for studies with nutrients, etc., added. treatment of PAH-contaminated soil is preferable to
For initial PAH concentrations, refer to Table 2. in-situ treatment, particularly if the soil is relatively
easily excavated.
high, although actual carbon turnover was greater than
in surface soils. Once creosote phenolics were exten- (3) Bioreactors
sively degraded (from eight to twelve weeks), there was Details of treatability and case studies are presented in
a significant increase in activity towards two- and Table 10. The use of bioreactors has increased recently,
three-ring PAHs. Nutrient additions appeared to have since optimum-degradation conditions are easier to main-
a positive effect on the extent of biodegradation of tain and good mixing of the soil with the microbial
HMW PAHs but no significant effect on that of LMW population and other additives, such as nutrients and
PAHs. Even so, there was still little loss of HMW surfactants, can be achieved. However, running costs
PAHs. are generally higher than in-situ and on-site treatments.
Full-scale treatment of soil on site has varied from Use of full-scale bioreactors to treat contaminated soil
open, rotovated mounds to, more recently, lined, enclosed is in its infancy, and many prototypes are currently
treatment beds, which are actively aerated. One of the under test (Parthen et al., 1990). Bioreactors have
first on-site treatment schemes was carried out at the tended to be used to treat the fine-soil fraction which is
Greenbank gasworks site in Blackburn, UK (Bewley et not so amenable to on-site treatment. Crushing and
al., 1989). Site conditions and treatment are presented mixing of the soil prior to introduction to the bioreactor
in Table 8. Reduction in EPAH during the full-scale are important. Most bioreactors in use are of the
treatment (see Table 2 for initial concentrations) from slurry-phase, drum, or airlift type.
an estimated initial mean of 22 000 mg kg ~ soil to a Bioreactors have been shown to increase the rate of
final mean of 148 mg k g ~ soil was reported. Bewley et PAH degradation in contaminated soil compared with
al. (1990) also applied a more advanced form of this on-site treatment of the same soil (DeKreuk & Annokkee,
treatment to a pilot-scale treatment bed at a disused 1988; Rittman & Johnson, 1989; Van den Munckhof &
refinery near Hanover, Germany (Table 8). Soil tests Veul, 1990). The Netherlands Organisation for Applied
showed a general decrease in hydrocarbons and vari- Scientific Research (TNO) has used a combination of
ability over the two-year sampling period (July physical and biological techniques to treat a variety of
1987-September 1989). Limited PAH analysis showed contaminated soils (Annokkee, 1990). A hydrocyclone
a decrease in ~PAH concentration from 155 mg kg-~ to is used to separate soils and sediments into fine and
28 mg kg-~ after ten months of treatment. Analysis for coarse fractions. Coarse fractions are then treated in a
individual PAHs was not undertaken. bioreactor or by landfarming. The fine fraction, not
244 S. C. Wilson, K. C. Jones

Table 10. Treatability studies and full-scale projects with bioreactors

Authors Treatability studies Case studies

Weissenfels Mueller et al. Van den Munckhof Annokkee

et al. (1990a) (1991c) and Veul (1990) (1990)

Location The Netherlands

Soil type Soil Slurry Sandy, Soil slurry Soil slurry

Surface &
subsoil

Contamination Contaminated soil from Creosote Soil Oil/PAHs

wood-impregnation and
coking plant

Description Airlift l-5-1itre bioreactors 25-m x 3.5-m diameter Rotating drum with
bioreactor batch mode axial baffles

Conditions 400 g soil Continuous mixing, Warm-air blower, Warm-air blower,

in 4 litre MSM,
pH 7-2, 30°C
surfactant Triton X,
air passed over C trap
pH 7.1, DO 9O% sprinkler, 30-35°C, nutrients added,
agitation/airflow, nutrients added 20°C, no inoculation
moisture = 10% w/w,
150 mg N fertiliser
kg l soil

Time 4 weeks 30 days

suitable for landfarming, is treated in a bioreactor or
aerated lagoon.
The biodegradability of two soils, one from a
wood-impregnation plant with higher concentrations of
L M W PAHs (Soil A) and a second from a former
coking plant with high concentrations of H M W PAHs
(Soil B), was investigated in a soil-slurry-system airlift
bioreactor (Weissenfels et al., 1990a). Results compare
favourably with those for in-situ treatment (Table 6).
The highest degradation rates were observed for PAHs
in soil from the wood-impregnation plant, particularly
for two-, three-, and some four-ring PAHs. The average
degradation of two-, three-, and four-ring PAHs in
soil A was 80% after four weeks compared with an
average degradation of 50% after eight weeks using
in-situ treatment. However, there was no significant
degradation of PAHs in soil B, except for some
two-, and three-ring compounds, even after inoculation
with a mixed bacterial culture known to degrade
PAHs.
Mueller et al. (1991c) used laboratory-scale bioreactors
to treat slurries prepared from creosote-contaminated
surface and subsurface soils that had been difficult to
treat effectively by on-site treatment. The soils con-
tained high concentrations of both H M W and L M W
PAHs (Table 2). Results after 30 days are summarised
in Table 9. The rate of degradation for both subsoil
and surface soil was: phenolics > heterocyclics >PAHs
> pentachlorophenol (PCP). In general, biodegradation
of PAHs in surface soils did not occur after fourteen
days (except naphthalene). Two-ring PAHs were
rapidly lost. After fourteen days o f incubation, 60% of
three-ring and 40% of four-ring PAHs or PAHs with
more than four rings were biodegraded. Degradation of
PAHs with four or more rings and PCP was not exten-
sive. Relatively high concentrations of H M W PAHs
( H M W PAHs composed 90% of the ~ P A H abiotic loss
into the sludge) and PCP were found in reactor sludge.
The rate of PAH biodegradation in subsurface soil was
more rapid, although the pattern was similar to that in
surface soils. After five days' incubation, the extent of
biodegradation of PAHS with two, three, and four or
more rings was approximately 90, 70, and 60%, respec-
tively. Although percentage removal of H M W PAHs
was significant, high concentrations were detected in
reactor sludge. In terms of percentage removal, this
abiotic loss consisted of 22°/,, fluoranthene, 36% pyrene,
29% benzo(b)fluorene, and 29% chrysene. Abiotic
losses therefore contributed greatly to PAH decrease in
soil. Nevertheless, extensive biodegradation of all
PAHs was evident.
These studies have shown that, although abiotic losses
of H M W PAHs may be significant when bioreactors are
used and some of the more recalcitrant PAHs are not
extensively degraded, use of the bioreactor has advan-
tages in terms of time and effectiveness when compared
with on-site and in-situ bioremediation treatment.
Although L M W PAHs are degraded, degradation of
H M W PAHs is still very limited in bioreactors.
EMERGING TECHNOLOGIES
A number of new or alternative bioremediation-treatment
technologies are currently emerging for decontamination
of PAH contaminated soil, although their effectiveness
for use in the field has not yet been proven.
Anaerobic degradation
Bioremediation of PAH-contaminated soil has primarily
been achieved under aerobic conditions, where molecular
 Bioremediation of soil contaminated with PAils: A review
oxygen is the electron acceptor. However, provision of
an adequate oxygen supply to enable mineralisation to
take place throughout the soil is frequently problematic
and expensive.
Anaerobic degradation of hydrocarbons, where micro-
organisms use alternative electron acceptors, such as
sulphate, nitrate, iron(Ill), manganese(II), or carbon
dioxide, has not received much attention owing to the
little success achieved in isolating from soil the microbes
that utilise hydrocarbons efficiently and difficulty main-
taining optimum conditions. However, these electron
acceptors may be present at a contaminated site, and,
in any case, their supply is often more economical than
that of oxygen. Recently, interest in anaerobic degrada-
tion has increased (Bertrand et al., 1989; Hutchins et
al., 1991).
Mihelic and Luthy (1988a, 1988b) have reported
degradation of LMW PAHs, namely, naphthalene and
acenaphthalene, in soil-water systems under denitrify-
ing, nitrate-excess conditions. Degradation was slower
than in an aerobic system. However, after an initial
acclimatisation period (12-24 days for naphthalene,
12-36 days for acenaphthalene) both PAHs were
degraded to non-detectable levels (<1 /~g litre-~) in
less than nine weeks. Microbial degradation depended
on the desorption-adsorption kinetics of the PAH
(Mihelic & Luthy, 1991; A1-Bashir et al., 1990), the
concentration of microbes present, and the competi-
tion for nitrate with mineralisation of soil organic
carbon.
Although anaerobic degradation of LMW PAHs has
been demonstrated, further research on biochemical
pathways, micro-organisms responsible, and limiting
factors is necessary before successful application in the
field can be achieved. Anaerobic degradation of H M W
PAHs has not yet been researched. However, potential
economic rewards may be forthcoming for clean-up of
certain wastes if success is achieved.
Other treatments
PAHs absorb ultra-violet (uv) light energy and are
subject to photolytic breakdown, although costs for
complete breakdown are prohibitively expensive. Irradi-
ation with uv light (300 nm) or natural sunlight in the
presence of a dilute oxidant, H202, has been shown to
degrade dilute solutions of benzo(a)pyrene in laboratory
tests (Miller et al., 1988). Photolysis may therefore be
used to render PAHs more susceptible to microbial
attack during biodegradation in some contaminated
soils.
In preliminary studies, Aprill and Sims (1990) have
used prairie grasses to enhance the degradation of
H M W PAHs in soil. Possible removal mechanisms
include increased microbial interaction due to rhizo-
sphere effects, increased abiotic incorporation of bio-
logically generated intermediate metabolites, and direct
incorporation into humic material. Further research is
required to investigate these mechanisms, but possible
future applications include the clean-up of low PAH
levels in soils.
245
CONCLUSIONS
Some success in the removal of two- and three-ring
PAHs has been achieved by using in-situ treatment.
However, this method of bioremediation is generally
ineffective for the degradation of many PAl-Is present
on contaminated land within a reasonable time scale.
The rate of PAH degradation in contaminated soil
during on-site treatment is higher than that for in-situ
treatment of the same soil, since conditions for degra-
dation can be enhanced. Generally, on-site treatment is
effective for soil contaminated with LMW PAHs, such
as medium-distillate fuels. On-site treatment of soil
contaminated with high concentrations of H M W PAHs
is less successful.
Degradation of PAHs has been achieved most
successfully by using bioreactors providing good soil
and micro-organism mixing. However, abiotic losses
need to be minimised, and techniques to remove toxic
materials and improve PAH availability and degrada-
tion must be developed to make bioremediation more
cost-effective. Even so, degradation of the H M W PAHs
has not been particularly successful.
A number of research areas need to be pursued
prior to the successful application of bioremediation
for clean-up of all PAH-contaminated soils. These are:
(i) isolation and successful in-field application of
micro-organisms that are able to use PAHs
with four or more rings as their sole carbon and
energy source;
(ii) further investigation and enhancement of the
co-metabolic mechanism of PAH degradation,
since most H M W PAHs are degraded by this
mechanism;
(iii) further investigation and enhancement of factors
controlling PAH availability and degradation
for use in all treatment methods, but particularly
bioreactors: these include alternative surfactants,
removal of materials toxic to micro-organisms,
and the addition of hydrogen peroxide without
excessive auto-decomposition;
(iv) investigation of factors controlling microbial
acclimatisation and techniques to decrease the
acclimatisation period;
(v) further investigation of innovative bioremedia-
tion techniques, such as anaerobic degradation,
and also polishing techniques, which may be
used to remove H M W PAHs from treated soil.
This review has highlighted the fact that conditions
encountered on every site are unique and that a full-site
assessment is necessary to identify the most appropriate
remediation scheme for a particular site. Treatability
and pilot-scale studies are also important to minimise
problems associated with application in the field and to
improve cost-effectiveness.
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