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© International Maritime Organization
 Bioremediation
in Marine Oil Spills
2004 edition

GUIDANCE DOCUMENT FOR DECISION MAKING AND

IMPLEMENTATION OF BIOREMEDIATION IN MARINE OIL SPILLS

B
INTERNATIONAL
MARITIME
ORGANIZATION
London, 2004

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 Published in 2004 by the
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Published in 2004 by the

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Copyright # International Maritime Organization 2005

Copyright # International Maritime Organization 2004

Acknowledgements
Pictures 1.3-1, 1.3-3, 1.3-4, 1.4-5 and 4.1-2 are reproduced by permission of DFO Canada.
Pictures 1.3-2 and 1.4-2 are reproduced by permission of Environment Canada.
Picture 1.3-5 is reproduced by permission of AEA Technology England.
Pictures 1.3-6, 1.3-7, 1.4-1, 1.4-4, 3.3-1, 3.3-2, 3.3-3, 3.3-4, 4.1-1
and 4.2-1 are reproduced by permission of CEDRE.
Picture 1.4-3 is reproduced by permission of TOTAL France.
Picture 2.4-1 is reproduced by permission of Indian Ocean Commission.
Picture A3-1 is reproduced by permission of Muséum national
d’histoire naturelle, Paris, France.

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Contents
Page

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Chapter 1 Introduction to bioremediation

1.1 What is bioremediation?. . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Why use bioremediation? . . . . . . . . . . . . . . . . . . . . . . . 3
Table 1: Pros and cons of bioremediation. . . . . . . . . 4
1.3 How bioremediation works. . . . . . . . . . . . . . . . . . . . . . 4
1.4 What are the main bioremediation strategies?. . . . . . 10
1.5 Scope for application of bioremediation . . . . . . . . . . . 14

Chapter 2 Contingency planning

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2 Bioremediation within the overall shoreline
clean-up response strategy . . . . . . . . . . . . . . . . . . . . . . 15
2.3 Selection of sites for bioremediation . . . . . . . . . . . . . . 16
2.4 Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.5 Logistics, identification of the requirements
for the implementation of bioremediation. . . . . . . . . . 18
2.6 Funding and claims . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.7 Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.8 Health and safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Chapter 3 Decision making

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.2 When, where and how to use bioremediation. . . . . . . 21
3.3 Guidance on the decision-making process . . . . . . . . . 21
Decision process flowchart. . . . . . . . . . . . . . . . . . . . . . 22
Table 2: Shoreline types and natural
cleaning times. . . . . . . . . . . . . . . . . . . . . . . . 24
Table 3: Criteria for assessing oxygen
limitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.4 Conclusion on decision making. . . . . . . . . . . . . . . . . . 28

III.

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Bioremediation in Marine Oil Spills

Chapter 4 Bioremediation guidelines implementation

4.1 Bioremediation treatment options . . . . . . . . . . . . . . . . 29
Table 4: Guidelines for the application
of bioremediation products
for nutrient enrichment . . . . . . . . . . . . . . . . 31
4.2 Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Appendix 1
Measurement and analysis of hydrocarbons
in marine sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Appendix 2
Indicative biodegradability of some petroleum oil products . . . . . . 40

Appendix 3
Assessing the biodegradation potential of an oil spill . . . . . . . . . . . 41

Appendix 4
Estimation of sediment permeability . . . . . . . . . . . . . . . . . . . . . . . . . 44

Appendix 5
Assessing oxygen content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Appendix 6
Assessing nitrogen concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Appendix 7
Basic recommendations for the sampling plan . . . . . . . . . . . . . . . . 48

iv

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 Preface

Major incidents such as the Amoco Cadiz (France, 1978), the Exxon Valdez
(USA, 1989), the Braer (UK, 1993), the Sea Empress (UK, 1996), the Erika
(France, 1999) and the Prestige (Spain, 2002) have provided the stimulus
for the development of alternative response techniques to tackle oil
pollution both at sea and on the shoreline. One such technique is
bioremediation. Although recognized as a potential response option 30
years ago, it is receiving renewed attention as more environmentally
acceptable clean-up methods are sought and as new claims of the potency
of bioremediation are made. During the second International Oil Spill
Research and Development Forum of the International Maritime Organ-
ization (IMO) in 1995, bioremediation was identified as a topic warranting
priority research to develop operational guidelines. An international
working group chaired by François Merlin of Centre de documentation
de recherche et d’expérimentations sur les pollutions accidentelles des
eaux (CEDRE), France and Kenneth Lee, Fisheries and Oceans Canada
was formed to address this issue.
The public often sees bioremediation as the ‘environmentally friendly’
response to an oil spill since it converts the oil into harmless products
such as carbon dioxide and water. Its potential has been demonstrated for
a number of years, as it has been used successfully to enhance the natural
degradation of oil in ex-situ methods as landfarming, composting and
biopiling.
The benefit of using bioremediation is dependent upon fulfilment of a
number of specific criteria. The scientific community is currently
researching many of these criteria in order to understand more fully the
processes involved and to improve the methods used.
Given the prominence that bioremediation has gained in oil spill response,
potential users need guidelines to help identify scenarios where this
technique could be environmentally beneficial if implemented into local
contingency plans. While there is little or no scientific evidence to show
that careful application of bioremediation strategy has caused any harm to
the environment, there is a need to be aware of situations in which
bioremediation would be unsuitable.
With a view to providing responders with a set of practical guidelines, the
47th session of the Marine Environment Protection Committee (MEPC) of
IMO decided that a guidance document for bioremediation use should be
developed and published by IMO. France agreed to act as the lead country
through CEDRE. During a workshop of oil spill response experts and
bioremediation specialists held in Brest, France, from 17 to 19 April 2001,
the first draft of the guidelines was prepared. This Working Group

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Bioremediation in Marine Oil Spills

completed a final draft document by the summer of 2001, and a short

version was submitted and accepted for publication in the IMO Manual on
Oil Pollution, as a chapter on bioremediation. The final draft documents for
decision-making and implementation of bioremediation in marine oil spills
submitted by France (MEPC 47/5/2 and MEPC 47/5/INF.9) were reviewed
during the 47th session of MEPC by the OPRC Working Group. The
Committee approved publication of the documents submitted.
The aim of these guidelines is to provide users with clear criteria to enable
them to evaluate the circumstances in which to consider the use of
bioremediation for shoreline cleanup. These guidelines are not intended to
address the treatment of waste generated at oil spills. They contain a
summary of the most important bioremediation processes and decision-
making criteria. The various strategies are discussed and some sugges-
tions as to how to monitor the effectiveness and check for possible adverse
consequences of the technique are made. Suggestions for further reading
are also provided for readers who wish to study this subject in greater
detail.
The Marine Environment Protection Committee of IMO expressed its
appreciation to:
* the Government of France and CEDRE for having taken the lead to
host the workshop to formulate the guidelines;
* Working Group members who contributed to the preparation of the
Guidance Document:
Anne Basseres (TOTAL, France)
David Bedborough (Consultant, United Kingdom)
Kevin Colcomb (MCA, United Kingdom)
Darko Domovic (REMPEC, Malta)
Michel Girin (CEDRE, France)
Kenneth Lee (DFO – Fisheries & Oceans, Canada)
Stephane Le Floch (CEDRE, France)
Sarah Macnaughton (AEA, United Kingdom)
Francois X. Merlin (CEDRE, France)
Jean Oudot (MNHN, France)
Karen Purnell (ITOPF, United Kingdom)
Svein Ramstad (SINTEF, Norway)
Richard P. Swannell (AEA, United Kingdom)
Dennis Vanderveen (TNO, Netherlands); and
* External reviewers:
Albert Venosa (EPA, United States of America)
Rebecca Hoff (NOAA, United States of America)
Ezio Amato (ICRAM, Italy)
Richard Santner (ITOPF, United Kingdom)
Roger Prince (Exxon/Mobil, United States of America)
David Fritz (BP Amoco, United States of America)

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 Chapter 1
Introduction to bioremediation

1.1 What is bioremediation?

Bioremediation is the use of biological processes

to accelerate the removal of contaminants
from the environment

In the above definition the application of bioremediation strategies is

associated with the stimulation of pollutant biodegradation. Biodegrada-
tion is based on metabolic processes by which micro-organisms, primarily
bacteria, break down a wide range of organic contaminants, such as oil,
that are susceptible to microbial degradation. Enhanced ecosystem
recovery is a consequence or goal of the practice of bioremediation.
In these guidelines the term bioremediation includes those techniques
used on site (e.g., biostimulation, bioaugmentation, phytoremediation,
monitored natural attenuation, composting/biopiling) and the extensions
to bioremediation that can be applied through combination with physical
or chemical clean-up methods (surf-washing, surfactant addition).

1.2 Why use bioremediation?

There is no single response technique that is suitable for all spill
circumstances. Therefore, a contingency plan should include consider-
ation of all current clean-up methods (see chapter 2).
A principal advantage of bioremediation over more conventional physical
and chemical methods is that it can result in the removal of the
contaminant from an environment by the enhancement of natural
biodegradation processes by conversion of contaminants to benign
substances such as water and carbon dioxide. Furthermore, it can
enhance the rate of habitat recovery, for example, by favouring plant
growth in wetlands. As such, it is more likely to be acceptable to the public
than the more invasive chemical or physical techniques.
Bioremediation, like all other methods, has advantages and disadvantages.
Table 1 shows the pros and cons of using bioremediation in comparison
with conventional response techniques.

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Bioremediation in Marine Oil Spills

Table 1 – Pros and cons of bioremediation

Pros Cons
Oil-degrading micro-organisms are Shoreline bioremediation
ubiquitous (present everywhere) strategies based on nutrient
and therefore bioremediation can enrichment will not work effectively
be used on a range of shoreline at sea due to the extent of dilution
types. that would occur in an open
There is evidence of successful system.
bioremediation operations based
on the addition of chemical
additives (e.g. nutrients and oil
dispersants) and/or habitat
alterations (e.g. surf-washing and/
or tilling).
Relatively non-intrusive method for Not recommended for use for the
final polishing. removal of bulk oil.
A natural process. Dependent on prevailing
Does not generate large volumes of environmental conditions and the
secondary waste. nature of the oil (i.e. limitations on
heavy fuel oils).
May enhance dispersion of oil
droplets.
Generally less labour-intensive Takes longer than other physical/
and more cost-effective than chemical techniques.
traditional clean-up methods
based on physical removal.
Has received a positive public Some concerns remain in regards
response. to potential adverse health effects
associated with the release of
bioremediation agents, particularly
bioaugmentation products, and
those resulting from the metabolic
by-products of biodegradation.

1.3 How bioremediation works

Micro-organisms metabolize (i.e. biodegrade) organic compounds for

energy and a source of carbon for cell growth (i.e. production of biomass).
Other elements, such as nitrogen and phosphorus, are required as well as
carbon for the synthesis of the molecules of life (e.g. proteins, enzymes,
amino acids and lipids). Organic contaminants that are susceptible to
biodegradation include oils (e.g. petrol, diesel, heating oil, crude oil,
lubricants, and some fuel oils), Polynuclear Aromatic Hydrocarbons
(PAHs), oxygenated hydrocarbons (e.g. glycols, surfactants, detergents),

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 Chapter 1 – Introduction to bioremediation

pesticides, BTEX components (benzene, toluene, ethylbenzene, xylene),

solvents, chlorinated solvents, amines, anilines, and even some explosives.
Microbial metabolism of organic contaminants, including oil, may follow
different mechanisms according to the environmental conditions. For
example, under aerobic conditions (i.e., in the presence of oxygen) many
organic molecules are eventually converted to carbon dioxide, water and
microbial cell mass (biomass) as illustrated by the following formula:
1 kg HCa þ 2.6 kg O2 þ 0.07 kg N þ 0.007 kg P
+
1.6 kg CO2 þ 1 kg H2O þ1 kg biomass
HCa = Hydrocarbon
Under anaerobic conditions (i.e., in the absence of oxygen), biodegradation
is usually much slower and therefore of less operational interest.

1.3-1: Bacteria at work (DFO Canada)

1.3.1 Mechanisms of biodegradation

Biodegradation will occur, along with other weathering processes,
immediately after the oil enters the environment. Petroleum hydrocarbons
can be divided into four major classes (and subclasses) whose potential for
biodegradation is highly variable. They can be listed in order of
biodegradability:
* alkanes (or saturates)
* aromatics, including Polycyclic Aromatic Hydrocarbons (PAHs)

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Bioremediation in Marine Oil Spills

* asphaltenes
* resins or polar compounds

Alkanes are degraded rapidly in the presence of oxygen by a wide range of

micro-organisms. Alkanes can be subdivided into normal paraffins
(straight-chain compounds, n-alkanes), branched-chain saturates and
cyclic saturates (or naphthenes or alicyclics). In general, the straight- or
branched-chain saturates may be degraded relatively quickly and
completely (degradation begins with straight-chained compounds) relative
to the cyclic compounds.

Aromatics are compounds with one or more aromatic rings or benzene

rings; they can also have substituents (e.g., benzenes, substituted
benzenes, two-, three-, four- and even five-ringed PAHs). Although the rate
of biodegradation of aromatic hydrocarbons is slower than for alkanes,
relatively rapid degradation rates have also been observed in aerobic
conditions. In general, light compounds (1 or 2 rings) degrade quite well
(and quickly), heavy compounds (with 5 or 6 rings) are highly resistant to
degradation. In terms of ecological significance, the mechanism of
biodegradation is of interest since some aromatic compounds tend to be
degraded into less toxic components.

For asphaltenes and resins, biodegradation has been shown to be slow

(and always incomplete) in comparison to the other hydrocarbon
components in crude oil. Moreover, both asphaltenes and resins may
contain compounds that are the by-products of crude oil degradation.
Although these chemicals make up a small proportion of petroleum
products, they are extremely persistent.

1.3.2 Factors affecting bioremediation

The success of bioremediation is heavily influenced by the nature of the

contaminated environment and the interactions between micro-organ-
isms. As a biological process, factors such as extreme temperatures, low
dissolved oxygen (DO) and low nutrient concentrations that impact micro-
organism growth can limit bioremediation. Such factors should be taken
into account in any decision-making process regarding the use of
bioremediation, described in chapter 3.

Temperature: Biodegradation rates are influenced by temperature. As a

result, temperature is often a limiting factor to bioremediation in colder
climates. Low temperature also increases oil viscosity, thereby reducing
bioavailability and volatilization of the toxic short-chain alkanes and
retarding the onset of biodegradation. However, Arctic studies have also
demonstrated that adaptation is a major factor that influences the natural
rates of microbial activity in the natural environment. Hence, suppression
of biodegradation activity has only been reported to occur at temperature
conditions close to freezing.

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 Chapter 1 – Introduction to bioremediation

1.3-2: Cold environment, Arctic Svalbard (SINTEF Norway)

Dissolved Oxygen (DO): Appropriate DO concentrations are vital for

bioremediation to occur. The surface layers in beach environments are
generally sufficiently oxygenated, as DO concentrations are usually
elevated in coastal areas due to wave action along the shore and primary
productivity in surface waters. However, reduced oxygen availability is of
greater concern for shorelines with fine-grained sediments, such as
saltmarshes or mudflats. Here, mass transfer of oxygen may not be
sufficient to replenish oxygen consumed by microbial metabolism.

1.3-3: Sandy Beach, Long Cove, Nova Scotia (DFO Canada)

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Bioremediation in Marine Oil Spills

1.3-4: Saltmarsh, Conrods Beach, Nova Scotia (DFO Canada)

1.3-5: Mudflat (AEA Technology, England)

Nutrient limitation: Bioremediation can only be sustained as long as there

are sufficient concentrations of nutrients (mainly nitrogen and phospho-
rus) available. With the spill of hydrocarbons supplying excess carbon,
nutrients must come from, or be added to, the environment for optimal
biodegradation to occur. Typical concentrations of nitrogen in coastal
seawaters fall in the range of 0.3–0.6 mg/L, which means that nitrogen
may be a limiting factor (see section 3.3.8), in spite of efficient water
exchange. Phosphorus is not normally considered to be a limiting factor in
the coastal marine environment. Nutrient concentrations may also be
limiting in pristine areas. Polluted waters, sediment-rich inland waterways,
estuaries and coastal waters usually have an adequate nutrient supply.

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 Chapter 1 – Introduction to bioremediation

1.3-6: High-nutrient site, Vietnam (CEDRE, France)

1.3-7: Low-nutrient site, Svalbard (CEDRE, France)

Pollutant accessibility and toxicity: Pollutant accessibility, or bioavailability

and potential toxicity are crucial to the success of bioremediation.
Bioavailability is influenced by a number of factors, including the
solubility of the contaminant and its sorption onto organic matter or
sediment particles. Research has shown that the longer contamination
remains in the sediment the less bioavailable it tends to be. Thus, for
‘weathered’ contamination an assessment of the bioavailability of the
pollutants is advisable prior to treatment. Moreover, weathering of oil on
shorelines will increase viscosity in the longer term, rendering it less
amenable to biodegradation.

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Bioremediation in Marine Oil Spills

Weathering preferentially removes shorter chain hydrocarbons that are

more volatile and easily biodegradable. If the hydrocarbon concentrations
are very high, biodegradation may be inhibited by the presence of toxic
organic molecules such as low-molecular-weight alkanes (heptane, hexane
and pentane, etc.), high levels of BTEX (benzene, toluene, ethylbenzene,
xylene) and substituted monoaromatics. This is rarely a problem after an
oil spill at sea as these toxic components tend to evaporate rapidly.

1.4 What are the main bioremediation strategies?

The bioremediation strategy of choice and its eventual success are
dependent on the nature of the contaminated shoreline, the probability
of migration and contamination to an unoiled area, and the environmental
impacts of the procedure itself. Presented below are in-situ (see paragraphs
1.4.1 to 1.4.3) and ex-situ (see paragraph 1.4.4) bioremediation strategies
that can be used directly on the polluted site (the beach, the beachhead
and the back of the beach).

1.4.1 Biostimulation
Since oil biodegradation is likely to be limited by something, several
mitigation procedures are based on biostimulation by amendments to
overcome limiting factors. As previously stated in section 1.3, oil
biodegradation requires oxygen, nitrogen and phosphorus to proceed
effectively. Most porous shorelines (sandy, gravel, pebble and cobble) are
carbon-limited, and the biodegrading micro-organisms respond rapidly to
the presence of hydrocarbon contamination by proliferation. At low oil
concentrations (less than 1 g/kg), the hydrocarbon toxicity will be minimal
and the oxygen availability as well as the nitrogen and phosphorus
ambient concentrations may be sufficient for rapid oil degradation. In
such circumstances, bioremediation should consist of monitoring the
natural processes. However, biostimulation would be appropriate at higher
oil concentrations since oxygen and nutrients would be the limiting
factors.
Generally speaking, bioremediation through biostimulation is time-
consuming and is used mainly as a polishing technique, after the bulk
of the oil has already been removed.
Oxygen stimulation: Oxygen limitation occurs when the sediment is not
permeable enough to allow oxygen to diffuse to the micro-organisms
themselves. This lack of permeability can be due to the presence of oil in
the sediment that clogs the interstitial spaces. In this case, oxygen can be
supplied by aerating the sediment, through periodic raking, tilling or
harrowing, in order to restore the sediment permeability and diffusion
capacity. However, during aeration, care should be taken not to increase
the depth of oil penetration in the sediment. Furthermore, such practices
are not recommended for use in saturated environments such as wetlands
or saltmarshes due to the destruction of plant material and root systems
which would result in the loss of habitat by erosion.

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 Chapter 1 – Introduction to bioremediation

1.4-1: A fresh spill, Brittany (CEDRE, France)

1.4-2: Tilling, Svalbard (SINTEF, Norway)

Nutrient stimulation: Nutrient amendments (primarily nitrogen) can be

made to ensure that concentrations are not limiting the rate of
biodegradation. Optimal ratios between the hydrocarbon and the nutrients
have been quantified in laboratory and field studies.
A great variety of products are available for use as nutrient biostimulants.
Appropriate inorganic and organic nutrient sources include briquettes,
granules and liquid fertilizers. While adverse effects have not been
observed in any case study of a coastal shoreline where bioremediation
has been implemented, some precaution should be taken to avoid an
excessive application of nutrients (such as ammonia) which could
theoretically result in eutrophication encouraging the development of
toxic algal blooms.

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Bioremediation in Marine Oil Spills

1.4-3: Nutrient addition from Exxon Valdez, Alaska (TOTAL France)

Alternative biostimulation strategies: Other clean-up procedures that

increase the surface area of the oil (e.g. surf-washing to facilitate oil
mineral aggregate formation or the use of chemical beach cleaning agents
that increase the rate of oil degradation) may be classified as biostimu-
lation techniques for bioremediation. Microbial attack occurs principally
at the oil–water interface. Thus, any physical and/or chemical methods
that facilitate an increase in the oil–water interface may effectively
enhance the rate and extent of biodegradation.

1.4-4: Surf-washing operation during the Prestige,

Pyrénées Atlantiques (CEDRE, France)

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 Chapter 1 – Introduction to bioremediation

1.4.2 Bioaugmentation
If oil-degrading micro-organisms are not indigenous to the contaminated
site then their addition (a process termed ‘‘bioaugmentation’’) may be
helpful, provided they can survive in their new environment. However, a
shoreline environment in which there are no recorded hydrocarbon-
degrading micro-organisms has yet to be found. Studies have shown that
the beneficial effects of bioaugmentation products may have been
attributed to nutrients within their formulation. There appears to be no
need for adding oil-degrading micro-organisms to the open environment.
Indeed it should be noted that, where indigenous oil-degrading micro-
organisms are present, they generally out-compete the introduced
microbes, and this results in the failure of bioaugmentation.

1.4.3 Phytoremediation
Freshwater wetlands and saltmarshes are among the most sensitive
ecosystems and are the most difficult to clean. Where traditional clean-up
techniques may exacerbate the damage, consideration is now being given
to the inherent capacity of wetland plant species to stimulate aerobic
biodegradation. This process of utilizing plant growth to accelerate the rate
of oil biodegradation and habitat recovery is called ‘‘phytoremediation’’. It
is based on stimulating the growth of existing tolerant plants by
fertilization, or replanting with species from the impacted region
(preferably those with phytoremediation attributes) when residual oil
concentrations have diminished to levels tolerated by the plants. There is
now evidence that some plant species in saltmarsh environments may
effectively stimulate aerobic oil biodegradation by aeration of the rhizo-
sphere and the release of plant by-products and enzymes that stimulate
microbial activity. Phytoremediation shows promise as an oil spill
countermeasure for coastal environments. However, below the plant
growth, oil will not be affected by phytoremediation.

1.4-5: Phytoremediation operation in Conrods Beach, Quebec (DFO Canada)

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Bioremediation in Marine Oil Spills

1.4.4 Ex-situ techniques

This guidance document is focused on in-situ techniques. Ex-situ
techniques such as landfarming, composting, and biopiling can be
conducted on or near contaminated sites. Landfarming and composting
are established techniques.
Composting involves the formation of large windrows of contaminated
material and the addition of nutrients. The windrows can be turned
periodically for aeration and homogenization. In essence, composting is a
slightly more intensive version of landfarming but is capable of treating
more material per unit area. Ex-situ techniques appear to offer an
appropriate means to deal with oily waste associated with spill clean-up
operations.
Engineered biopiling is a more intensive version of composting, where a
greater effort is made to optimize the biodegradation processes. Air is
forced through the pile either continuously or periodically to ensure that
the biopile is completely aerated. Suction of air through the biopiles has
the advantage of concentrating any volatiles in a fixed volume of air such
that they may be treated using other equipment, whereas blowing can lead
to the dispersal of volatiles and odours in the atmosphere and cause a
nuisance. The biopile may be covered and heated in periods of low
temperature, thereby maintaining the optimal temperature range for
biodegradation (20–30 8C). The leachate can be collected and sprayed back
onto the pile to keep the soil moist.

1.5 Scope for application of bioremediation

In summary, application of the appropriate technique for oil clean-up is
dependent on a number of factors. The first question to ask is whether
clean-up is required at all. Oil-contaminated sites may recover most rapidly
without intervention. A range of factors that influence the choice of
treatment strategy include the likely environmental impact of leaving the
contaminated site alone; the impact of environmental damage from
movement and use of personnel, equipment and chemicals; accessibility
of the shoreline; and cost/benefit analyses (chapters 2 and 3).
Where shoreline clean-up is required, a number of different physical and
chemical as well as biological techniques have been developed. In heavily
populated locations with easy public access to the coast, physical removal
procedures may be appropriate. However, when access for personnel or
heavy equipment is limited, or when natural clean-up via biodegradation is
limited by low oxygen levels or low nutrient concentration, bioremediation
may be an appropriate clean-up strategy (chapter 3, paragraph 3.2).

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 Chapter 2
Contingency planning

2.1 Introduction
As with other response techniques, bioremediation requires careful
planning in order to achieve the desired results. As it is influenced by
environmental factors, bioremediation is not an oil spill response tool that
is suitable for all scenarios. Identification of potential sites for its
application on an operational scale will require detailed analysis and
consideration before inclusion in the contingency plan. Contingency plans
for bioremediation use should contain clear policy statements regarding
its use. This document should address the procedures applicable for the
approval of bioremediation agents. At present, specific national regulations
exist in only a few countries, primarily for the evaluation and approval of
bioremediation agents. Implementation of the technique may fall under
regulations dealing with waste disposal, agricultural practices or environ-
mental pollution control, and should be considered when undertaking
strategic and local contingency planning. Details for implementation
should be specified and incorporated in the appropriate local plans (tier 1
and 2; refer to the IMO Manual on Oil Pollution: Section II, ‘‘Contingency
Planning’’ 1995 edition, chapter 1).

2.2 Bioremediation within the overall shoreline

clean-up response strategy
Response to oil spills usually comprises response operations at sea
(containment and recovery, or chemical dispersion), protection of threat-
ened shorelines, and shoreline clean-up. Bioremediation techniques (by
nutrient enrichment) can be effectively applied to lightly oiled beaches and
other contaminated shoreline sites after the majority of the free (bulk) oil
has been removed using one or more of the standard shoreline clean-up
techniques (e.g. manual or mechanical removal, flushing). While it is not a
primary response method for the removal of bulk oil, bioremediation
should be considered as an important technique that complements
standard shoreline clean-up procedures.
It can be used to mitigate long-term environmental impacts of residual oil
deposited on the shoreline after an oil spill, and to restore the affected
shoreline ecosystems to their original state as far as is practical. The
immediate application of bioremediation strategies on lightly oiled beaches
and its use as a polishing tool in the final stages of other shoreline clean-
up operations may significantly speed up the removal of residual oil and
hence the time taken for restoration of the shoreline.

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Bioremediation in Marine Oil Spills

2.3 Selection of sites for bioremediation

Identification of candidate sites suitable for the adoption of bioremediation
can be carried out at the planning stage. This level of detail is specific to
the local plan. The process of identifying suitable sites is outlined in a
decision tree under chapter 3. The selection process can take time and
may require specialist expertise.
It is important for the contingency plan to provide clear guidance to the
strategic response team on site selection, which should also identify
sections of shoreline where bioremediation would be a less appropriate
strategy. Contingency plan authors may find it useful to consult with
bioremediation specialists to assist in the preparation of this part.
Agreement should be sought with all parties who have an interest in the
adoption of bioremediation as a response tool.
The most important criteria to examine when determining site suitability
are:
* Geomorphologic characteristics, i.e. the shoreline substrate
* Oceanographic features, i.e. local energy regimes
* Climate, i.e. typical ambient temperatures
* Tidal range
* Shoreline usage
* Environmental sensitivities
* Socio-economic factors
* Cost/benefit analysis

2.4 Mapping
Maps are the best way of presenting information in a contingency plan.
Characterization with mapping of areas that are (or are not) suitable for
bioremediation should therefore be carried out as part of the contingency
planning process.
National contingency plan maps will only indicate the suitability for
bioremediation of different coastal areas, while those attached to local
plans should provide more detailed information required for its imple-
mentation. Consideration should be given to the interaction of environ-
mental factors that may influence the loss of habitat by erosion. Thus, in
addition to the information normally included in such maps (i.e. the type of
coastal formations, tidal movements, access to the shoreline, shoreline
habitats, sensitive areas, etc.), specific information on biological and
physical factors controlling the effectiveness of bioremediation techniques
(chapter 3) should also be included. Examples include:
* Sediment grain size
* Exposure (high/low energy)
* Background nutrient concentrations (mg N/litre)

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 Chapter 2 – Contingency planning

* Oxygen availability (dissolved interstitial oxygen)

* Seasonal ambient temperatures
* Tidal range
* Shoreline gradient
* Vegetation
* Near-shore current regimes

It is helpful to supplement maps with photographs of the area concerned.

These might be particularly useful for the evaluation of the degree of
recovery following bioremediation after a spill. Photographs should also
show seasonal variations in coastal vegetation if appropriate. The use of
electronic maps facilitates the integration of images into maps. External
specific expertise can be useful for this task.

The process of the preparation of maps can be long; however, the time
dedicated to it will be fully justified in the case of an emergency.
Fundamental baseline data collected during the preparation of these
maps will not only facilitate the decision-making process in case of
pollution incidents, but also simplify post-spill environmental and socio-
economic evaluations.

2.4-1: Example of mapping, Mauritius (Indian Ocean Commission)

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Bioremediation in Marine Oil Spills

2.5 Logistics, identification of the requirements

for the implementation of bioremediation
When the decision for bioremediation use has been agreed upon, the
resources required should be identified based on the nature and extent of
the incident. Contingency plans should include a description of likely
resource requirements and guidance on the procurement of:
* Local and national expertise for overall supervision of operations
* Manpower/operators
* Bioremediation agents (specifically developed products, agricultural
fertilizers)
* Laboratory facilities and analytical capabilities
* Field equipment

2.6 Funding and claims

It is likely that parts of the contingency plan dealing with funding of clean-
up operations and the compensation of costs will address bioremediation
as an element of overall spill response efforts.
Since bioremediation is closely related to the restoration of habitats and
environmental recovery of polluted areas, there is high potential for
controversial issues in the context of compensation of costs. Thus,
bioremediation operations may need more considerations than traditional
clean-up techniques, including extensive monitoring of environmental
parameters as well as treatment progress (changes in pollutant composi-
tion and amounts).
The costs of bioremediation techniques will be considered for compensa-
tion under the present international regimes if the criteria for claims
admissibility have been applied satisfactorily, and can be supported by
relevant technical and scientific evidence. During the response to a
specific incident, insurers should be given the opportunity to become
involved in the discussions on the use of bioremediation at an early stage if
compensation is to be claimed.
The application of bioremediation techniques should be closely supervised
and the rationale and progress precisely recorded in order to provide
sufficient evidence for potential claims for compensation. As such, the plan
should include information on the analytical resources and the expertise
available to undertake this task.

2.7 Training
Implementation of bioremediation techniques requires only basic skills,
similar to those regularly used in farming. Training of operators can
therefore be included in routine oil spill responder training programmes,
planned at national and local levels.

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 Chapter 2 – Contingency planning

On the other hand, the level of training required for the proper supervision
of the implementation of bioremediation techniques and controlling their
progress is more specific and is considered to be too extensive to be
provided through standard training courses for on-scene commanders and
supervisors.
Supervisory staff need to have at least a basic scientific training and
certain experience in chemistry and biology, and ideally should be
recruited from institutions with expertise in microbiology, environmental
chemistry, or related disciplines.
If the required expertise is not available locally, it may be brought in from
specialized national or external sources, which may include industry.
Such an approach may provide the opportunity for the training of local
scientific and/or technical staff without a background in bioremediation
technology.

2.8 Health and safety

Bioremediation may involve the use of products that could potentially
impact human health, so the contingency plan should address the issue of
health and safety. This should include the health and safety data sheets
for substances likely to be used. Ideally, generic activity risk assessments
for the implementation and monitoring techniques should be carried out
for inclusion in the contingency plan.

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 Chapter 3
Decision making

3.1 Introduction
In this chapter the decision-making process for determining whether to
use bioremediation and selecting the most appropriate approach is
explained. A flowchart outlining when, where and how to use bioremedia-
tion is given in section 3.2. Detailed guidance on each decision point is
given in section 3.3.
Bioremediation is not a simple technique; however, as it is a secondary
clean-up technique, there is time to establish an experts group to validate
the choices. Should highly sensitive sites be affected, the involvement of
specialists should be encouraged (e.g. botanists in operations within
wetlands).

3.2 When, where and how to use bioremediation

The decision-making process begins at the moment oil impacts the
shoreline. Bioremediation is a long-term remediation technique and is only
applicable for contaminated shorelines. See page 22 for a flowchart.

3.3 Guidance on the decision-making process

3.3.1 Objective of bioremediation and criteria for success

The objective of the bioremediation is to minimize the damage resulting
from the shoreline pollution. This can be achieved by:
* reducing the oiling and/or
* encouraging habitat recovery.
Whatever the objective, clear criteria and target levels should be
established to monitor success, for example:
* reduction of oil concentration to an agreed level, or
* re-establishing the main structure and functions of the habitat.
These criteria and target levels should be established in consultation with
local stakeholders.

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Determine criteria for

successful bioremediation

Yes Leave oil to be

Site exposed? removed naturally or
use standard method

No

Yes
Apply other
Free oil present?
(removal) methods

No

Yes Yes
Phytoremediation Apply
Wetland?
suitable? phytoremediation

No No

No Additional
Treatment completed clean-up
required?

Yes

Oil sufficiently No
biodegradable?
Consider
Yes alternative
clean-up
options

Temperature No
high enough?

Yes

Oxygen No No
Aeration feasible?
sufficient?

Yes Yes Yes

Aerate

Nitrogen Yes Leave to biodegrade Monitor until cleanup

sufficient? naturally target met

No

Add nitrogen

No Further
Stop bioremediation bioremediation
Consider other option appropriate?
Monitor bioremediation
performance

Reconsider No Clean-up
clean-up criteria criteria met?

Yes
03019

Treatment completed

3.2-1: Decision process flowchart

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 Chapter 3 – Decision making

3.3.2 Is the site exposed?

In practice, bioremediation is considered suitable for sheltered shorelines

where the polluted sediments are not likely to be removed by tidal action.
These areas are also generally the most environmentally sensitive (table 2).

In certain cases, bioremediation could be used on a partially exposed

beach if the pollutant is buried deeply enough to become trapped.

3.3-1: Sheltered site, Vietnam (CEDRE, France)

3.3-2: Exposed site (CEDRE, France)

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Bioremediation in Marine Oil Spills

Table 2 – Shoreline types and natural cleaning times

(Underlined ¼ most suitable for bioremediation)
Typical time required
Exposure Type of coast
for natural cleaning
HIGH Wave-cut cliffs, seawalls and piers Weeks
ENERGY Wave-cut rock platforms Weeks–months
Pebble beaches Months–years
Mixed sand and gravel beaches Months–years
Coarse-grained sand beaches Years
Fine-grained sand beaches Years
Coral reefs Years
LOW Cliffs, seawalls and piers Months
ENERGY Rock platforms Months
Pebble beaches Months–years
Mixed sand and gravel beaches Months–years
Coarse-grained sand beaches Months–years
Fine-grained sand beaches Years
Tidal mudflats Years
Saltmarsh and freshwater marsh Years
Mangroves Years

3.3.3 Is free oil present?

Bioremediation is not suitable for shorelines that are saturated with oil.
Saturation is clearly indicated by:
* visible significant seepage of oil (not sheen) which can be observed
spontaneously when the tide is coming up, at high tide or when
mild pressure is exerted on the sediment (e.g. by a boot or a tool),
* formation of pools of oil along the shoreline.

Oil saturation is highly dependent on the sediment type and oil properties,
but generally speaking occurs at concentrations higher than about
25,000 mg/kg (or ppm).

In the case of oil-saturated sediment, it is necessary to consider other

standard appropriate clean-up methods to remove this free oil. Bioreme-
diation should be considered at the end of this first cleaning if further
clean-up is required or if the impacted site must be restored.

3.3.4 Is the site a wetland?

Wetlands (saltmarshes and freshwater marshes) are particularly sensitive
areas. When oiled, there are a limited number of response options:
* Leave undisturbed to recover naturally (often preferred),
* Gentle flooding (or flushing) to remove the free oil,

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 Chapter 3 – Decision making

* In-situ burning, or
* Phytoremediation (enhancement of oil degradation and/or habitat
recovery by plant growth).

3.3-3: Example of wetland: During the Prestige, marsh Barrañan,

Galicia, Spain (CEDRE, France)

3.3.5 Is oil biodegradable?

If oil reduction is the main objective, bioremediation can only be applied if
the biodegradability of the oil is significant, in rate and extent.
Information about biodegradability can be obtained either from the
technical literature or directly from chemical analysis. After spillage, oil
is subjected to a number of weathering processes including evaporation,
emulsification, photo-oxidation and dissolution. Thus, the potential for
bioremediation alters as the oil becomes more highly weathered. Therefore,
it may be prudent to chemically analyse samples of oil taken from the
shoreline, in order to accurately determine the actual potential for
bioremediation (i.e. what can be degraded in a reasonable time).
Methods for analysis of oil are given in appendix 1. Examples of the extent
of biodegradability of a range of crude oils are given in appendix 2.
Appendix 3 provides guidelines to estimate the biodegradation potential of
a specific oil according to its composition.
As a general rule, oil with a higher proportion of low-molecular-weight
hydrocarbon (diesel oil, and light crudes) will be more easily biodegraded
than oils containing a significant amount of high-molecular-weight
compounds such as heavy fuel oils and residual oils.
Both physical and biological processes remove oil stranded on coastal
shorelines. Based on the pollutant’s overall biodegradability, a decision

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Bioremediation in Marine Oil Spills

can be made about whether or not bioremediation is a worthwhile option

as a means to remove residual oil. As a guide, if the weathered oil (or
mixtures of oils) stranded on the shorelines is less than 30% biodegrad-
able, it may be justified to question the net benefit of bioremediation
treatments. In summary, the application of bioremediation treatments is
recommended for use on sheltered shorelines where it can enhance the
degradation of >30% of the residual oil mass (see section 3.3.2). However,
consideration should also be given to its ability to promote habitat
recovery, as the removal of some oil may be all that is required to reduce
the residual oil concentration below a toxicity threshold concentration. In
addition, phytoremediation use should be considered as a means to
enhance recovery in wetland environments (see section 3.3.4).

3.3.6 Is the temperature high enough (458C)?

Bioremediation is sensitive to the ambient (sediment/water) temperature.

When the temperature drops, e.g. during seasonal changes, the biode-
gradation activity slows down. As a rule, bioremediation is less likely to be
beneficial if the sediment temperature is below 58C. At lower temperatures
the degradation process will be slower and additional justification for the
application of bioremediation treatments may be required. However, it still
offers some advantage, as it is probably still faster than natural
attenuation. Thus, bioremediation in cold conditions may be considered
in certain circumstances (e.g. for extensive restoration in remote areas
with limited resources).

3.3.7 Is the oxygen concentration sufficient?

Rapid biodegradation of hydrocarbons is an aerobic process and requires

a great deal of oxygen. Therefore, the polluted sediment must be well
oxygenated to sustain optimal biodegradation rates.

Fine, muddy sediments have a low permeability and are often anoxic. The
presence of petroleum in fine-grained sediment often reduces permeability
by clogging spaces between sediment particles. A method for estimating
sediment permeability is given in appendix 4. Organic matter, whether
from a biogenic (plant- or animal-derived) or petroleum source (crude oil),
generally increases the oxygen deficit because the microflora consume
oxygen during metabolism of it. Consequently, oxygen availability is often
a limiting factor in biodegradation, so that the primary goal of a
bioremediation operation may be to rectify this situation if possible.

The oxygen content of the sediment might be estimated in several ways,

and some methods that are commonly used are described in appendix 5.
Criteria for assessing oxygen limitations are provided in Table 3.

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 Chapter 3 – Decision making

Table 3 – Criteria for assessing oxygen limitation

Assessing Criteria for estimating
Decision
oxygen content oxygen limitation
Visual and olfactory Black sediment with sulphidic Consider the
observation odour feasibility of
overcoming
Measurement of An Eh of 5 –50 mV oxygen
oxidation–reduction limitation
potential (redox)
Measurement of Dissolved oxygen 5 0.2 mg/L
dissolved oxygen in
the interstitial water

In terms of sediment permeability, a water flow rate of 0.2 mL/min/cm2 is

insufficient to allow bioremediation without pre-treatment (i.e. physical
mixing) of the sediment to improve permeability. The application of
fertilizer in an effort to employ biostimulation may reduce the available
oxygen even further by increasing microbiological activity which, in turn,
causes oxygen to be consumed at a faster rate.

3.3-4: Measuring the O2 level in the interstitial water (CEDRE, France)

The main techniques for overcoming oxygen limitation are mechanical.

These methods improve sediment permeability by mixing or agitation
(e.g. raking, tilling/harrowing). This must be done carefully to avoid
burying the pollutant even deeper in the sediments. These techniques may
also release oil from the sediment (see section 4). Due to the impact of
mechanical aeration on vegetation, these methods of aeration are not
feasible in vegetated habitats.

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Bioremediation in Marine Oil Spills

The feasibility of aeration techniques depends upon:

* logistical considerations (e.g. equipment availability and accessi-
bility of the site)
* sediment type (i.e. soft mudflat sediments cannot be tilled)
If aeration is not feasible then other remedial strategies should be
considered (See other IMO publications chapter 4 clean-up methods).

3.3.8 Is the nitrogen concentration sufficient?

Hydrocarbon biodegradation requires significant concentrations of nitro-
gen to proceed at an optimal rate. Therefore it is important to quantify the
amount of nitrogen available for biodegradation. This can be determined
by measuring the nitrogen content of the sediment interstitial water.
Methods for assessing nitrogen levels are given in appendix 6.
At a level of 2 mg total N/L or higher, nitrogen is not considered to be
limiting biodegradation; hence, there is no need for fertilization with
nitrogen products. Under these conditions, on aerobic shorelines, the oil
may be left to remediate naturally. In order to estimate the completion of
the bioremediation, monitoring the in-situ nitrogen level is required (see
chapter 4). If the concentration of nitrogen is lower than 2 mg total N/L,
then fertilizer should be considered.
When applying nutrients, it is possible that phosphorus limitation may
occur. Therefore typically a small amount of phosphate is applied with
nitrogen (1:10 ratio). Most fertilizer mixtures already contain phosphorus.

3.4 Conclusion on decision making

If, as the result of working through the structured decision process
flowchart, limitations have been clearly identified, some of the following
actions can be considered:
* Biostimulation by fertilization (nitrogen addition) for lack of
nutrients;
* Biostimulation by sediment aeration for lack of oxygen or sediment
permeability;
* Phytoremediation in marshes or wetlands; and
* Other bioremediation options such as bioaugmentation (section
1.4.2) and enhanced dispersion (section 4.1.5).
Whatever treatment is chosen, the situation will need to be periodically
reviewed to monitor changes in conditions resulting from natural
processes or consequences of the treatment. The application of bioreme-
diation and subsequent monitoring are described in more detail in the
next chapter.

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 Chapter 4
Bioremediation guidelines
implementation

4.1 Bioremediation treatment options

There are two main approaches towards in-situ oil spill bioremediation.
(1) Bioaugmentation: the addition of oil-degrading bacteria to
supplement existing microbial populations.
(2) Biostimulation: the addition of oxygen, nutrients or growth-
enhancing co-substrates, or modification of habitat quality, in
order to stimulate the growth of indigenous oil degraders.

4.1.1 Bioaugmentation
There is a perception that marine oil spills may be effectively treated by the
addition of oil-degrading bacteria. In reality, there is little or no need to add
micro-organisms to oil-contaminated ecosystems. Microbial ecologists
have conclusively demonstrated that oil-degrading bacteria within the
environment increase in numbers following exposure to oil. Furthermore,
field trials have shown that the addition of commercial mixtures or
enriched cultures of indigenous oil-degrading bacteria do not significantly
enhance the rates of oil biodegradation over that achieved by nutrient
enrichment alone.

4.1.2 Biostimulation by nutrient addition

The potential capability of indigenous microflora to degrade oil is a
function of the physical and chemical properties of the seawater and oil,
the environmental conditions, and the biota themselves. It is generally
accepted that nutrient availability is one of the limiting factors that it is
possible to correct. Fertilizing with nitrogen and phosphorus offers great
promise as a countermeasure against marine spills. The ratio of carbon,
nitrogen, and phosphorus to support optimal oil degradation is approx-
imately C:N:P ¼ 100:10:1. Controlled studies suggest that optimal rates of
degradation could be sustained by retaining moderate, non-toxic, renew-
able concentrations of nutrients within the interstitial pore water.
Field and laboratory beach microcosm studies point to interstitial
concentrations of nitrogen of approximately 2 mg/L for optimal biostimu-
lation. Liquid inorganic fertilizers had proven to be effective, but require
frequent application and are comparatively labour-intensive and expen-
sive. Field trials have demonstrated the feasibility of applying commercial
agricultural fertilizers on a periodic basis as a cost-effective bioremediation
treatment. Other advantages of this protocol include product availability
and ease of application.

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Slow-release briquettes tend to decompose through hydrolysis and tidal

action. Because briquettes are moved independent of the oil by tidal action
and waves, it is important that the briquettes are of sufficient density and
appropriately secured for maximum benefit. Slow-release granules are
easily applied, releasing the nutrient when in contact with seawater or
rain. However, in energetic tides, small granules may be washed away
before dissolving, and therefore be ineffective. While bags of nutrients have
been tethered to the beach in mesh containers in field studies to overcome
these limitations, the technique may not be appropriate in full-scale
operations. The use of agricultural slow-release fertilizers may decrease
the cost of fertilizer application. In summary, to date, spreading granular
fertilizer appears to be the most cost-effective way to remediate a site at full
scale.
Proprietary oleophilic nutrient formulations including other organic
products have also been developed. These partition preferentially with
the oil to promote growth of local microbial hydrocarbon degraders at the
oil–water interface.
Product availability and environmental conditions must be considered in
the selection and application of bioremediation agents. For example, low
temperatures (510 8C) and surface oil coatings were shown to reduce the
permeability of the coating of a slow-release fertilizer formulation,
effectively suppressing nutrient release.
The successive actions that are normally undertaken when using
biostimulation by nutrient addition are summarized in table 4.

4.1.3 Biostimulation by oxygen addition

As microbial oil degradation rates within sediments are very slow under
oxygen-limited conditions, increasing the concentration and depth of
oxygen penetration by mechanical means has been shown to improve both
the rate of the natural biodegradation and the efficacy of bioremediation
treatments. If field surveys indicate oxygen limitation within the oiled
sediment, agricultural procedures (e.g. raking, tilling and disking) can be
used to increase the permeability of the sediment.
Precautions must be taken to ensure that any oil which might be released
from the sediment during mechanical treatment is contained (use of
floating booms, sorbent padding) and to avoid transfer of oil to deeper
layers of sediment, particularly when those remain anaerobic. Mechanical
treatments are unlikely to be suitable for use in sensitive habitats with
vegetation, such as marsh or wetlands, since their application would result
in vegetative destruction.
The use of chemical oxidant can be considered for improving oxygen
availability. However, in addition to its high cost, consideration must be
given to potential product toxicity.
As recommended for all site remediation protocols, monitoring pro-
grammes must be implemented to ensure minimal damage from physical
disturbance and chemical toxicity.

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 Chapter 4 – Bioremediation guidelines implementation

Table 4 – Guidelines for the application of bioremediation

products for nutrient enrichment

1 ) Is the product pre-approved for use?

Check safety data sheet.
To ensure treatment success and minimize environmental impacts,
use of commercial bioremediation products should be limited to
those that have passed regulatory screening procedures for
performance and toxicity.

2 ) Product testing
For products that have not been pre-approved, small-scale tests
should be conducted under the direction of the responder to
determine effective application rates that are below the toxicity
threshold for the product.

3 ) Determine the quantity to be applied

Use manufacturer’s recommended dosage. Otherwise, aim to achieve
nitrogen concentration in the interstitial waste of approximately
2 mg/L.

4 ) Select application equipment

Fertilizers should be applied directly to the surface of the site using
standard agricultural procedures. Care should be taken to ensure
that there is no runoff due to over-application of liquid fertilizers.
Slow-release fertilizers may be secured by burying in fine mesh bags.

5 ) Determine schedule and criteria for re-application

Nutrients should be re-applied when the concentration approaches
background levels. The frequency of re-application should be
determined by analysis of the nutrient concentrations in the pore
water.

6 ) Check tidal conditions

Tidal conditions should be considered to facilitate nutrient
penetration in the sediment. This will most likely be at low or falling
tide.

7 ) Consider preparation of the sediment

The sediment surface may be physically prepared to facilitate
nutrient delivery or penetration (e.g. raking).

8 ) Control the application

Apply the product using a constant and controlled application rate.

9 ) Consider post-application measures

Re-apply the product as necessary, according to the result of the
analytical nitrogen level monitoring.

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4.1-1: Beach aeration (CEDRE, France)

4.1.4 Phytoremediation
Freshwater wetlands and saltmarshes are among the most sensitive
ecosystems and the most difficult to clean. Application of traditional oil
spill clean-up techniques within this habitat may cause more damage
than the oil itself. Consideration is now being given to on-site remediation
procedures for impacted wetland sites. Phytoremediation takes advantage
of the inherent capacity of tolerant wetland plant species to aerate the
rhizosphere (region around the roots) as a means of stimulating aerobic
biodegradation. Plants also may release exudates and enzymes that
stimulate microbial activity. In addition, stimulated growth or replanting
can help to control coastal erosion and loss of habitat. Operations should
incorporate the advice of biologists with experience in wetland ecology.

4.1-2: Phytoremediation operation, Quebec (DFO Canada)

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 Chapter 4 – Bioremediation guidelines implementation

4.1.5 Enhanced dispersion

Microbial attack of oil spilled in the marine environment principally occurs
at the oil–water interface. Thus, facilitating an increase in the oil–water
interface may enhance the rate and extent of biodegradation as the oil
becomes more accessible to nutrients, oxygen and bacteria. Increases in
microbial activity and oil biodegradation have been correlated with the
addition of chemical dispersants, surface-active agents, biosurfactants,
and the facilitation of oil–mineral aggregate formation. Only pre-approved
products should be used and the manufacturer’s instructions for use of
the product should be followed. Application of the product should facilitate
the transport of oil stranded within coastal sediments into the water
column at concentrations below the threshold value which will cause
significant toxic effects. Controlled feasibility studies (i.e. plot studies)
should be conducted prior to full response operations to ensure that the
chosen procedure will not transport oil deeper into the sediment. For surf-
washing operations, where oil dispersion is facilitated by mechanical
procedures to accelerate the interaction between oil and mineral fines,
consideration must be given to the ecological impacts associated with
physical disturbance of the site. For protection of near-shore habitat, a
simultaneous biological monitoring programme should be implemented.

4.2 Monitoring
Monitoring programmes are needed to verify ongoing treatment success
without detrimental effects on the environment. Treatment success can be
assessed by chemical analysis to illustrate the reduction in residual oil
concentrations or changes in composition. Biological studies can be used
to show a reduction in oil-induced effects. Detrimental effects include any
changes to ecosystem structure and function as a result of bioremediation
treatment.
Monitoring programmes are also needed to identify operational endpoints
for the remediation operation. Since some trace of hydrocarbons will be
found at all spill-impacted sites, regardless of the treatment process used,
operational endpoints for bioremediation should be based on evidence of
attaining an acceptable level of residual oil and/or habitat recovery.
Monitoring programmes should document the net benefit of bioremedia-
tion over natural attenuation.
Heterogeneity within the natural environment is the major obstacle to
overcome in the design of programmes to monitor bioremediation success.
To ensure that the data accurately reflect reality, it is paramount that all
survey/sampling plans are based on standard statistical procedures.
Efforts should be made to ensure that an adequate number of samples are
taken to resolve significant differences, if any, to illustrate treatment
success. Appendix 7 gives basic recommendations for a sampling plan.
A comprehensive monitoring programme will cover changes in environ-
mental factors that can influence bioremediation rates, the efficiency of
treatments, evidence of oil biodegradation, toxicity reduction, and habitat
recovery. For operational guidance, the monitoring programme must be

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Bioremediation in Marine Oil Spills

capable of identifying detrimental treatment effects (e.g. toxicity of the

bioremediation agent or oil degradation by-products). For example, by
using biological tests which employ plants and animals appropriate for the
affected environment. The ecological significance of these biotests is
improved by the use of a multi-trophic-level test battery approach, which
would produce results for bacteria, plants, invertebrates and vertebrates.
An integrated interpretation of this kind of data with physical and
chemical data should provide a more accurate picture of the progress of
the bioremediation process.

4.2.1 Monitoring of treatments

Analysis of nitrogen can be done either on or off site. For off-site analysis of
total nitrogen (e.g. Kjeldahl nitrogen), sediment samples should be kept
frozen (–20 8C) until analysed. For analysis of organic nitrogen, samples of
interstitial water should be taken using nutrient wells. On-site measure-
ments can be done using colorimetric kits or electronic probes, following
manufacturer’s guidelines. Nutrients should be monitored weekly at a few
representative sites to determine the time required for nitrogen depletion.
The sampling strategy can be modified depending on results.
Dissolved oxygen concentrations can also be measured on site with
colorimetric test kits and electronic probes. The requirement for oxygen
analysis depends on the treatment. Where oxygen is the limiting factor and
aeration is part of the bioremediation strategy, levels of dissolved oxygen
should be measured to determine the effectiveness of aeration. Both
oxygen and carbon dioxide concentrations may be used as an indicator of
metabolic activity by micro-organisms.
Total Petroleum Hydrocarbon (TPH) can be measured using gravimetry,
spectrometry or chromatography methods to determine oil loss. The
progress of degradation can be monitored by detailed chemical analysis
(GC–MS) on selected sediment samples according to TPH levels. To
measure the benefit of the treatments, the results should be compared
to similar untreated areas. Samples should be taken before treatment and
then every two months. The samples should be stored frozen until
analysis.
Environmental assessments that take into account potential changes in
ecosystem structure and function should be conducted to ensure the safe
application of bioremediation agents and to verify product efficacy. Two
complementary approaches have evolved for environmental assessment:
bioassessment and bioassay. Consultation with the appropriate regulating
bodies and experts is recommended.
For bioassessment, changes in benthic community structure can be used
as a means of assessing ecosystem response to contaminated sediments
in aquatic ecosystems. Of particular importance are the macrobenthic
invertebrates because of their basic longevity, sedentary lifestyles,
proximity to sediments, influence on sedimentary processes, and trophic
importance. The bioassessment process can readily include potential
impacts on vegetation.

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 Chapter 4 – Bioremediation guidelines implementation

If the aim of oil spill bioremediation is to return a site to its pre-spill

condition, recolonization of impacted areas should be a primary process to
monitor in bioassessment.
Bioassays are toxicity tests that measure organisms’ response on
exposure to a sample. A single-species biotest cannot represent the range
of sensitivity of all biota within an ecosystem. To improve ecological
relevance, a test battery approach with species from different trophic levels
may be required. While any living organisms can be used, toxicity tests
with fish and macroinvertebrates have been standardized by environ-
mental agencies to assess the hazards of industrial wastes to aquatic
systems. Major criteria to consider in the selection of species for sediment
toxicity testing include behaviour in sediment, sensitivity to test material,
ecological and economic relevance, availability and geographical distribu-
tion, taxonomic relation to indigenous animals, acceptability for use in
toxicity measurement (e.g. standardized test method) and tolerance to
natural sediment characteristics such as grain size. In general, assays
using whole sediment samples and larval or juvenile life stages are the
most sensitive and are recommended.

4.2-1: Sediment sampling (CEDRE, France)

4.2.2 Operational endpoints for bioremediation

Bioremediation treatments should be terminated when it is deemed that
contaminant concentrations are reduced to acceptable levels, according to
the usage and environmental specificity of the site, or if detrimental effects
from the treatment strategy are identified. Cost/benefit analysis should be
considered in the choice of acceptable levels. Like most oil spill counter-
measures, it is futile to expect bioremediation techniques to remove all
traces of residual hydrocarbons. In terms of ecological relevance, clear
evidence of habitat recovery, such as toxicity limits within regulatory
guidelines and return of original community structure, should suffice.

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Bioremediation in Marine Oil Spills

FURTHER READING AND BIBLIOGRAPHY

Atlas, R. M. and Bartha, R. 1972. Degradation and mineralisation of
petroleum in seawater: Limitations by nitrogen and phosphorus. Biotech-
nology Bioengineering, 14, 309–317
Atlas, R. M. and Bartha, R. 1992. Hydrocarbon biodegradation and oil spill
bioremediation advances. Microbial Ecology, 12, 287–338.
Bragg, J. R., Prince, R. C., Harner, E. J. and Atlas, R. M. 1994. Effectiveness
of bioremediation for the Exxon Valdez oil spill. Nature, 368, 413–418.
Lee, K. and Levy, E. M. 1987. Enhanced biodegradation of a light crude oil in
sandy beaches. Proceedings of the 1987 Oil Spill Conference, American
Petroleum Institute, Publication Number 4452, pp. 411–416.
Lee, K. and Merlin, F. X. 1999. Bioremediation of oil on shoreline
environment: development of techniques and guidelines. Pure and Applied
Chemistry, 71, 161–172.
Le Floch, S., Merlin, F. X., Guillerme, M., Dalmazzone, C. and Le Corre, P.
1999. A field experimentation on bioremediation: BIOREN. Environmental
Technology, 20, 897–907.
Merlin, F. X., Lee, K., Swannell, R. P. J., Oudot, J., Basseres, A., Reilly, T.,
Chaumery, C., Dalmazzone, C. and Sveum, P. 1994. Protocol for exper-
imental assessment of bioremediation agents on a petroleum polluted
shoreline. Proceedings of the 17th AMOP Technical Seminar, June 8–10,
Vancouver, British Columbia, Canada, pp. 465–479.
Merlin, F. X., Pinvidic, P., Chaumery, C. Oudot, J., Swannell, R. P. J., Lee, K.,
Reilly, T., Ducreux, J. and Dalmazzone, C. 1995. Bioremediation: results of
the field trials of Landevennec (France). Proceedings of the International Oil
Spill Conference, Long Beach, US, pp. 917–918.
Prince, R. C. 1993. Petroleum spill bioremediation in marine environments.
Critical Reviews in Microbiology, 19, 217–242.
Swannell, R. P. J., Lee, K. and McDonagh, M. 1996. Field evaluations of oil
spill bioremediation. Microbiology Reviews, 60, 342–365.
Swannell, R. P. J., Mitchell, D. J., Dunn, R. M., Groenewoud, H., Foekema, E.,
Scholten, M., Merlin, F., Le Floch, S., Lee, K. and Borseth, J. F. 1996. A field
evaluation of bioremediation strategies for mudflats. Proceedings of the
Coastal Zone Canada. Rimouski, Quebec, Canada, 12–15th August.
Venosa, A. D., Suidan, M. T., Wrenn, B. A., Strohmeier, K. L., Haines, J. R.,
Eberhart, B. L., King, D. and Holder, E. L. 1996. Bioremediation of an
experimental oil spill on the shoreline of Delaware Bay. Environmental
Science and Technology, vol. 30, no. 5, pp. 1764–1775.

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 Appendix 1
Measurement and analysis
of hydrocarbons in
marine sediments

Total hydrocarbons (THCs)

After drying of the sample at 560 8C (optional), hydrocarbons are extracted
by sonication or in a soxhlet apparatus with dichloromethane (DCM).
Following optional purification on a florisil column that retains most
biogenic lipids, THCs can be determined by one of these three methods:
* microgravimetry,
* infrared spectrophotometry, and
* gas chromatography using flame ionization detection (GC–FID) – the
total area of the chromatogram is calculated relative to the area
under the curve for a known amount of added internal standard.

Biodegradation
The extent and rate of biodegradation can be determined by GC–FID or by
gas chromatography-mass spectrometry (GC–MS) with reference to a
biodegradation-resistant compound (biomarker) in the oil, like nor-hopane
or hopane (m/z 191).

Biomarkers, in particular hopanes and steranes, present as trace

components in crude oil, are recalcitrant to environmental alterations
and often have compositions characteristic of sources of petroleum and,
therefore, are used as source identifiers of petroleum pollution. These
compounds are used to study the fate and effects of oil released in the
environment that undergoes extensive physical, chemical, and biological
alterations, collectively known as weathering. Quantitative measurement
and the use of compositional indices of these compounds can be used to
determine the rate of degradation, natural or man-induced (bioremedia-
tion), in environmental samples.

Hopanes are widely used as internal markers in environmental and

petroleum remediation studies, due to the persistency of this class of
natural products. Hopanes present in sediments range from C27 to C35,
usually with the C30 isomers predominant.

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Bioremediation in Marine Oil Spills

Methodology used to isolate petroleum residues from sediments

Extraction
A variety of extraction techniques can be employed to isolate petroleum
residues in sediments. Soxhlet extraction, sonication, shake-out and
supercritical fluid chromatography represent a few techniques that are
most commonly used. The most common technique is soxhlet extraction
for isolation of complex groups of compounds. In this procedure, as well as
the other techniques mentioned, various solvents are used to isolate the
contaminants of interest. The type of solvent used depends on the polarity
of the compounds of interest. For the extraction of petroleum hydro-
carbons, dichloromethane is a typical and convenient solvent.

Purification of sediment extracts

If the sediment extracts contain compounds that will interfere with the
measurement of the analytes of interest, then a purification step is
essential. Interfering compounds may behave in a similar way chemically
to the compounds of interest or any compound that comes through the
extraction intact. A number of chromatography support materials are used
to remove these interfering compounds. The most common ones are silica
gel and alumina. Other techniques may be employed as well, provided that
they are adequate in removing the interference. A variety of solvents,
ranging from non-polar (hexane) to polar (methanol), may be used to purify
extracts prior to gas chromatography–mass spectrometry (GC–MS)
analyses. These supports have been applied using column chromatog-
raphy or in recent years through the use of solid-phase extraction (SPE).
SPE cartridges are available commercially through suppliers of chroma-
tography products.
As an example, the fractionation into saturated, aromatic and resins
fractions can be achieved by using column chromatography (activated
silica gel 60–100 mesh); successive elutions with hexane, hexane–
dichloromethane (3/2 v/v) and methanol enable the saturates, aromatics
and then the polar fraction (i.e. resins plus asphaltenes plus biogenic
lipids) to be obtained sequentially.

Gas chromatography–mass spectrometry analyses

Quantitative analyses of sediment extracts are performed by comparing
the detector signal produced by the sample with that of defined standards.
The use of a flame ionization detector (FID) sensitive to petroleum
compounds or, which is more generally applicable, a mass-selective
detector (MSD) or even a mass spectrometer (MS) is essential. Due to
incomplete separation, several co-eluting compounds can be present
under a single detector signal. Therefore, the shape and size of the signal
have to be critically examined. By using an MSD or an MS as the detector,
either the molecular mass or characteristic mass fragments are
adequately recorded. If only FID is available, the relative retention time
and the signal size should be confirmed on columns with stationary

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 Appendix 1

phases of different polarities or by the use of multi-dimensional gas

chromatography techniques.
The aliphatic and aromatic fractions can be analysed by GC–MS or FID.
The reasons mentioned above should be considered before the use of FID.
A selective ion monitoring (SIM) method is employed, using GC–MS to
increase sensitivity and to eliminate interferences from compounds that
may have similar ions and cause difficulty in the quantification process.
The GC–MS data provide identification (using retention time and spectral
matching against reference standards) and quantification of analytes
present in the fractions.
The reproducibility of injection is controlled by the use of internal
standards not present in the sample. For GC–MS, 13C-labelled or
perdeuterated aliphatics and PAHs must be used as internal standards.
These internal standards behave in a similar way chemically and therefore
would co-elute with the parental compounds and could not be used with
GC–FID.
It is essential, regardless of the method used to extract, purify and analyse
sediment extracts for petroleum residues, to have adequate quality-
assurance protocols that include inter-laboratory calibration exercises.
To report the biodegradation of THCs, molecular classes or individual
compounds are expressed in percent of the initial values in the original oil.

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 Appendix 2
Indicative biodegradability
of some petroleum oil products

Gasoline 100%

Jet fuel 100%

Diesel oil 85%

Crude oil 30–70%

Heavy fuel 10–20%

Asphalts 55%

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 Appendix 3
Assessing the biodegradation
potential of an oil

The composition of a petroleum product is an indication of the

biodegradation rate that can realistically be obtained within a reasonable
timeframe (i.e., about one to two years).

The biodegradability of a petroleum product is estimated by GC–FID*,

GC–MS{ (with internal standards) and SARA{ fractionation; the potential
biodegradability of the different oil fractions is given in table A3-1 (bold
characters); unresolved is considered here as the sum of GC-unresolved
complex mixture (UCM) and the compounds which are not detected by GC;
these can be quantified by comparison of the SARA fractionation results
and the quantification of the GC UCM; (as a rough indication, total GC-
detected compounds are 100% for a diesel oil, 60–80% for crude oil, and
around 50% for heavy fuel oil). See table A3-1: Assessment of the
biodegradability of three petroleum products.

Based on the rough estimate of a pollutant’s overall biodegradation rate, a

decision can be made as to whether or not bioremediation is a worthwhile
option. If the hydrocarbon is already highly degraded, the prospects for
biodegradation are very poor, and the evaluation method proposed above
cannot be applied. If there are few or no tall peaks on the chromatogram of
saturated fractions (tall peaks correspond to straight-chain linear
saturated fractions which are preferentially biodegraded by micro-
organisms), this generally indicates that the hydrocarbon is already quite
degraded.

Global assessment of degradability of different petroleum products is also

given in appendix 2.

Figure A3-1 shows the change in the chromatograms of saturated and

aromatic fractions of a petroleum product as a function of biodegradation
status.

* GC–FID: Gas Chromatography with Flame Ionization Detector

{
GC–MS: Gas Chromatography with Mass Spectrometry detector
{
SARA: Saturates, Aromatics, Resins, Asphaltenes separation.

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Bioremediation in Marine Oil Spills

Total Hydrocarbons Aromatics

A3-1: Change in the gas chromatograms of saturated

and aromatic fractions of hydrocarbons from drilling cuttings as a function
of their biodegradation status. The numbers represent the carbon numbers
of n-alkanes. UCM, unresolved complex mixture; IS, internal standard
n-1-icosene; NPH, naphthalenes; FLU, fluorenes; PHN, phenanthrenes;
DBT, dibenzothiophenes; T0, initial; D30–D270, day of experiment.
(Muséum national d’histoire naturelle, Paris, France)

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 Table A3-1 – Assessment of biodegradability of three petroleum products
LIGHT to MEDIUM CRUDE OIL
DIESEL OIL HEAVY FUEL
(e.g. arabian light crude)
example of Biodegradation example of example of Biodegradation example of example of Biodegradation example of
composition rate % biodegradability composition rate % biodegradability composition rate % biodegradability
% % % % % %

SATURATES

n + branched 24 95 23 10 85 8.5 4 60 2.5

unresolved 36 75 27 24 50 12 21 15 3

AROMATICS

GC-resolved 3 95 3 8 90 7.2 5 50 2.5

unresolved 27 70 19 31 55 16.5 49 8 4

RESINS + 10 10 1 27 15 4 21 0 0
ASPHALTENES

TOTAL 100 73 100 48 100 12

Appendix 3
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 Appendix 4
Estimation of sediment permeability

The permeability of sediment at and above the polluted layer can be

estimated by digging small holes in the sediment and checking how much
time it takes them to refill with interstitial water or how much time it takes
the water to escape after the holes are filled with water.
A more detailed estimate can be obtained by using a small cylinder (about
10 cm in diameter and height) that is open at both ends. The cylinder must
be pushed into the sediment up to its midpoint, while keeping the cylinder
straight and disturbing the sediment as little as possible. A known volume
(250 mL) of water should be poured into the cylinder. The time it takes the
water to flow through the sediment should be measured. Water flow
relative to the surface area unit can be used as a measure of sediment
permeability.
These analyses should be carried out at several locations on the site, as
there are likely to be variations in sediment composition or grain size, and
runoff may be occurring. When sediment permeability is less than
0.20 mL/min/cm2, optimal oil degradation rates may be limited.

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 Appendix 5
Assessing oxygen content

The oxygen content of the sediment can be assessed in several ways and
replicate analyses should be carried out at several locations on the site,
especially if there are variations in sediment composition and grain size, or
if runoff is thought to be occurring.

1 Observation
During digging in fine-grained sediment, if a black layer of sediment and/or
a sulphidic odour associated with hydrogen sulphide (H2S) production is
observed, it is advantageous to determine the depth of the anoxic sediment
layer. However, it must first be confirmed that the black colour is not due
to the petroleum itself. The odour of the sediment is a good indicator in this
regard. Except in clear-cut situations (muddy and sand-mud environ-
ments), it is insufficient to observe that the oxygen supply is low, and
measurements are required to assess the degree of oxygenation.
Dissolved oxygen in the interstitial water can be measured with
colorimetric test kits or an oxygen meter. A hole is dug in the sediment.
The water is removed with a syringe. The hole is allowed to fill again with
interstitial water. The probe is placed in the interstitial water without
using the probe to mix the water, and the reading is recorded. The
presence of petroleum in the water can foul the membrane of the oxygen
meter. It is recommended that, between each measurement, a reading be
taken in the open water at the site to check whether instrument drift is
occurring. The probe and its membrane should be sprayed periodically
with clean water from the site, using a pipet. An oxygen concentration of
0.2 mg/L or less in the interstitial water suggests that oxygen may be a
limiting factor.

2 Measurement of oxidation–reduction potential

The oxidation–reduction potential (or redox potential) of sediment
indicates its degree of oxygenation (aerobic/anaerobic conditions); it is
based on the proportion of reduced or oxidized compounds present in the
sediment. The redox potential is measured at various depths by placing an
AgCl reference electrode directly in the water above the sediment, and a
platinum electrode in the sediment to a depth that includes the polluted
layer. If a combination redox electrode is used, simply insert it in the
sediment to the depth of oil penetration. Record the millivoltage. By taking
measurements at various locations, profiles of redox potential are obtained
for the surface layer of the sediments. A potential of less than –50 mV is
considered indicative of a lack of oxygen.

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 Appendix 6
Assessing nitrogen concentration

In the coastal environment, nitrogen is available to microflora in mineral

form, mostly nitrate (NO3–) and ammonium (NH4+), as well as in organic
form and possibly nitrite (NO2–). Nitrate concentrations in seawater range
from 0 to 50 mmol/L, with the highest concentrations found in deep water
layers. In the surface layer of the ocean, wide seasonal variations occur
which are linked to phytoplankton growth; low concentration in summer
below method detection limits, and high concentration in winter (up to
40 mmol/L). Ammonia nitrogen (NH4+) comes from animal excretions and
bacterial decomposition of organic nitrogen compounds. It serves as an
effective tracer for urban and agricultural pollution. Near the coast,
nutrient concentrations can be appreciably higher because of inputs from
zones with a high biological productivity (algal beds, marshes, etc.) or from
human activities, predominantly agricultural and possibly urban develop-
ment. For example, high nitrogen and phosphorus concentrations
(nutrient enrichment associated with household and agricultural
effluents) may cause eutrophication.

Nitrates are transformed fairly slowly in the natural environment and are
not particularly toxic. By contrast, ammonium ions and nitrites are toxic
but are rapidly converted. Thus, substantial concentrations of nitrite and
ammonium are rarely encountered in nature, in contrast to nitrate ions
that sometimes accumulate. One of the environmental concerns associ-
ated with bioremediation is that of high nitrite and/or ammonium ion
concentration that may suppress the activity of microflora. However,
ammonia concentrations above toxicity threshold limits are rarely
exceeded in near-shore waters as a result of bioremediation operations
due to natural dispersion processes (e.g. waves, tides, currents).

Phosphorus is rarely a limiting nutrient since demand for phosphorus

(minimum concentration in the order of 2 mmol/L) is ten times lower than
that for nitrogen.

Nutrient levels should be measured at the site, in the interstitial water,

possibly in the sediment, and especially in the open water near the site, to
determine whether nitrogen (and phosphorus) availability is a limiting
factor for biodegradation. The following analyses should be performed on
the interstitial water and the open water surrounding the site:
* determination of nitrates, ammonia nitrogen and total organic
nitrogen (Kjeldahl), and
* phosphorus and nitrite levels may be determined as well.

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 Appendix 6

At a level of 2 mg/L or higher (that is, 140 mmol/L), nitrogen is not

considered to be a limiting factor for biodegradation, hence there is no
need for nitrogen fertilization. Owing to seasonal variations, nutrient
concentrations may sometimes be limiting in summer and excessive in
winter, and this should be considered in deciding whether or not to apply
nutrients.

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 Appendix 7
Basic recommendations
for the sampling plan

Much of the information needed to decide whether or not bioremediation is

appropriate and the subsequent planning and monitoring of the operation
can be derived from measurements and sampling at the proposed site.
To ensure that the resulting data accurately reflect reality, it is paramount
that the measurements and sampling be conducted logically and in
accordance with a pre-defined and well-designed survey or sampling plan.
In theory, the more extensive the sampling and analyses (measurement
and sample collection using a grid encompassing the entire site), the more
accurate is the information. In fact, the greater the variability in the
parameter being measured, the finer the grids should be in order to obtain
an accurate picture of the site.
The most objective method consists of selecting within the grid a certain
number of measurement and collection points (random survey plan). It is
important that the co-ordinates of these points be randomly located within
the grid to avoid any operator bias. Measurements obtained in this way are
used to determine the mean value and the dispersion of observed values
about the mean.
The optimum number of points can be determined by: (1) tracking changes
in the dispersion of measured values relative to the number of measure-
ments made, or (2) statistical analysis of analytical variability. In the first
procedure, sample numbers are gradually increased until they no longer
produce an associated decrease in the standard deviation, at which point
additional measurements are not required, as they will not produce greater
precision. The alternative is to establish a priori a given level of acceptable
variability and then determine statistically the minimum number of
samples to collect and analyse to achieve that level of variability. For
example, if the number of samples to analyse to give a standard deviation
of 5% is 30, how many fewer samples can be taken to have a standard
deviation of 10%?
Often, when the targeted parameters present considerable spatial
variability, it may be worthwhile dividing the site into more homogeneous
subunits, which will be measured or sampled in the same way but
independently (stratified random survey plan). For example, when the
petroleum content in sediments needs to be determined, and a major part
of the pollution is concentrated in a band situated near the high-water
mark, the site should be divided into the upper beach and the rest of the

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 Appendix 7

site, for evaluation purposes. For each of these zones, both the mean
petroleum concentration and its dispersion can be determined.
In practice, it is convenient to do this work in two stages.
(1) Carry out an initial survey aimed solely at obtaining a rapid,
general idea of how the parameter of interest is distributed, and
divide the site into smaller subunits if necessary. This initial
step can be carried out whenever possible, either through
simple observation when the parameter being studied is visible
(e.g. colour of petroleum-impregnated sediment) or by using
rapid, inexpensive field measurement methods (e.g. an infrared
spectrophotometer to measure sediments impregnated with a
colourless refined petroleum product). When observations
cannot be made and no practical field measurement technique
exists, the investigator’s judgement and experience can be used
in gauging the potential range of variability to determine
whether and how the site should be divided. For example, to
study nutrient concentrations in interstitial water, factors
related to potential variability, such as runoff from upstream,
sediment type and permeability, are taken into account in
dividing the site into more homogeneous subunits.
(2) Every subunit is then accurately measured and sampled
(suitable measurement or analysis method) according to a
random survey plan (random measurement/sample co-ordi-
nates on a pre-defined grid). For every subunit, the mean value
and dispersion are determined (standard deviation with 95%
confidence limits) for the parameter under study.
When transporting samples from the site for further analyses, specific
storage conditions for the samples may be required to preserve their
quality.

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