GUIDELINES FOR THE BIOREMEDIATION OF MARINE SHORELINES 

AND FRESHWATER WETLANDS 

Prepared by: 

1

Xueqing Zhu, 2Albert D. Venosa, 1Makram T. Suidan, and 3Kenneth Lee 

1

University of Cincinnati 

Department of Civil and Environmental Engineering 

Cincinnati, OH 45221 

2

U.S. Environmental Protection Agency 

National Risk Management Research Laboratory 

Cincinnati, OH 45268 

3

Department of Fisheries and Oceans-Canada 

Bedford Institute of Oceanography 

Marine Environmental Sciences Division 

Dartmouth, Nova Scotia B2Y 4A2 

September, 2001 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Risk Management Research Laboratory 

Land Remediation and Pollution Control Division 

26 W. Martin Luther King Drive 

Cincinnati, OH 45268 
Acknowledgments 

The preparation of this guidance document was performed under the direction of 

Albert D. Venosa of EPA’s Treatment and Destruction Branch, Land Remediation and Pollution 

Control Division, National Risk Management Research Laboratory, Cincinnati, OH. The report 

was prepared under EPA Contract 68-C7-0057, Task Order 23 by the University of Cincinnati 

with the assistance of the Department of Fisheries and Oceans-Canada. 

Disclaimer 

This report has been reviewed and approved for publication by the Land Remediation and 

Pollution Control Division, National Risk Management Research Laboratory of the U.S. 

Environmental Protection Agency. Mention of company names, trade names, or commercial 

products does not constitute EPA endorsement or recommendations for use. 

Further Information 

For further information, contact: 

Albert D. Venosa, Ph.D. 

U.S. EPA 

26 W. Martin Luther King Drive 

Cincinnati, OH 45268 

Tel: 513-569-7668 

Fax: 513-569-7105 

Email: venosa.albert@epa.gov
EXECUTIVE SUMMARY 

The objective of this document is to present a detailed technical guidance document for use by 

spill responders for the bioremediation of marine shorelines and freshwater wetlands 

contaminated with oil and oil products. Technical personnel who are responsible for designing 

and operating field bioremediation processes as well as consultants and equipment manufacturers 

will also find it useful. This manual presents a rational approach for the design of bioremediation 

processes pertinent to cleanup of oil-contaminated marine shorelines and freshwater wetlands. 

This document evaluates current practices and state-of-the-art research results pertaining to 

bioremediation of hydrocarbon contamination relative to types and amounts of amendments 

used, frequency of application, assessment of the extent of bioremediation, sampling, and 

analysis. The scope of the document is limited to marine shorelines and freshwater wetlands 

because of definitive results from recently completed, EPA-sponsored field studies. The final 

product is presented in a report form that is understandable by responders, on-scene coordinators, 

and remediation specialists. This report includes a thorough review and critique of the literature 

and theories pertinent to oil biodegradation, nutrient dynamics in shorelines, and analytical 

chemistry of oil and remediation nutrients. 

A planning approach to site identification, evaluation, and selection along with information on 

field investigations is also presented. The manual includes examples of bioremediation options 

and case studies of bioremediation applied to marine shorelines and freshwater wetland 

environments. 

The contents of this document are arranged in a logical sequence first to provide basic 

information for the evaluation of bioremediation as a spill response option followed by 

guidelines for application that includes methods to monitor its effectiveness. Thus, Chapter 1 

presents an overall introduction and background discussion of bioremediation including 

occurrence of oil spills, response methodologies, and a summary of the scope, organization, and 

objective of the manual. Chapter 2 covers the basic information about oil, shorelines, 

mechanisms of oil biodegradation, and a state-of-the-art review of controlled laboratory 

experiments and field trials of oil biodegradation and nutrient dynamics in shoreline 

environments. For additional background information, Chapter 3 provides a more thorough 

review and critique of current analytical methods used to monitor and verify oil spill 

bioremediation success. Chapter 4 summarizes major biostimulatory and bioaugmenting 

amendment methods and their application strategies. Chapter 5 is the heart of the document and 

presents the actual guidelines for designing, planning, and implementing oil bioremediation in 

the field, including site characterization, evaluation of appropriate bioremediation technologies, 

and the selection of the most appropriate technology for a specific site. Finally, Chapter 6 

provides guidelines for assessment and interpretation of field results and provides help in 

assessing endpoints of bioremediation (i.e., when treatment is considered complete). 

The overall conclusions reached by the guidance manual are as follows. First, with respect to 

marine sandy shorelines, natural attenuation may be appropriate if background nutrient 

concentrations were high enough that intrinsic biodegradation would take place at close to the 

expected maximum rate. The Delaware study proved this clearly. Certainly in nutrient-limited 

places like Prince William Sound, Alaska, nutrient addition should accelerate cleanup rates 
many-fold. However, the decision to use the natural attenuation approach may be tempered by 

the need to protect a certain habitat or vital resource from the impact of oil. For example, using 

Delaware as the model, every spring season, horseshoe crabs migrate to the shoreline of 

Delaware for their annual mating season. Millions of eggs are laid and buried a few mm below 

the surface of the sand. Migrating birds making their way from South America to Arctic Canada 

fly by this area and feed upon these eggs to provide energy to continue their long flight. If an oil 

spill occurred in February or March, it would certainly be appropriate to institute bioremediation 

to accelerate the disappearance of the oil prior to the horseshoe crab mating season despite the 

expected high natural attenuation rate. So, even in the case where background nutrients are high 

enough to support rapid biodegradation, addition of more nutrients would help protect such a 

vital resource. If the spill occurred during the summer, and no vital natural resources were 

threatened by the spill, then reliance on natural attenuation might be the wisest course of action. 

Of course, removal of free product and high concentrations of oil should still be conducted by 

conventional means even if a no bioremediation action is warranted by the circumstances. 

With respect to freshwater wetlands, the St. Lawrence River study demonstrated that, if 

significant penetration of oil takes place into the subsurface, biodegradation would take place 

very slowly and ineffectively. This is because of the anaerobic conditions that quickly occur in 

these types of saturated environments, and anaerobic biodegradation of petroleum oils is much 

slower and less complete than under aerobic conditions. One of the objectives of the St. 

Lawrence River experimental design was to determine the amenability of wetlands to 

biodegradation when oil has penetrated into the sediment. The oil was artificially raked into the 

sediment to mimic such an occurrence. Consequently, no significant treatment effects were 

observed because all the nutrients in the world would not stimulate biodegradation if oxygen 

were the primary limiting material. If penetration did not take place beyond a few mm, then 

bioremediation might be an appropriate cleanup technology, since more oxygen would be 

available near the surface. It is clear that whatever oxygen gets transported to the root zone by 

the plants is only sufficient to support plant growth and insufficient to support the rhizosphere 

microorganisms to degrade contaminating oil. 

However, if ecosystem restoration is the primary goal rather than oil cleanup, the St. Lawrence 

River study strongly suggested that nutrient addition would accelerate and greatly enhance 

restoration of the site. Abundant plant growth took place in the nutrient-treated plots despite the 

lack of oil disappearance from the extra nutrients. Furthermore, the stimulation lasted more than 

one growing season even though nutrients were never added after the first year. Clearly, the 

plants took up and stored the extra nitrogen for use in subsequent growing seasons, so restoration 

of the site was abundantly evident in a few short months. 

Thus, in conclusion, the decision to bioremediate a site is dependent on cleanup, restoration, and 

habitat protection objectives and whatever factors that are present that would have an impact on 

success. Responders must take into consideration the oxygen and nutrient balance at the site. If 

the circumstances are such that no amount of nutrients will accelerate biodegradation, then the 

decision should be made on the need to accelerate oil disappearance to protect a vital living 

resource or simply to speed up restoration of the ecosystem. If there is no immediate need to 

protect a vital resource or restore the ecosystem, then natural attenuation may be the appropriate 

response action. These decisions are clearly influenced by the circumstances of the spill. 
TABLE OF CONTENTS 

Chapter 1 INTRODUCTION ..............................................................................................1 

1.1 Occurrence of Oil Spills ..................................................................................1 

1.2 Response to Oil Spills to Marine Shorelines and 

Freshwater Environments ..................................................................................3 

1.2.1 Natural methods ..................................................................................3 

1.2.2 Physical methods ..................................................................................4 

1.2.3 Chemical methods ..................................................................................5 

1.3 Bioremediation as an Oil Spills Cleanup Technology ..................................5 

1.4 Scope of This Document ..................................................................................7 

1.4.1 Objectives ..............................................................................................7 

1.4.2 Organization of the guidance document ..............................................7 

Chapter 2 FACTORS AFFECTING NATURAL OIL BIODEGRADATION 

AND BIOREMEDIATION SUCCESS ..........................................................9

2.1 Physical-Chemical Properties of Crude Oil and Oil Products ......................9 

2.1.1 Chemical composition of crude oils and oil products ......................9 

2.1.2 Physical properties of oil ....................................................................12 

2.2 Behavior of Oil in the Environment ....................................................................13 

2.2.1 Weathering processes ................................................................................13 

2.2.2 Oil and shoreline interactions ....................................................................16 

2.2.3 Shoreline sensitivities ................................................................................16 

2.3 Biodegradation of Oil ............................................................................................17 

2.3.1 Mechanism of oil biodegradation: a microbiological perspective ........17 

2.3.2 Environmental factors affecting oil biodegradation ................................21 

2.3.3 Evaluation of oil biodegradation: application of biomarkers ....................23 

2.4 Laboratory Studies on Bioremediation of Oil ............................................24 

2.4.1 Bioaugmentation ................................................................................25 

2.4.2 Biostimulation ................................................................................26 

2.5 Demonstrations of Oil Bioremediation Under Field Conditions ....................28 

2.5.1 Mesocosm studies ................................................................................29 

2.5.2 Field demonstration ................................................................................30 

2.5.3 Kinetics of oil bioremediation ........................................................40 

2.6 Nutrient Hydrodynamics ................................................................................41 

2.6.1 Nutrient transport in beaches: a mesocosm study ................................42 

2.6.2 Nutrient transport in beaches: field trials ............................................43 

Chapter 3 METHODS USED IN MONITORING OIL BIOREMEDIATION ........47 

3.1 Analytical Methods ............................................................................................47 

3.1.1 Microbiological analysis ....................................................................47 

3.1.1.1 Enumeration of hydrocarbon-degrading microorganisms: 

culture based techniques ........................................................47 

3.1.1.2 Culture-independent population/community techniques ........48 

3.1.2 Chemical analysis of nutrients ........................................................50 
3.1.3 Chemical analysis of oil and oil constituents ............................................51 

3.1.3.1 Total petroleum hydrocarbon (TPH) techniques ....................52 

3.1.3.2 Analysis of specific oil constituents ............................................53 

3.2 Biomarkers ........................................................................................................57 

3.2.1 Commonly used biomarkers ....................................................................57 

3.2.2 The effect of contaminant redistribution 

on observed remediation rates ........................................................61 

3.3 Sampling in the Field ............................................................................................63 

3.3.1 Sampling strategies ................................................................................64 

3.3.2 Field sampling experiences ....................................................................65 

3.4 Monitoring General Site Background Conditions ............................................66 

3.5.1 Oxygen ............................................................................................67 

3.5.2 pH ........................................................................................................67 

3.5.3 Temperature ............................................................................................67 

3.5.4 Salinity ............................................................................................68 

3.5 Monitoring of Biological Impacts ....................................................................68 

3.5.1 Bioassessment ............................................................................................68 

3.5.2 Bioassays ............................................................................................71 

3.5.2.1 Benthic invertebrates ....................................................................72 

3.5.2.2 Microtox ................................................................................72 

3.5.2.3 Fish ............................................................................................73 

3.5.3 Application of Bioassays to Assess Bioremediation 

in Marine Environments ....................................................................74 

Chapter 4 TYPES OF AMENDMENTS AND CONSIDERATIONS 

IN THEIR APPLICATIONS ....................................................................75 

4.1 Nutrient Amendment ............................................................................................75 

4.1.1 Water-soluble nutrients ....................................................................75 

4.1.2 Granular Nutrients (slow-release) ........................................................76 

4.1.3 Oleophilic Nutrients ................................................................................76 

4.2 Microbial Amendments ................................................................................78 

4.3 Plant Amendments (Phytoremediation) ........................................................80 

4.3.1 Mechanisms of phytoremediation ........................................................80 

4.3.2 Considerations in application of oil phytoremediation ....................81 

4.3.3 Applications in marine shoreline and freshwater wetlands ....................82 

4.4 Oxygen Amendment ............................................................................................83 

4.4.1 Tilling ........................................................................................................84 

4.4.2 Forced aeration ................................................................................84 

4.4.3 Chemical methods ................................................................................84 

Chapter 5 GUIDELINES FOR BIOREMEDIATION OF MARINE SHORELINES 

AND FRESHWATER WETLANDS: DECISION-MAKING AND 

PLANNING ........................................................................................................86 

5.1 Pre-treatment Assessment ................................................................................88 

5.1.1 Oil type and concentration ....................................................................88 
5.1.2 Background nutrient content ....................................................................89 

5.1.3 Type of shorelines ................................................................................90 

5.1.4 Other factors ............................................................................................92 

5.1.5 Summary of pretreatment assessment........................................................92 

5.2 Selection of Nutrient Products ....................................................................93 

5.2.1 Nutrient selection based on efficacy and toxicity ................................93 

5.2.2 Environmental factors affecting nutrient selection ................................97 

5.3 Determination of the Optimal Nutrient Loading and Application Strategy ........97 

5.3.1 Concentration of nutrients needed for optimal biostimulation ........97 

5.3.2 Nutrient application strategies ........................................................99 

5.4 Sampling and Monitoring Plan ..................................................................101 

5.4.1 Important variables ..............................................................................101 

5.4.2 Statistical considerations in sampling plan ..........................................103 

5.5 Considerations for Freshwater Wetland Bioremediation ..............................104 

5.5.1 Characteristics of freshwater wetlands ..........................................104 

5.5.2 Bioremediation strategies in freshwater wetlands ..............................106 

Chapter 6 GUIDELINES FOR ASSESSMENT OF FIELD RESULTS AND 

TERMINATION OF TREATMENT ......................................................109 

6.1 Assessment of Oil Biodegradation Efficacy ......................................................109 

6.1.1 Verification of oil biodegradation ......................................................109 

6.1.2 Assessment of physical removal ......................................................113 

6.1.3 Operational endpoints based on oil biodegradation ..............................115 

6.2 Environmental Assessments ..............................................................................115 

6.2.1 Operational guidance from environmental assessments 

for treatment application ..................................................................116 

6.3 Case Study: Environmental Assessment of Bioremediation Treatments 

in a Tidal Freshwater Marsh ..............................................................................117 

6.3.1 Alterations in ecosystem structure ......................................................117 

6.3.2 Alterations in ecosystem function ......................................................118 

6.3.2.1 Microbial response ..................................................................118 

6.3.2.2 Microtox solid phase test ......................................................119 

6.3.2.3 Algal solid phase assay ......................................................119 

6.3.2.4 Cladoceran survival test ......................................................121 

6.3.2.5 Amphipod survival test ......................................................121 

6.3.2.6 Gastropod survival/histopathology ..........................................122 

6.3.2.7 Acute and chronic effects on fish ..........................................124 

6.4 Ecotoxicological Tests for Risk Assessment ......................................................128 

6.5 Ecotoxicological Tests to Identify Operational Endpoints ..............................129

REFERENCES ..................................................................................................................130 
Chapter 1 INTRODUCTION 

1.1 Occurrence of Oil Spills 

Modern society continues to rely on the use of petroleum hydrocarbons for its energy needs. 

Despite recent technological advances, accidental spills of crude oil and its refined products 

occur on a frequent basis during routine operations of extraction, transportation, storage, refining 

and distribution. It is estimated that between 1.7 and 8.8 million metric tons of oil are released 

into the world’s water every year (NAS, 1985), of which more than 90% is directly related to 

human activities including deliberate waste disposal. Contrary to popular perception, only one 

eighth of the oil released into the aquatic environment is from tanker accidents. It is also 

estimated that about 30% of the spilled oil enters freshwater systems (Cooney, 1984). These 

figures are rather uncertain and can vary greatly from year to year depending on sources of 

estimation and spill incidents. Table 1.1 summarizes the number of oil spills and the quantities 

of release from 1970 to 1999 based on International Tanker Owners Pollution Federation’s oil 

spill database (ITOPF, 2001). These data include oil spills of over seven tons from tankers, 

combined carriers, and barges. Although the data suggest a reduction in oil spills, the trend may 

only represent a temporary downward fluctuation that is part of erratic cycling over the long term 

(Etkin and Welch, 1997). 

Marine shorelines are important public and ecological resources that serve as a home to a variety 

of wildlife and provide public recreation. Marine oil spills, particularly large scale spill 

accidents, have posed great threats and cause extensive damage to the marine coastal 

environments. For example, the spill of 37,000 metric tons (11 million gallons) of North Slope 

crude oil into Prince William Sound, Alaska, from the Exxon Valdez in 1989 led to the mortality 

of thousands of seabirds and marine mammals, a significant reduction in population of many 

intertidal and subtidal organisms, and many long term environmental impacts (Spies et al., 

1996). In 1996, the Sea Empress released approximately 72,000 tons of Forties crude oil and 360 

tons of heavy fuel oil at Milford Haven in South Wales and posed a considerable threat to local 

fisheries, wildlife and tourism (Edwards and White, 1999; Harris; 1997). 

Compared to marine oil spills, inland oil spills have received much less attention. However, 

freshwater spills are very common, with more than 2000 oil spills, on average, taking place each 

year in the inland waters of the continental United States (Owens et al., 1993). Although 

freshwater spills tend to be of a smaller volume than their marine counterparts (Stalcup, et al., 

1997), they have a greater potential to endanger public health and the environment because they 

often occur within populated areas and may directly contaminate surface water and groundwater 

supplies. For example, in 1988, an Ashland Oil Company storage tank in Pittsburgh ruptured 

and spilled about 2,500 tons (750,000 gallons) of diesel oil in the Monongahela River, which 

contaminated drinking water intakes and led to downstream water shortages as far as 200 miles 

(Miklaucic and Saseen, 1989). 

These catastrophic accidents, especially the Exxon Valdez spill, have increased public awareness 

about the risks involved in the storage and transportation of oil and oil products and have 

prompted more stringent regulations, such as the enactment of the 1990 Oil Pollution Act by 

1
Congress. However, because oil is so widely used, despite all the precautions, it is almost certain 

that oil spills and leakage will continue to occur. Thus, it is essential that we have effective 

countermeasures to deal with the problem. 

Table 1.1 Oil spills from tankers and other vessels into world water (adapted from ITOPF’s Oil 

Spill Database, http://www.itopf.com/stats.html) 

Number of Oil Spills 

Year 

< 700 metric tons > 700 metric tons 

Quantities of Oil Spills 

(x 103 metric tons) 

1970 

1971 

1972 

1973 

1974 

1975 

1976 

1977 

1978 

1979 

1980 

1981 

1982 

1983 

1984 

1985 

1986 

1987 

1988 

1989 

1990 

1991 

1992 

1993 

1994 

1995 

1996 

1997 

1998 

1999 

6 

18 

49 

25 

91 

97 

67 

65 

54 

59 

51 

49 

44 

52 

25 

29 

25 

27 

11 

32 

50 

27 

31 

30 

27 

21 

20 

27 

22 

19 

29 

14 

24 

32 

26 

19 

25 

16 

23 

34 

13 

6 

3 

11 

8 

8 

7 

10 

10 

13 

13 

8 

9 

11 

7 

2 

3 

10 

4 

5 

301 

167 

311 

166 

222 

342 

369 

298 

395 

608 

206 

44 

11 

384 

28 

88 

19 

30 

198 

178 

61 

435 

162 

144 

105 

9 

79 

67 

10 

24 

2
1.2 Response to Oil Spills in Marine Shorelines and Freshwater Environments 

Strategies for cleaning up an oil spill are greatly affected by a variety of factors, such as the type 

of oil, the characteristics of the spill site, and occasionally political considerations. A number of 

approaches and technologies have been developed for controlling oil spills in marine shorelines 

and freshwater environments. These methods have been reviewed and described extensively in a 

number of technical documents, such as: Shoreline Countermeasure Manual (NOAA, 1992), 

Options for Minimizing Environmental Impacts of Freshwater Spill Response (NOAA and API, 

1994), Understanding Oil Spills and Oil Spill Response (U.S. EPA, 1999), and Oil Spill 

Response in the Marine Environment (Doerffer, 1992). The most commonly used shoreline 

cleanup options (Table 1.2) are briefly described in the following text. 

Table 1.2. Conventional Shoreline Clean-up Options 

Category of Response Options Example Technology 

Natural method Natural attenuation 

Physical method Booming 

Skimming 

Manual removal (Wiping) 

Mechanical removal 

Washing 

Sediment relocation/Surf-washing 

Tilling 

In-situ burning 

Chemical method Dispersants 

Demulsifiers 

Solidifiers 

Surface film chemicals 

1.2.1 Natural methods 

Natural attenuation or natural recovery is basically a no-action option that allows oil to be 

removed and degraded by natural means. For some spills, it is probably more cost-effective and 

ecologically sound to leave an oil-contaminated site to recover naturally than to attempt to 

intervene. Examples of such cases are spills at remote or inaccessible locations when natural 

removal rates are fast, or spills at sensitive sites where cleanup actions may cause more harm 

than good. It should also be noted that when natural attenuation is used as a clean up method, a 

monitoring program is still required to assess the performance of natural attenuation. Major 

natural processes that result in the removal of oils include: 

3
• 	 Evaporation: Evaporation is the most important natural cleansing process during the early 

stages of an oil spill, and it results in the removal of lighter-weight components in oil. 

Depending on the composition of the oil spilled, up to 50 percent of the more toxic, lighter 

weight components of an oil may evaporate within the first 12 hours following a spill (U.S. 

EPA, 1999). 

• 	 Photooxidation: Photooxidation occurs when oxygen under sunlight reacts with oil 

components. Photooxidation leads to the breakdown of more complex compounds into 

simpler compounds that tend to be lighter in weight and more soluble in water, allowing 

them to be removed further through other processes. 

• 	 Biodegradation: Various types of microorganisms that are capable of oxidizing petroleum 

hydrocarbons are widespread in nature. Biodegradation is a particularly important 

mechanism for removing the non-volatile components of oil from the environment. This is a 

relatively slow process and may require months to years for microorganisms to degrade a 

significant fraction of an oil stranded within the sediments of marine and/or freshwater 

environments. 

1.2.2 Physical methods 

Physical containment and recovery of bulk or free oil is the primary response option of choice in 

the United States for the cleanup of oil spills in marine and freshwater shoreline environments. 

Commonly used physical methods include: 

• 	 Booming and skimming: Use of booms to contain and control the movement of floating oil 

and use of skimmers to recover it. The environmental impact of this method is minimal if 

traffic of the cleanup work force is controlled. 

• 	 Wiping with absorbent materials: Use of hydrophobic materials to wipe up oil from the 

contaminated surface. While the disposal of contaminated waste is an issue, the 

environmental effect of this method is also limited if traffic of cleanup crew and waste 

generation is controlled. 

• 	 Mechanical removal: Collection and removal of oiled surface sediments by using 

mechanical equipment. This method should be used only when limited amounts of oiled 

materials have to be removed. It should not be considered for cleanup of sensitive habitats or 

where beach erosion may result. 

• 	 Washing: washing of the oil adhering along the shorelines to the water’s edge for collection. 

Washing strategies range from low-pressure cold water flushing to high-pressure hot water 

flushing. This method, especially using high-pressure or hot water, should be avoided for 

wetlands or other sensitive habitats. 

• 	 Sediment relocation and tilling: Movement of oiled sediment from one section of the beach 

to another or tilling and mixing the contaminated sediment to enhance natural cleansing 

4
processes by facilitating the dispersion of oil into the water column and promoting the 

interaction between oil and mineral fines. Tilling may cause oil penetration deep into the 

shoreline sediments. The potential environmental impacts from the release of oil and oiled 

sediment into adjacent water bodies should also be considered. 

• 	 In-situ burning: Oil on the shoreline is burned usually when it is on a combustible substrate 

such as vegetation, logs, and other debris. This method may cause significant air pollution 

and destruction of plants and animals. 

1.2.3 Chemical methods 

Chemical methods, particularly dispersants, have been routinely used in many countries as a 

response option. For some countries, such as the United Kingdom, where rough coastal 

conditions may make mechanical response problematic, dispersants are the primary choice 

(Lessard and Demarco, 2000). However, chemical methods have not been extensively used in 

the United States due to the disagreement about their effectiveness and the concerns of their 

toxicity and long-term environmental effects (U.S. EPA, 1999). Major existing chemical agents 

include: 

• 	 Dispersants: dispersing agents, which contain surfactants, are used to remove floating oil 

from the water surface to disperse it into the water column before the oil reaches and 

contaminates the shoreline. This is done to reduce toxicity effects by dilution to benign 

concentrations and accelerate oil biodegradation rates by increasing its effective surface area. 

• Demulsifiers: Used to break oil-in-water emulsions and to enhance natural dispersion. 

• 	 Solidifiers: Chemicals that enhance the polymerization of oil can be used to stabilize the oil, 

to minimize spreading, and to increase the effectiveness of physical recovery operations. 

• 	 Surface film chemicals: Film-forming agents can be used to prevent oil from adhering to 

shoreline substrates and to enhance the removal of oil adhering to surfaces in pressure 

washing operations. 

1.3 Bioremediation as an Oil Spill Cleanup Technology 

Although conventional methods, such as physical removal, are the first response option, they 

rarely achieve complete cleanup of oil spills. According to the Office of Technology 

Assessment (OTA, 1990), current mechanical methods typically recover no more than 10-15 

percent of the oil after a major spill. Bioremediation has emerged as one of the most promising 

secondary treatment options for oil removal since its successful application after the 1989 Exxon 

Valdez spill (Bragg et al., 1994; Prince et. al., 1994). Bioremediation has been defined as “the act 

of adding materials to contaminated environments to cause an acceleration of the natural 

biodegradation processes” (OTA, 1991). This technology is based on the premise that a large 

percentage of oil components are readily biodegradable in nature (Atlas, 1984, 1981; Prince, 

1993). The success of oil spill bioremediation depends on our ability to establish and maintain 

5
conditions that favor enhanced oil biodegradation rates in the contaminated environment. There 

are two main approaches to oil spill bioremediation: 

• 	 bioaugmentation, in which known oil-degrading bacteria are added to supplement the 

existing microbial population, and 

• 	 biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition 

of nutrients or other growth-limiting cosubstrates, and/or by alterations in environmental 

conditions (e.g. surf-washing, oxygen addition by plant growth, etc.). 

Both laboratory studies and field tests have shown that bioremediation, biostimulation in 

particular, can enhance oil biodegradation on contaminated shorelines (Prince, 1993; Swannell, 

et al., 1996). Recent field studies have also demonstrated that biostimulation is a more effective 

approach because the addition of hydrocarbon degrading microorganisms will not enhance oil 

degradation more than simple nutrient addition (Lee et al, 1997a; Venosa et al., 1996; see 

Chapter 2 in detail). Bioremediation has several advantages over conventional technologies. 

First, the application of bioremediation is relatively inexpensive. For example, during the 

cleanup of the Exxon Valdez spill, the cost of bioremediating 120 km of shoreline was less than 

one day’s costs for physical washing (Atlas, 1995). Bioremediation is also a more 

environmentally benign technology since it involves the eventual degradation of oil to mineral 

products (such as carbon dioxide and water), while physical and chemical methods typically 

transfer the contaminant from one environmental compartment to another. Since it is based on 

natural processes and is less intrusive and disruptive to the contaminated site, this “green 

technology” may also be more acceptable to the general public. 

Bioremediation like other technologies also has its limitations. Bioremediation involves highly 

heterogeneous and complex processes. The success of oil bioremediation depends on having the 

appropriate microorganisms in place under suitable environmental conditions. Its operational 

use can be limited by the composition of the oil spilled. Bioremediation is also a relatively slow 

process, requiring weeks to months to take effect, which may not be feasible when immediate 

cleanup is demanded. Concerns also arise about potential adverse effects associated with the 

application of bioremediation agents. These include the toxicity of bioremediation agents 

themselves and metabolic by-products of oil degradation and possible eutrophic effects 

associated with nutrient enrichment (Swannell et al., 1996). Bioremediation has been proven to 

be a cost-effective treatment tool, if used properly, in cleaning certain oil-contaminated 

environments. Few detrimental treatment effects have been observed in actual field operations. 

Currently, one of the major challenges in the application of oil bioremediation is the lack of 

guidelines regarding when and how to use this technology. Although extensive research has 

been conducted on oil bioremediation during the last decade, most existing studies have 

concentrated on either evaluating the feasibility of bioremediation for dealing with oil 

contamination, or testing favored products and methods (Mearns, 1997). Only limited number of 

pilot-scale and field trials, which may provide the most convincing demonstrations of this 

technology, have been carried out. To make matters worse, many field tests have not been 

properly designed, well controlled or correctly analyzed, leading to skepticism and confusion 

6
among the user community (Venosa, 1998). There is an urgent need for a detailed and workable 

set of guidelines for the application of this technology for oil spill responders that answers 

questions such as when to use bioremediation, what bioremediation agents should be used, how 

to apply them, and how to monitor and evaluate the results. Scientific data for the support of an 

operational guidelines document has recently been provided from laboratory studies and fields 

trials carried out by the U.S. Environmental Protection Agency (EPA), University of Cincinnati, 

and Fisheries and Oceans Canada. 

1.4 Scope of This Document 

1.4.1 Objectives 

The objective of this manual is to provide detailed and applicable technical guidelines for use by 

spill responders for the bioremediation of marine shorelines and freshwater wetlands that are 

contaminated with crude oil and its refined products. The document will also provide guidance to 

scientists, other technical personnel, and manufacturers with an interest in the design and 

implementation of field bioremediation processes. The document evaluates current practices and 

state-of-the-art research results pertaining to bioremediation of hydrocarbon contamination and 

presents a rational procedure for the design of bioremediation processes pertinent to clean-up of 

oil stranded within sediments of shoreline environments. 

The scope of this document is limited to marine shorelines and freshwater wetlands where the 

effectiveness of bioremediation has been quantified in controlled field and case studies following 

spill incidents. To date, there is no conclusive evidence of successful oil spill clean up 

operations in the open sea by the application of bioremediation strategies (Atlas, 1995; Swannell 

et al., 1996). With respect to freshwater environments, an oil spill is most likely to have the 

greatest impact on sensitive wetlands or marshes rather than running rivers. To provide insight 

into the feasibility of bioremediation strategies in these habitats, the information presented in this 

manual is based on experimental oil spills conducted in the 1990s jointly by EPA, the University 

of Cincinnati, and Fisheries and Oceans-Canada. The first study was a field experiment on a 

sandy beach in Delaware in 1994. In 1999 and 2000, a similar field investigation was conducted 

on a freshwater wetland along the shoreline of the St. Lawrence River, Quebec, Canada. Another 

field study was conduced in Dartmouth, Nova Scotia in 2000 on a salt marsh dominated by 

Spartina alterniflora. This document will provide oil bioremediation guidelines based on current 

practices and research with the emphasis on the findings of these fields studies. Because the 

study conducted on the shoreline of Nova Scotia has not been concluded at the time of this 

writing, guidelines of oil bioremediation in salt marshes will be available as a supplementary 

document upon the conclusion of this investigation. 

1.4.2 Organization of the guidance document 

For ease of use, the contents of this document are arranged in a logical sequence first to provide 

basic information for the evaluation of bioremediation as a spill response option followed by 

guidelines for application that includes methods to monitor its effectiveness. Thus, Chapter 2 

covers the basic information about oil, shorelines, mechanisms of oil biodegradation, and a state7
of-art review of controlled laboratory experiments and field trials of oil biodegradation and 

nutrient dynamics in shoreline environments. For additional background information, Chapter 3 

provides a more thorough review and critique of current analytical methods used to monitor and 

verify oil spill bioremediation success. Chapter 4 summarizes major biostimulatory and 

bioaugmenting amendment methods and their application strategies. Chapter 5 presents 

guidelines for designing, planning, and implementing oil bioremediation in the field, including 

site characterization, evaluation of appropriate bioremediation technologies, and the selection of 

the most appropriate technology for a specific site. Finally, Chapter 6 provides guidelines for 

assessment and interpretation of field results. 

8
Chapter 2 FACTORS AFFECTING NATURAL OIL BIODEGRADATION AND 

BIOREMEDIATION SUCCESS 

Oil bioremediation is a complex process involving interactions of oil and microorganisms under 

the conditions of the prevailing environment. To understand the scope and strategies of oil 

bioremediation, it is essential to first understand the properties of oil, the environment of concern 

(e.g., marine shorelines and freshwater wetlands), the fate of oil in that environment, the 

mechanisms of oil biodegradation and the factors that control its rate. 

2.1 Physical-Chemical Properties of Crude Oil and Oil Products 

Crude oil and petroleum products are very complex and variable mixtures of thousands of 

individual compounds that exhibit a wide range of physical properties. Understanding these 

properties is important in determining behavior of spilled oil and the appropriate response option. 

The composition and properties of various petroleum hydrocarbons have been described in detail 

by Clark and Brown (1977) and the National Academy of Sciences (1985). Large oil property 

databases also exist such as the one posted on the Internet by Environment Canada 

(www.etcenttre.org/spills), which contains information on over 400 oils (Jokuty, et al., 2000). 

2.1.1 Chemical composition of crude oils and oil products 

Crude Oil 

Crude oil is comprised of both hydrocarbon compounds (accounting for 50–98% of total 

composition) and non-hydrocarbon compounds (containing sulfur, nitrogen, oxygen, and various 

trace metals) in a wide array of combinations (Clark and Brown, 1977). The chemical 

composition and physical characteristics of several crude oils is illustrated in Table 2.1. Some

representative organic compounds found in crude oil are illustrated in Figure 2.1. Petroleum

components may be classified into four major groups based on their differential solubility in 

organic solvents (Leahy and Colwell, 1990). 

1. Saturated hydrocarbons: Include normal and branched alkanes with structures of CnH2n+2 

(aliphatics) and cyclic alkanes with structures of CnH2n (alicyclics), which range in chain 

length from one carbon to over 40 carbons. Saturates usually are the most abundant 

constituents in crude oils. 

2. Aromatic hydrocarbons: Include monocyclic aromatics (e.g., benzene, toluene, and xylenes) 

and polycyclic aromatic hydrocarbons (PAHs) (e.g., naphthalene, anthracene, and 

phenanthrene), which have two or more fused aromatic rings. PAHs are of particular 

environmental concern because they are potential carcinogens or may be transformed into 

carcinogens by microbial metabolism. 

9
3. Resins: Include polar compounds containing nitrogen, sulfur, and oxygen (e.g., pyridines 

and thiophenes). They are often referred to as NSO compounds. 

4. Asphaltenes: Consist of poorly characterized high molecular weight compounds that include 

both high molecular weight and poorly characterized hydrocarbons and NSOs. Metals such 

as nickel, vanadium, and iron are also associated with asphaltenes. 

Table 2.1 Chemical composition and physical properties of representative crude oils 

(adapted from Clark and Brown, 1977) 

Characteristic or Component Prudhoe Bay South Louisiana Kuwait 

API gravity (20o

C) 

Sulfur (wt%) 

Nitrogen (wt%) 

Nickel (ppm) 

Vanadium (ppm) 

Naphtha fraction (wt%)a 

Saturates 

Aromatics 

Resins & Asphaltenes 

High-boiling fraction (wt%)b 

Saturates 

Aromatics 

Resins & Asphaltenes 

27.8 34.5 31.4 

0.94 0.25 2.44 

0.23 0.69 0.14 

10 2.2 7.7 

20 1.9 28 

23.2 18.6 28.0 

19.9 16.5 20.3 

3.2 2.1 2.4 

-- -- --

76.8 81.4 77.3 

47.7 56.3 34.0 

25.0 16.5 21.9 

4.1 8.6 21.4 

These analyses represent values for one typical crude oil from three distinct geographical 

regions; variations in composition can be expected for oils produced from different formations or 

fields within each region. 

a Fraction boiling from 20o

 to 205oC

b Fraction boiling above 205o

C

10
SATURATES AROMATICS

H3CC C C C CH3

H2 H2 H2 H2 

n-hexane 

n-heptadecane (n-C17H36) 

pristane (C19H40) 

H H 

17α(H),21β(H)-hopane 

RESINS 

N CH3 

2-methylpyridine 

dibenzothiophene 

S 

CH3 

toluene 

naphthalene 

chrysene 

benzo[a]pyrene 

ASPHALTENES 

(C79H92N2S2O)3 

Figure 2.1 Representative organic compounds found in crude oils 

11
Refined oil products 

Refined petroleum products, such as gasoline, kerosene, jet fuels, fuel oils, and lubricating oils, 

are derived from crude oils through processes such as catalytic cracking and fractional 

distillation. These products have physical and chemical characteristics that differ according to 

the type of crude oil and subsequent refining processes. They contain components of crude oil 

covering a narrow range of boiling points. In addition, during catalytic cracking operations, 

unsaturated compounds, or olefins (alkenes and cycloalkenes), which are not present in crude 

oils, can be formed. The concentrations of olefins are as high as 30% in gasoline and about 1% 

in jet fuel (NAS, 1985). A list of chemical compositions of the fractions of crude oils and the 

refined products is shown in Table 2.2. 

Table 2.2 Chemical compositions of refined petroleum products (adapted from Clark and 

Brown, 1977) 

Distillation Hydrocarbon Range of Typical Refined 

Fraction Types Carbon Atoms Products 

Gasoline & naphtha Saturates 4-12 Gasoline 

Olefins 

Aromatics 

Middle distillate Saturates 10-20 Kerosene 

Olefins Jet fuel 

Aromatics Heating oils 

Diesel oils 

Wide-cut gas oil Saturates 18-45 Wax 

Aromatics Lubricating oil 

Residum Resins >40 Residual oils 

Asphaltenes Asphalt 

2.1.2 Physical properties of oil 

Important physical properties of oil that affect its behavior in the environment and spill cleanup 

responses include: 

1. Density: Two types of density expressions for oils are often used: specific gravity and 

American Petroleum Institute (API) gravity. Specific gravity is the ratio of the mass of a 

substance to the mass of the equivalent volume of water at a specified temperature. The API 

gravity arbitrarily assigns a value of 10° to pure water at 10°C (60°F). The API gravity can 

be calculated from the specific gravity using the formula: 

12
o 141.5 API Gravity ( ) = 

Specific Gavity (16 oC / 60 oF ) − 131.5 ( 2.1 )

Oils with low densities or low specify gravities have high API gravities. Crude oils have 

specific gravities in the range of 0.79 to 1.00 (equivalent to API Gravities of 10 to 48) (Clark 

and Brown, 1977). Oil density is an important index of oil composition that is frequently 

used to predict its fate in water. 

2. Viscosity: Viscosity is the property of a fluid that describes how it resists a change in shape 

or movement. The lower the viscosity a fluid has, the more easily it flows. The viscosity of 

petroleum is related to oil compositions and the ambient temperature. It is an important 

index of the spreading rate of a spilled oil. 

3. Pour Point: The pour point of an oil is the temperature at which it becomes semi-solid or 

stops flowing. The pour point of crude oils varies from –57°C to 32°C. It is another 

important characteristic with respect to oil fate and cleanup strategies. 

4. Solubility in water: The solubility of oil in water is extremely low and depends on the 

chemical composition of the petroleum hydrocarbon in question and temperature. For a 

typical crude oil, solubility is around 30 mg/L (NAS, 1985). The most soluble oil 

components are the low molecular weight aromatics such as benzene, toluene and xylene. 

This property is important with respect to oil fate, oil toxicity and bioremediation processes. 

Other important physical properties of oils include flash point, vapor pressure, surface tension, 

and adhesion. 

2.2 Behavior of Oil in the Environment 

2.2.1 Weathering processes 

When oil is introduced into the environment, it immediately goes through a variety of physical, 

chemical and biological changes (Figure 2.2). These weathering processes will alter oil 

composition and properties in ways that may affect spill response strategies. Bioremediation is a 

relatively slow process that is often used as a polishing step after conventional cleanup options 

have been applied. Thus the residual target oil may be extensively weathered prior to the 

deployment of bioremediation strategies. 

Weathering processes, including biodegradation, have been reviewed and described extensively 

in the literature (Clark and MacLeod, 1977; Jordan, R.E. and Payne, J.R., 1980; National 

Academy of Sciences, 1985). Major physical and chemical fates of oil are briefly summarized in 

this section and the biological fate will be discussed in section 2.3. 

13
Spreading 

The spreading of oil on water is one of the most important processes during the first hours of a 

spill, provided that the oil pour point is lower than the ambient temperature. The principal forces 

influencing the spreading of oil include gravity, inertia, friction, viscosity and surface tension. 

This process increases the overall surface area of the spill, thus enhancing mass transfer via 

evaporation, dissolution, and later biodegradation. 

Evaporation 

In terms of environmental impacts, evaporation is the most important weathering process during 

the early stages of an oil spill in that it can be responsible for the removal of a large fraction of 

the oil including the more toxic, lower molecular weight components. For oil on water, 

evaporation removes virtually all the normal alkanes smaller than C15 within 1 to 10 days. 

Volatile aromatic compounds, such as benzene and toluene, can also be rapidly removed from an 

oil slick through evaporation. However, these volatile oil components may be more persistent 

when oil is stranded in sediments. The volatile components make up 20-50% of most crude oils, 

about 75% of No. 2 fuel oil, and about 100% of gasoline and kerosene. As a result, the physical 

properties of the remaining slick change significantly (e.g., increased density and viscosity). 

Major factors influencing the rate of evaporation include composition and physical properties of 

the oil, wave action, wind velocity, and water temperature (Clark and MacLeod, 1977; Jordan, 

R.E. and Payne, J.R., 1980). 

Dissolution 

Although dissolution is less important from the viewpoint of mass loss during an oil spill, 

dissolved hydrocarbon concentrations in water are particularly important due to their potential 

influence on the success of bioremediation and the effect of toxicity on biological systems. The 

extent of dissolution depends on the solubility of the spilled oil, weather conditions, and the 

characteristics of the spill site. The low molecular weight aromatics are the most soluble oil 

components, and they are also the most toxic components in crude and refined oils. Although 

many of them may be removed through evaporation, their impact on the environment is much 

greater than simple mass balance considerations would imply (NAS, 1985). Dissolution rates are 

also influenced by photochemical and biological processes. 

Photooxidation 

Photooxidation is another weathering process that may have important biological consequences. 

In the presence of oxygen, natural sunlight has sufficient energy to transform many complex 

petroleum compounds such as high molecular weight aromatics and polar compounds into 

simpler compounds through a series of free-radical chain reactions. This process may increase 

the solubility of oil in water, due to the formation of polar compounds such as hydroperoxides, 

aldehydes, ketones, phenols, and carboxylic acids. Detrimental effects may be associated with 

this increase in the solubility of oil in water (i.e., bioavailability) and the formation of toxic 

compounds mediated by photooxidation. On the other hand, the formation of polar compounds 

may increase the rate of biodegradation of petroleum, particularly at lower concentrations where 

acute toxicity effects are limited (Nicodem et al. 1997). 

14
Dispersion 

Dispersion, or formation of oil-in-water emulsions, involves incorporating small droplets of oil 

into the water column, resulting in an increase in surface area of the oil. In general, oil-in-water 

emulsions are not stable. However, they can be maintained by continuous agitation, interaction 

with suspended particulates, and the addition of chemical dispersants. Dispersion may influence 

oil biodegradation rates by increasing the contact between oil and microorganisms and/or by 

increasing the dissolution rates of the more soluble oil components. 

Emulsification 

The process of emulsification of oils involves a change of state from an oil-on-water slick or an 

oil-in-water dispersion to a water-in-oil emulsion, with the eventual possible formation of a 

thick, sticky mixture that may contain up to 80% water, commonly called “chocolate mousse”. 

The formation and stability of emulsions are primarily related to the chemical composition of the 

oils and are enhanced by wax and asphaltic materials. Surface-active materials generated 

through photochemical and biological processes are also involved in formation of the emulsions. 

The formation of emulsions makes oil clean-up operations more difficult by decreasing the 

effectiveness of physical oil spill recovery procedures and suppressing the natural rates of oil 

biodegradation. 

Other important physical and chemical weathering processes that influence the rates of oil 

degradation include adsorption onto suspended particulate materials, sinking and sedimentation, 

and tar ball formation. 

Photooxidation 

Evaporation 

Emulsification 

Spreading 

Dispersion 

Sinking & Sedimentation 

Adsorption and 

Penetration 

Migration 

and Release 

Dissolution 

Biodegradation 

Biodegradation 

Fine Particle Interaction 

Figure 2.2 Major weathering processes after an oil spill 

15 
2.2.2 Oil and shoreline interactions 

When oil spills occur in marine or freshwater shoreline (e.g., freshwater wetlands) environments, 

interactions between the spilled oil and the shore further complicate the weathering processes. It 

is very important to understand these interactions in determining the scope and limitations of oil 

spill bioremediation. 

The behavior of spilled oil in shoreline environments is primarily dependent on the properties of 

the shoreline, such as the porosity of the substrate and the energy of the waves acting on a 

shoreline. Higher wave exposure enhances both physical removal and weathering processes. 

Wave-swept rocky shores tend to recover from oil spills within a matter of months whereas 

mangroves and marshes may act as a petroleum sink for many years. However, tidal pumping is 

also a factor promoting oil penetration into the sediments. The rate and depth of oil penetration 

depend primarily on the porosity of the substrate. On coarse-grained shorelines like cobble and 

sandy beaches, oil can penetrate deeper and remain longer (when it is trapped below the limit of 

wave action), compared to finer grained sediments such as silts and clay. However, oil is more 

easily removed by water flushing from coarse-grained sediments. Interactions of oil with tidal 

action, waves, and shoreline substrate may also form asphalt-like oil-sediment mats that are 

resistant to further biological and photochemical weathering. 

Recent studies have shown that the interactions between oil and fine mineral particles also play 

an important role in natural oil cleansing in marine shorelines (Bragg and Owens, 1995; Lee et 

al., 1997b). This process of oil and fine-particle interaction reduces the adhesion of oil to 

intertidal shoreline substrates through the formation of oil-mineral fine flocs that are easily 

dispersed by tidal action and currents. More importantly, oil-mineral fine flocs enhance the 

availability of oil for biodegradation, and thus oil biodegradation rates are accelerated by this 

process (Lee et al., 1997c). 

2.2.3 Shoreline sensitivities 

Marine shorelines and freshwater environments have a wide range of sensitivities to oil and 

clean-up activities. The National Oceanic & Atmospheric Administration and the American 

Petroleum Institute have developed the Environmental Sensitivity Index (ESI) to classify 

shoreline types for spill response (NOAA, 1992; NOAA and API, 1994). This classification 

scheme (e.g., Table 2.3) has been used in oil spill contingency planning and spill response 

operations (Hayes et al., 1995). Major factors considered in ranking habitat sensitivity include 

shoreline type (substrate, grain size, tidal elevation), exposure to wave and tidal energy, 

biological productivity and sensitivity, and ease of cleanup. Bioremediation may be effective 

and cause the least damage on both the moderately and the most sensitive shoreline types. 

The Environmental Sensitivity Index for freshwater shorelines is shown in Table 2.4, based on 

NOAA & API (1994) and Hayes et al. (1995 & 1997). Major factors considered in ranking ESI 

for these habitats include degree of exposure to natural removal processes, biological 

16
productivity and sensitivity, human use of the habitat, and ease of oil removal. Bioremediation 

may be feasible and cause the least damage on both the moderately sensitive and the most 

sensitive shoreline types, although its effectiveness is still uncertain due to the lack of sufficient 

research and field efficacy demonstrations. 

Table 2.3 Shoreline ESI ranking for habitats in marine shorelines (where 1 is least sensitive 

and 10 is most sensitive to oil and clean up actions) 

Environmental Sensitivity Index (ESI) Shoreline Type 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Exposed rocky shores 

Sea walls and piers 

Exposed wave-cut platforms 

Fine-grained sand beaches 

Coarse-grained sand beaches 

Mixed sand and gravel beaches 

Gravel beaches and riprap 

Exposed tidal flats 

Sheltered rocky shores 

Sheltered tidal flats 

Salt marshes and Mangroves 

2.3 Biodegradation of Oil 

Biodegradation of oil is one of the most important processes involved in weathering and the 

eventual removal of petroleum from the environment, particularly for the nonvolatile 

components of petroleum. Numerous scientific review articles have covered various aspects of 

this process an d the environmental factors that influence the rate of biodegradation (Zobell, 1946 

& 1973; Atlas, 1981 & 1984; NAS, 1985; Focht and Westlake, 1987; Leahy and Colwell, 1990). 

2.3.1 Mechanism of oil biodegradation: a microbiological perspective 

Distribution of hydrocarbon-degrading microorganisms 

Microorganisms capable of degrading petroleum hydrocarbons and related compounds are 

ubiquitous in marine, freshwater, and soil habitats. Over 200 species of bacteria, yeasts and 

fungi have been shown to degrade hydrocarbons ranging from methane to compounds of over 40 

carbon atoms (Zobell, 1973). In the marine environment, bacteria are considered to be the 

predominant hydrocarbon-degraders with a distribution range that even covers extreme cold 

Antarctic and Arctic environments (Floodgate, 1984; Jordan and Payne, 1980). In the freshwater 

environment, yeast and fungi may also play a significant role in degrading petroleum 

hydrocarbons (Cooney, 1984). Some of the most important hydrocarbon-degrading 

microorganisms in both marine and freshwater environments are listed in Table 2.5. 

17
Table 2.4 Shoreline ESI ranking for habitats in freshwater shorelines (where 1 is least 

sensitive and 10 is most sensitive to oil and clean up actions) 

ESI Lacustrine a

 Large Rivers a 

1 Exposed rocky cliffs Exposed rocky banks 

Exposed man-made structures Vertical, solid revetments banks 

Quiet pools with low-sensitive 

2 Shelving bedrock shores Rocky shoals, bedrock Small, nonnavigable channel 

Ledges with moderate currents and 

low-sensitive banks 

3 Eroding scarps in Exposed, eroding banks in Navigable channel with 

unconsolidated sediments Unconsolidated sediments moderate currents and lowsensitive banks 

4 Sand beaches Sandy bars and gently Small, nonnavigable channel 

Sloping banks with rapids over bedrock 

5 Mixed sand and gravel Mixed sand and gravel bars Navigable channel with rapids 

beaches over bedrock 

6 Gravel beaches and riprap Gravel bars and riprap Channel with associated lowvulnerable upper bottomland 

hardwoods 

7 Exposed flats Not present Navigable streams with 

associated wide swamps on 

one side 

8 Sheltered rocky shores Vegetated, steeply sloping Navigable streams with 

Sheltered man-made Bluffs associated wide swamps on 

structures Sheltered man-made both sides 

Structures 

9 Sheltered vegetated low Vegetated low banks Meandering channel with 

banks Muddy substrates abundant leakage points into 

(unvegetated) associated swamps and oxbows 

10 Sheltered sand flats Freshwater marshes and Navigable anastomosing 

Small Rivers b 

channel with abundant leakage 

points into associated swamps 

Freshwater marshes and 

swamps 

Swamps 

a ESI adapted from Hayes et al. (1995) 

b RSI (Reach Sensitivity Index) adapted from Hayes et al. (1997) 

18
Table 2.5 Representative microorganisms capable of degrading petroleum hydrocarbons (Based 

on Atlas, 1984; Focht and Westlake, 1987; Jordan and Payne, 1980; Leahy and Colwell, 1990) 

Bacteria Yeast and Fungi 

Achromobacter 

Acinetobacter 

Alcaligenes 

Arthrobacter 

Bacillus 

Brevibacterium 

Cornybacterium 

Flavobacterium 

Nocardia 

Pseudimonas 

Vibrio 

Aspergillus 

Candida 

Cladosporium 

Penicillium 

Rhodotorula 

Sporobolomyces 

Trichoderma 

The distribution of hydrocarbon-utilizing microorganisms is also related to the historical 

exposure of the environment to hydrocarbons. Those environments with a recent or chronic oil 

contamination will have a higher percentage of hydrocarbon degraders than unpolluted areas. In 

“pristine” ecosystems, hydrocarbon utilizers may make up less than 0.1% of the microbial 

community; and in oil-polluted environments, they can constitute up to 100% of the viable 

microorganisms (Atlas, 1981). 

It should be noted that there is no single strain of bacteria with the metabolic capacity to degrade 

all the components found within crude oil. In nature, biodegradation of a crude oil typically 

involves a succession of species within the consortia of microbes present. Microorganisms 

classified as non-hydrocarbon utilizers may also play an important role in the eventual removal 

of petroleum from the environment. Degradation of petroleum involves progressive or 

sequential reactions, in which certain organisms may carry out the initial attack on the petroleum

constituent; this produces intermediate compounds that are subsequently utilized by a different 

group of organisms, in the process that results in further degradation (Karrick, 1977). 

Biodegradation of oil components 

As described earlier, petroleum components can be classified into four major groups: saturates, 

aromatics, resins, and asphaltenes. Major metabolic pathways for many of these compounds 

have been well studied and documented (Atlas, 1981 & 1984; Cerniglia, 1992; Watkinson and 

Morgon, 1990) to explain their differences in susceptibility to biodegradation. 

Saturates In general, the n-alkanes are the most readily degraded components in a 

petroleum mixture (Zobell, 1946; Atlas, 1981). Biodegradation of n-alkanes with molecular 

weights up to C44 has been demonstrated (Haines and Alexander, 1974). Alkanes in the C10 to 

C26 range are considered the most readily and frequently utilized hydrocarbons (Atlas 1995b; 

19
NAS, 1985). The predominant mechanism of n-alkane degradation involves terminal oxidation 

to the corresponding alcohol, aldehydes, or fatty acid functional group. Branched alkanes are less 

readily degraded in comparison to n-alkanes. Methyl branching increases the resistance to 

microbial attack because fewer alkane degraders can overcome the blockage of beta-oxidation 

(NAS, 1985). Highly branched isoprenoid alkanes, such as pristane and phytane, which were 

earlier thought to be resistant to biodegradation, have also been shown to be readily 

biodegradable. Cycloalkanes, however, are particularly resistant to biodegradation. Complex 

alicyclic compounds such as hopanes and steranes are among the most persistent compounds of 

petroleum spills in the environment (Atlas, 1981). 

Aromatics Although the aromatics are generally more resistant to biodegradation, some low 

molecular weight aromatics such as naphthalene may actually be oxidized before many saturates 

(Focht and Westlake, 1987). Monoaromatic hydrocarbons are toxic to some microorganisms due 

to their solvent action on cell membranes, but in low concentrations they are easily 

biodegradable under aerobic conditions. PAHs with 2-4 rings are less toxic and biodegradable at 

rates that decrease with the level of complexity. PAHs with five or more rings can only be 

degraded through co-metabolism, in which microorganisms fortuitously transform non-growth 

substrates while metabolizing simpler hydrocarbons or other primary substrates in the oil. 

Alkylated aromatics are degraded less rapidly than their parent compounds; the more highly 

alkylated groups are degraded less rapidly than less alkylated ones. The metabolic pathways for 

the biodegradation of aromatic compounds have been the subject of extensive study (Atlas, 1981; 

Prince, 1993; Cerniglia, 1992). The bacterial degradation of aromatics normally involves the 

formation of a diol, followed by ring cleavage and formation of a di-carboxylic acid. Fungi and 

other eukaryotes normally oxidize aromatics using mono-oxygenases, forming a trans-diol. 

Resins and asphaltenes Compared to saturates and aromatics, very little is known about 

biodegradation of resins and asphaltenes; this is due to their complex structures, which are 

difficult to analyze. Resins and asphaltenes have previously been considered to be refractory to 

degradation. However, there is recent evidence of asphaltene degradation through cometabolism 

(Leahy and Colwell, 1990). Some resins, particularly low-molecular-weight resin fractions, can 

also be biodegraded at low concentrations (NAS, 1985). Further research is still needed to 

understand the biodegradation of these compounds. 

In summary, the susceptibility of petroleum hydrocarbons to microbial degradation is generally 

in the following order: n-alkanes > branched alkanes > low-molecular-weight aromatics > cyclic 

alkanes. However, this pattern is not universal (Perry, 1984). The compositional heterogeneity 

among different oils greatly affects the biodegradation rate of their constituents. The degradation 

rate for the same oil constituents may vary significantly for different oils. Cometabolism also 

plays an important role in oil biodegradation. Many complex branched, cyclic, and aromatic 

hydrocarbons, which otherwise would not be biodegraded individually, can be oxidized through 

cometabolism in an oil mixture due to the abundance of other substrates that can be metabolized 

easily within the oil (Atlas, 1981). The biological fate of oil components in an oil mixture still 

requires further research. Particularly, effort should be made to establish a database regarding 

the biodegradability of different types of oils and petroleum products. 

20
2.3.2 Environmental factors affecting oil biodegradation 

When oil spills occur in the environment, the rate of oil biodegradation is also greatly influenced 

by the characteristics of the contaminated environment. Major environmental factors affecting 

oil biodegradation include weathering processes, temperature, availability and concentration of 

nutrients, availability and concentration of oxygen, and pH. 

Weathering processes 

The weathering processes described in section 2.2.1 have profound effects on oil biodegradation. 

Evaporation of volatile oil components may benefit microorganisms by removing more toxic 

low-molecular-weight components such as benzene and smaller n-alkanes. However, this 

process also leads to a lower biodegradable percentage of oil, since these components in general 

are readily biodegraded (Atlas, 1981; NAS, 1985). The oil surface area is important because 

growth of oil degraders occurs almost exclusively at the oil-water interface (Atlas and Bartha, 

1992). Formation of water-in-oil emulsions or mousses reduces the surface area, therefore 

decreasing biodegradation. Tarballs, which are large aggregates of weathered and undegraded 

oil, also restrict access to microorganisms because of their limited surface area (Leahy and 

Colwell, 1990). Dispersion of hydrocarbons in the water column in the form of oil-in-water 

emulsions increases the surface area of the oil and thus its availability for microbial attack. The 

formation of oil-in-water emulsions through the microbial production and release of 

biosurfactants has also been found to be an important process in the uptake of hydrocarbons by 

bacteria and fungi (Singer and Finnerty, 1984). In contrast, the application of chemical 

dispersants has produced mixed results and has not been shown to be an effective way to 

enhance oil biodegradation. Photooxidation leads to the formation of more soluble compounds, 

which are often more biodegradable. However, the effect of photooxidation processes on 

biodegradation is still not well understood (Nicodem et al., 1997). 

Biodegradation rates are also influenced by concentrations of individual oil constituents, which 

may be affected by various weathering processes. For example, microbes may attack very low 

concentrations of pollutants in the environment inefficiently (Focht and Westlake, 1987). 

However, high concentrations of hydrocarbons may cause inhibition of biodegradation by 

nutrient or oxygen limitations or toxic effects. There would seem to be, for many hydrocarbons, 

an optimum concentration range for metabolism below which degradation is not stimulated and 

above which inhibition occurs. Weathering processes will affect the ultimate concentrations of 

petroleum hydrocarbons in the environment in different ways. Evaporation may reduce the 

concentrations of volatile compounds but concentrate some other constituents. Sorption and 

emulsification may concentrate the pollutants, while dispersion and dissolution tend to dilute 

them. 

Temperature 

The ambient temperature of an environment affects both the properties of spilled oil and the 

activity or population of microorganisms. At low temperatures, the viscosity of the oil increases, 

while the volatility of toxic low-molecular-weight hydrocarbons is reduced, delaying the onset of 

21
biodegradation (Atlas, 1981). Some hydrocarbons are more soluble at lower temperatures (e.g., 

short-chain alkanes), and some low-molecular-weight aromatics are more soluble at the higher 

temperature (Focht and Westlake, 1987). Although hydrocarbon biodegradation can occur over 

a wide range of temperatures, the rate of biodegradation generally decreases with decreasing 

temperature. Highest degradation rates generally occur in the range of 30 to 40°C in soil 

environments, 20 to 30°C in some freshwater environments, and 15 to 20°C in marine 

environments (Bossert and Bartha, 1984; Cooney, 1984; Jordan and Payne, 1980). 

The effect of temperature is also complicated by other factors such as the composition of the 

microbial population. In environments where a psychrophilic population has been established, 

degradation can occur at significant rates under cold conditions. Hydrocarbon biodegradation 

has been observed at temperature as low as 0-2°C in seawater and –1.1°C in a soil. Colwell et 

al. (1978) reported greater degradation of Metula crude oil at 3°C than at 22°C with a mixed 

culture in beach sand samples. Westlake et al. (1974) also found that bacteria capable of 

degradation at 4°C would metabolize oil at 30°C, but those populations that developed at 30°C 

had a limited activity at 4°C. 

Oxygen 

Aerobic conditions are generally considered necessary for extensive degradation of oil 

hydrocarbons in the environment since major degradative pathways for both saturates and 

aromatics involve oxygenases (Atlas, 1981; NAS, 1984; Cerniglia, 1992). Many studies have 

shown that oxygen depletion leads to sharply reduced biodegradation activities in marine 

sediments and in soils (Atlas, 1981; Bossert and Bartha, 1984; Hambrick III et al., 1980). 

Conditions of oxygen limitation normally do not exist in the upper levels of the water column in 

marine and freshwater environments and in the surface layer of most beach environments. It 

may become limiting in subsurface sediments, anoxic zones of water columns, and most finegrained marine shorelines, freshwater wetlands, mudflats, and salt marshes. Factors affecting the 

availability of oxygen also include the action of wave and water flow, the physical state of the 

oil, and the amount of available substrates. 

Anaerobic oil degradation has been shown in some studies to occur only at negligible rates, as 

reviewed by Atlas (1981), leading to the conclusion that the environmental importance of 

anaerobic hydrocarbon degradation can be discounted. However, recent studies have shown that 

anaerobic hydrocarbon metabolism may be an important process in certain conditions (Head and 

Swannell, 1999). The biodegradation of some aromatic hydrocarbons, such as BTEX 

compounds, has been clearly demonstrated to occur under a variety of anaerobic conditions 

(Krumholz et al., 1996; Leathy and Colwell, 1990). Studies have also demonstrated that in some 

marine sediments, PAHs and alkanes can be degraded under sulfate-reducing conditions at 

similar rates to those under aerobic conditions (Caldwell et al., 1998; Coates et al., 1997). The 

importance of anaerobic biodegradation of oil in the environment still requires further studies. 

Nutrients 

In theory, approximately 150 mg of nitrogen and 30 mg of phosphorus are utilized in the 

conversion of 1 g of hydrocarbon to cell materials (Rosenberg and Ron, 1996). When a major 

22
oil spill occurs in marine and freshwater environments the supply of carbon is dramatically 

increased and the availability of nitrogen and phosphorus generally becomes the limiting factor 

for oil degradation (Atlas, 1984; Leahy and Colwell, 1990). In marine environments, nutrient 

limitation is generally correlated to the low background levels of nitrogen and phosphorus in 

seawater (Floodgate, 1984). Nutrient concentrations are more variable in freshwater systems 

where lakes and wetlands range from oligotrophic to eutrophic; rivers can be nutrient-poor at the 

source, but generally become nutrient-rich downstream after receiving industrial and domestic 

effluents and agricultural runoff (Cooney, 1984). Freshwater wetlands are typically considered to 

be nutrient limited, due to heavy demand for nutrients by the plants. They are also viewed as 

being nutrient traps, as a substantial amount of nutrients may be bound in biomass (Mitsch and 

Gosselink, 1993). Both freshwater lakes and wetlands may also exhibit seasonal variations in 

nutrient levels, which will affect the performance of oil biodegradation. Ward and Brock (1976) 

found that in an oil-contaminated lake, oil biodegradation was at the highest rate during early 

spring when the nutrient content (i.e., N and P) was also high. As N and P levels decreased in the 

summer (probably due to algal productivity) oil biodegradation also decreased. Another potential 

limiting nutrient is iron, which was found to limit oil degradation in clean offshore seawater, but 

is not likely to be limiting in freshwater (Focht and Westlake, 1987). 

Other factors 

Other important factors affecting biodegradation of petroleum hydrocarbons include pH and 

salinity. The pH of seawater is generally stable and slightly alkaline (Bossert and Bartha, 1984). 

In contrast, the pH of freshwater and soil environments can vary widely. Organic soils in 

wetlands are often acidic, while mineral soils have more neutral and alkaline conditions. Most 

heterotrophic bacteria and fungi favor a neutral pH, with fungi being more tolerant of acidic 

conditions. Studies have shown that degradation of oil increases with increasing pH, and that 

optimum degradation occurs under slightly alkaline conditions (Dibble and Bartha, 1979; Focht 

and Westlake, 1987). 

Changes in salinity may affect oil biodegradation through alteration of the microbial population. 

Dramatic variation in salinity may occur in estuarine environments where marine organisms 

mingle with freshwater forms. Many freshwater organisms can survive for long periods in 

seawater although few can reproduce. In contrast, most marine species have an optimum salinity 

range of 2.5 to 3.5% and grow poorly or not at all at salinity lower than 1.5 to 2% (Zobell, 1973). 

In a study of hypersaline salt evaporation ponds, Ward and Brock (1978) showed that rates of 

hydrocarbon metabolism decreased with increasing salinity in the range of 3.3 to 28.4%. More 

studies are required to understand the effect of salinity on oil biodegradation. 

2.3.3 Evaluation of oil biodegradation: application of biomarkers 

The evaluation of oil biodegradation is a difficult task, especially in the field, due to the 

complication of weathering processes and the heterogeneity of contaminated sites. As described 

earlier, physical and chemical weathering can significantly affect the composition and 

concentrations of oils. Oil contaminated sites are often highly heterogeneous, where oil 

concentrations can vary greatly within a small area. Consequently, variability associated with 

23
field studies can be so high as to preclude or interfere with one’s ability to discern significant 

treatment differences. Non-biodegradable or slowly biodegradable components in oil - often 

called biomarkers - have been used successfully to mitigate the high variability associated with 

field studies (Bragg et al., 1994; Venosa et al., 1996; Lee et al., 1997a). This approach estimates 

the extent of biodegradation by evaluating the ratios of target hydrocarbon concentrations 

relative to the concentration of these recalcitrant biomarkers. Studies have shown that use of 

biomarkers reduces spatial variability of oil data when compared to other mass balance 

approaches and allows biodegradation to be monitored effectively by reducing the number of 

samples required (Douglas et al., 1994). 

Commonly used biomarkers for evaluation of biodegradation of crude oils include the 

isoprenoids pristane and phytane, steranes, and the pentacyclic triterpanes such as the hopanoids 

(Peters and Moldowan, 1993). While the isoprenoids pristane and phytane are somewhat more 

resistant to biodegradation than n-alkanes with similar boiling points (C17, C18), they should only 

be used to monitor the earliest stages of a biodegradation treatment program, as they are known 

to be biodegradable under natural conditions (Prince et al., 1994b). Hopanes have become the 

biomarker of choice as they are much more resistant to microbial biodegradation (Atlas, 1981; 

Peters and Moldowan, 1993). The compound 17α(H), 21β(H)-hopane was successfully used to 

determine the efficacy of bioremediation field trials coordinated with the Exxon Valdez spill 

clean up operations (Douglas et al., 1994; Mearns, 1997; Prince, et al., 1994 a&b). However, 

caution must be taken with their use as biomarkers since they are also very resistant to physical 

and chemical weathering processes that affect many alkanes and aromatics. Therefore, hopane 

normalization is more useful in reducing the variability associated with heterogeneous oil 

distribution or in cases where the effects of physical and chemical weathering are negligible. 

Biodegradation can also be verified as the main removal mechanism by determining the relative 

degradation rates for homologous series of alkylated PAHs (Elmendorf et al., 1994; Venosa et 

al., 1997a). 

2.4 Laboratory Studies on Bioremediation of Oil 

Biodegradation as a natural process may proceed slowly, depending on the type of oil (i.e., light 

crude oils degrade faster than heavier oils). Bioremediation strategies are based on the 

application of various methodologies to increase the rate or extent of the biodegradation process. 

The success of oil spill bioremediation depends on our ability to optimize various physical, 

chemical, and biological conditions in the contaminated environment. As described in previous 

sections, the most important requirement is the presence of microorganisms with the appropriate 

metabolic capabilities. If these microorganisms are present, then optimal rates of growth and 

hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of 

nutrients and oxygen are present and that the pH is between 6 and 9 (Atlas and Bartha, 1992). 

The physical and chemical characteristics of the oil and oil surface area are also important 

determinants of bioremediation success. Obviously, some of these factors can be manipulated 

more easily than others. For example, on an operational scale, there is nothing that can be done 

to alter the chemical composition of the oil. 

24
There are two main approaches to oil spill bioremediation. 1) bioaugmentation, in which oildegrading microorganisms are added to supplement the existing microbial population, and 2) 

biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of 

nutrients or other growth-limiting cosubstrates and/or habitat alteration. Both these approaches 

have been extensively studied, on a laboratory scale as well as in the bioremediation of oil 

contaminated shorelines. 

2.4.1 Bioaugmentation 

Although hydrocarbon-degrading microorganisms are widespread in nature, bioaugmentation has 

been considered as a potential strategy for oil-bioremediation since the 1970s. The rationale for 

adding oil-degrading microorganisms is that indigenous microbial populations may not be 

capable of degrading the wide range of potential substrates present in complex mixtures such as 

petroleum (Leahy and Colwell, 1990). Other conditions under which bioaugmentation may be 

considered are when the indigenous hydrocarbon-degrading population is low, the speed of 

decontamination is the primary factor, and when seeding may reduce the lag period to start the 

bioremediation process (Forsyth et al., 1995). 

Many vendors offer microbial agents claiming to enhance oil biodegradation (Prince, (1993). 

However, laboratory studies on bioaugmentation have produced mixed results. Aldrett et al. 

(1997) tested 12 commercial microbial cultures for bioremediation of Alaska North Slope crude 

oil in the lab. After 28 days, four products showed an enhancement of oil biodegradation with 

significantly higher degradation rates of alkanes and aromatics when compared to a nutrient 

control. In another shaker-flask experiment, Hozumi et al. (2000) investigated the effectiveness 

of a microbial product in treating a heavy oil spilled from Nakhodka using the thin layer 

chromatography-flame ionization detection (TLC-FID) analysis. They found that approximately 

35% of the oil was degraded with addition of the microbial product compared to no oil loss for a 

control during a three-week test period. Surprisingly, the asphaltene fraction showed the highest 

loss among the four major oil components, which raises the question whether this oil loss was 

actually due to biodegradation rather than some quality control problem with the chemical 

analysis. Some laboratory studies found that microbial seeding may enhance oil degradation in 

seawater but not in freshwater environments (Leahy and Colwell, 1990). To examine whether 

microbial products can compete with the indigenous populations, Venosa et al. (1991) tested 10 

different commercial microbial products using weathered Alaskan crude oil in shaker flask 

microcosms. Although two products showed enhancement compared to a nutrient control, better 

degradation was observed in every case when the commercial products were first sterilized, 

suggesting that indigenous Alaskan microorganisms were primarily responsible for the oil 

biodegradation and seeded microorganisms seemed to compete poorly with the indigenous 

population in the closed flask environment. Thus, bioaugmentation may be effective in benchscale studies where environmental conditions are well controlled, but this will not guarantee its 

effectiveness in the field. 

Creation of a “superbug” that combines the genetic information from many organisms and the 

ability to degrade a variety of different types of hydrocarbons has also been considered. Friello 

25
et al. (1976) successfully produced a multiplasmid-containing Pseudomonas strain capable of 

oxidizing aliphatic, aromatic, terpenic, and polyaromatic hydrocarbons. Thibault and Elliot 

(1980) also developed a multiplasmid P. putida strain that can simultaneously degrade some 

lighter alkanes and aromatics. However, the survival of such a strain in the environment could is 

questionable. More importantly, the issues of safety, containment, and the potential for 

ecological damage must be fully resolved before field testing of these organisms can be 

conducted (Leahy and Colwell, 1990). There is also the problem of public perception over the 

release of “foreign” and especially “genetically engineered” microorganisms into the 

environment. 

2.4.2 Biostimulation 

Biostimulation involves the addition of rate-limiting nutrients to accelerate the biodegradation 

process. In most shoreline ecosystems that have been heavily contaminated with hydrocarbons, 

nutrients are likely the limiting factors in oil biodegradation. The main purpose of bench-scale 

treatability studies is to determine the type, concentration, and frequency of addition of 

amendments needed for maximum stimulation in the field (Venosa, 1998). 

Most laboratory experiments have shown that addition of growth limiting nutrients, namely 

nitrogen and phosphorus, has enhanced the rate of oil biodegradation. However, the optimal 

nutrient types and concentrations vary widely depending on the oil properties and the 

environmental conditions. Wrenn et al. (1994) studied the effects of different forms of nitrogen 

on biodegradation of light Arabian crude oil in respirometers. They found that in poorly 

buffered seawater, nitrate is a better nitrogen source than ammonia because acid production 

associated with ammonia metabolism may inhibit oil biodegradation. When the culture pH was 

controlled, the performance of oil biodegradation was similar for both amendments with a 

shorter lag time for ammonia addition. Ramstad and Sveum (1995) also compared the effect of 

nitrate, ammonia, and an organic nitrogen-containing nutrient on biodegradation of topped 

Statfjord crude oil in a continuous-flow seawater column system. With no control of pH in this 

study, nitrate was found to have the most pronounced effect in stimulating oil degradation when 

using pristane as a biomarker. However, in a microcosm study, Jackson and Pardue (1999) found 

that addition of ammonia appeared to be more effective than nitrate in stimulating degradation of 

crude oil in salt marsh soils. On a weight basis, the amount of ammonia required to achieve the 

same increase in biodegradation as nitrate was only about 20%. This was attributed to the fact 

that ammonia is less likely to be lost from the system by washout due to its higher adsorptive 

capacity to organic matter. 

Oil biodegradation largely takes place at the interface between oil and water. Therefore, the 

effectiveness of biostimulation depends on the nutrient concentration in the interstitial pore water 

of oily sediments (Atlas and Bartha, 1992; Bragg et al., 1994). The nutrient concentration 

should be maintained at a level high enough to facilitate bacterial growth. However, caution 

must be exercised as excessive concentrations of nutrients, such as ammonia, induce toxic 

responses in many marine species (Pritchard et al., 1991). Using nitrate as a biostimulation 

agent, Venosa et al. (1994) determined that approximately 1.5 to 2.0 mg N/L supported near 

26
maximal biodegradation of heptadecane immobilized onto sand particles in a microcosm study. 

Du et al. (1999) investigated the optimal nitrogen concentration for the biodegradation of Alaska 

North Slope crude oil in continuous flow beach microcosms at a loading of 5g-oil/kg sand. The 

results showed that nitrate concentrations below approximately 10 mgN/L limited the rate of oil 

biodegradation. The higher nutrient requirement was attributed to the more complex substrate 

(crude oil). Ahn (1999) further studied the effect of nitrate concentrations under tidal flow 

conditions using the same microcosms, oil, and oil loading as Du et al. (1999). Nitrate 

concentrations ranging from 6.25 to 400 mg N/L were supplied semi-diurnally to simulate tide 

flow. The results from both oil analysis (hopane as a biomarker) and microbial growth 

(phospholipid analysis) showed that the optimal nitrate concentration fed under these conditions 

was approximately 25 mgN/L. However, the data showed that at the end of the test (after one 

month), an approximately 80% degradation of all target alkanes and PAHs was achieved in all 

test cases that covered a range of nitrate concentrations in the test solutions added. This result 

suggested that nitrate concentration might only affect the rates and not the extent of oil 

degradation. Further research in this regard is required to optimize bioremediation strategies. 

One of the main challenges associated with biostimulation in oil-contaminated coastal areas is 

maintaining optimal nutrient concentrations in contact with the oil. Oil from offshore spills 

usually contaminates the intertidal zone, where the washout rate for water-soluble nutrients can 

be very high, and this can adversely impact the effectiveness of biostimulation. Many attempts 

have been made in the design of nutrient delivery systems that overcome the washout problems 

characteristic of intertidal environments (Prince, 1993). These include oleophilic and slowrelease fertilizer formulations, as well as systems that rely on the subsurface flow of water 

through the beach (Wise et al., 1994). Several papers have compared the effectiveness of these 

nutrient products to stimulate hydrocarbon biodegradation rates. Croft et al. (1995) tested the 

efficiency of an oleophilic organic (Inipol EAP22) and a slow-release inorganic fertilizer (Max 

Bac) and found that the oleophilic fertilizer was much more effective at stimulating oil 

degradation on sand than the slow-release product. Sveum and Ramstad (1995) also found that 

organic products such as fish meal and stick water (a fish meal by-product) were more effective 

than a slow-release fertilizer (Mac Bac). The failure for this slow-release fertilizer was attributed 

to the nutrient release rate being too slow to affect oil biodegradation. However, in some other 

studies, application of organic fertilizers such as Inipol EAP22 also failed to stimulate oil 

biodegradation (Sveum and Ladousse, 1989; Safferman, 1991). Safferman (1991) investigated 

the rates of nutrient release from several slow-release products and found that Inipol EAP22 was 

rapidly washed out from oiled cobble before becoming available to hydrocarbon-degrading 

bacteria. However, no attempt was made in most of these studies to estimate the steady-state 

nutrient concentrations that would result in an intertidal environment. The variable results from 

the laboratory studies indicate that the performance of these products greatly depends on the 

nutrient release rates and the prevailing environmental conditions. 

Although much research has been carried out on the bioremediation of oil-contaminated marine 

shorelines, few studies have been conducted on oil bioremediation in wetland environments. 

Purandare et al. (1999) conducted the only reported microcosm study on biostimulation in 

freshwater wetland. They investigated different inorganic mineral nutrients for their ability to 

27
enhance biodegradation of a crude oil. Aquaria of 10-gallon capacity, filled with wetland soil 

and planted with species of emergent wetland plants, were used to simulate natural wetlands. 

Two levels of water coverage were studied: (1) water level even with soil surface, and (2) water 

level 10 cm above the soil surface. Six treatments were evaluated for each water level: unoiled, 

no-nutrient control; oiled + no nutrient control; oiled + nitrate addition; oiled + nitrate + 

phosphate addition; oiled + ammonia addition; and oiled + ammonia + phosphate addition. 

Approximately 14g of weathered light Arabian crude oil was added to each column to form 

about a 2mm oil layer. The results showed that for both flooded and unflooded wetland 

conditions, the addition of nitrate and phosphate seemed to enhance the degradation of oil above 

the natural attenuation rate. The highest biodegradation rates of alkanes and PAHs occurred in 

the high water level microcosms receiving nitrate and phosphorus, in which a 90% alkane 

reduction and 50% PAH removal was observed, compared to only 50% alkane reduction and 

40% PAH removal in the control microcosms. Higher degradation of alkanes and PAHs in the 

high water level relative to that in the low water level suggested an increased hydrocarbon 

bioavailability. However, a microcosm study conducted by Garcia-Blanco et al. (2001a) in a 

simulated tidal salt marsh found that addition of nutrients did not stimulate the biodegradation of 

a No. 2 fuel oil. Low oxygen availability was suggested to be the limiting factor for oil 

degradation in salt marshes. 

In summary, laboratory studies have shown that biostimulation and, in some cases 

bioaugmentation, can enhance the rates of oil biodegradation, particularly in marine 

environments. Oxygen may become a limiting factor in oil biodegradation under certain 

circumstances, such as salt mashes and freshwater wetlands. However, these conclusions still 

need to be verified through field evaluations. 

2.5 Demonstrations of Oil Bioremediation Under Field Conditions 

Field studies can provide the most convincing demonstration of the effectiveness of oil 

bioremediation since laboratory studies are not always able to account for numerous real world 

conditions such as spatial heterogeneity, biological interactions, and mass transfer limitations. 

Compared to laboratory investigations, relatively few tests have been carried out to evaluate the 

effectiveness of oil bioremediation in the field because such trials are both difficult and 

expensive to conduct. Swannell et al. (1996) conducted the most extensive review available on 

field evaluations of oil bioremediation in marine environments. Venosa (1998) presented an indepth critical review emphasizing problems in the design and control of the existing field tests. A 

review by Lee (2000) addressed the potential significance of enhancing plant growth (i.e. 

phytoremediation) and oil mineral aggregate formation as biostimulation treatments. Other 

reviews are also available (Prince, 1993; Leahy and Colwell, 1990). This section will summarize 

the latest findings from recent field studies on marine shorelines and freshwater wetlands, as well 

as major points identified in the previous reviews. 

28
2.5.1 Mesocosm studies 

Mesocosms or pilot-scale systems can help to simulate actual conditions at relatively low cost, 

and are frequently used as bridges between microcosms and field systems. Mesocosms have 

been used to evaluate the effectiveness of numerous bioremediation strategies. 

Bioaugmentation 

Unlike results from bench-scale tests, numerous mesocosm studies have demonstrated the 

ineffectiveness of bioaugmentation treatments. For example, Tagger et al. (1983) overlaid two 

mesocosms with crude oil. One was inoculated with an acclimated culture, while only 

indigenous populations were used in the other. Five months after inoculation, no statistically 

significant change in oil composition occurred between the two treatments. Neralla et al. (1995) 

investigated the effect of seeding in salt marsh conditions. The greenhouse experiment was 

conducted with 19-L buckets filled with marsh sediments and actively growing Spartina 

alterniflora. There were 10 duplicated treatments in a total of 20 mesocosms. Results showed 

that the addition of bioaugmentation products did not enhance the degradation of weathered 

Arabian Lube crude oil. The conclusion was not surprising since the soil used in the experiment 

came from microcosms used in a similar study, and a large population of hydrocarbon-degrading 

microorganisms was already present in the sediment. These results again indicate that oil 

biodegradation is unlikely limited by availability of hydrocarbon degraders and that seeded 

microorganisms could not compete with indigent populations. 

Biostimulation 

Contrary to the mesocosm bioaugmentation studies, the addition of nutrients has proven to be an 

effective strategy for oil bioremediation. Basseres et al. (1993) conducted a mesocosm trial on 

bioremediation of light Arabian crude oil using two 600-liter tanks filled with sandy beach 

materials at a site near the Mediterranean. Animal meal containing 60% protein was added at 10 

% w/w to one tank. A second was left untreated as a control. Over the 60-day period of the test, 

40% of the aliphatic fraction in the treated oil was degraded whereas only 25% was degraded in 

the control. The number of hydrocarbon degraders was found to be higher in the meal-treated 

mesocosm than in the control. However, no replicate treatments and nutrient measurements 

were performed during the study, so it was not possible to determine if the observed differences 

were statistically significant. 

Mendelssohn et al. (1995) conducted a greenhouse mesocosm study to determine the effect of oil 

bioremediation products on salt marsh ecosystems. A randomized block design was used with 

three treatments (fertilizer, microbial products and control) at two levels of oil dosage. Each 

treatment combination was replicated five times for a total of 30 sods of marsh. The results 

demonstrated that the addition of a fertilizer product significantly increased the growth response 

of a salt marsh grass (Spartina alterniflora) and the rate of soil respiration, while the microbial 

products did not significantly affect either of these processes, suggesting the bioremediation 

products had neither toxic nor stimulatory effects on the salt marsh environments. 

29
Purandare (1999) conducted a mesocosm study following the microcosm study described earlier 

(Purandare et al., 1999) to further test the effectiveness of bioremediation of an oil-contaminated 

freshwater wetland. Outdoor mesocosms each measuring 6 m x 4.5 m, filled with wetland 

sediments, and planted with three species of emergent wetland plants were used in the study to 

investigate the effect of different inorganic mineral nutrients on biodegradation of a Louisiana 

crude oil. A total of 12 of these mesocosms were used (3 replicates of 4 treatments). The four 

treatment strategies included a no-nutrient control, phosphate addition, nitrate + phosphate 

addition, and ammonia + phosphate addition. Biodegradation rates were computed from hopanenormalized analyte data. The results showed that addition of nutrients seemed to enhance oil 

biodegradation initially. However, beyond 12 weeks, the untreated control achieved a 

comparable degree of biodegradation. No conclusion as to which nutrients were actually limiting 

the biodegradation process was reached due to the high variability in the data. The study also 

found that addition of nutrients led to better plant and root growth, which suggested that, 

although biostimulation may not significantly enhance oil degradation in freshwater wetlands, it 

may encourage a faster recovery of the ecosystem. It also suggested that the wetland plants may 

have out-competed the oil-degrading microorganisms for nutrients and may have used substrates 

other than oil hydrocarbons for their growth (soil organic matter). 

2.5.2 Field Demonstrations 

Bioremediation field studies are reviewed here, first with respect to bioaugmentation and 

biostimulation, then by a more detailed discussion of four case studies that cover a wide range of 

shoreline types. 

Bioaugmentation 

The effectiveness of seeding has been studied in only a few field trials. Venosa et al. (1992) 

conducted a field test in Prince William Sound following the Exxon Valdez spill to investigate 

the effectiveness of two commercial microbial products vis-à-vis natural attenuation and nutrient 

addition alone. These products were selected based on a previous laboratory study (Venosa et 

al., 1991). This field trial failed to demonstrate enhanced oil biodegradation by these products. 

No biostimulation occurred in the nutrient control plots either. There were no significant 

differences between the treatment and control plots during the 27-day trial period. However, the 

site where the project took place (Disk Island) was characterized as having highly weathered 

(degraded) oil and very calm waters, so dissolved oxygen may have been limiting, thus 

precluding effective biodegradation by any means. 

Other studies (Lee and Levy, 1987; Tagger et al., 1983) suggested that exogenous microbial 

inocula are not able to compete successfully with indigenous populations. One approach in 

overcoming this competition has been proposed by Rosenberg et al. (1992). They developed a 

product that combined a polymerized ureaformaldehyde (F-1) with a selected oil-degrading 

culture capable of using this fertilizer as a nitrogen source. Thus, the bacteria had a selective 

advantage over the indigenous population unable to utilize F-1 as nutrient source. A field trial 

conducted at an Israeli beach showed that this approach seemed to be successful in enhancing oil 

30
biodegradation. However, conclusions were confounded by the lack of adequate controls in the 

study (Swannell et al., 1996; Venosa, 1998). 

Studies comparing the performance of bioaugmentation and biostimulation have suggested that 

nutrient addition alone had a greater effect on oil biodegradation than did the addition of 

microbial products (Lee et al., 1997a; Venosa et al., 1996). ). This is probably because the 

microbial population is rarely a limiting factor as compared to the nutrients since the size of the 

hydrocarbon-degrading bacterial population usually increases rapidly in response to oil 

contamination. Lee et al. (1997a) conducted a 129-day field trial to compare the effect of four 

treatments on biodegradation of weathered Venture Condensate in a sandy beach. The four 

treatments included: inorganic nutrient and oil, a microbial product and oil, inorganic nutrient, a 

microbial product and oil, and oiled control. C2-chrysenes was used as a biomarker due to the 

low concentration of hopane in the condensate. The results showed that periodic addition of 

inorganic nutrients was the most effective strategy for enhancing oil degradation and reducing 

sediment toxicity, and that the full potential of the microbial product was limited by nutrient 

availability. A similar study conducted in a wetland by Simon et al. (1999) also show that 

addition of bioaugmentation agents did not enhance biodegradation of an Arabian medium crude 

oil. However, nutrient addition did not demonstrate any significant effect in their study either, 

suggesting other factors, such as oxygen, were limiting oil degradation. 

Several other possible reasons for the failure of inocula in degrading contaminants in nature were 

summarized by Goldstein et al. (1985), which include: (1) the concentration of the contaminant 

may be too low to support the growth of the inoculated species, (2) the natural environment may 

contain substances inhibiting growth or activity of the inocula, (3) the growth rate of the 

inoculated species may be limited by predation such as protozoa, (4) the added species may use 

other substrates in nature rather than the targeted contaminants, and (5) the seeded 

microorganisms may be unable to move through the pores of the sediment to the contaminants. 

A few field trials did claim success in demonstrating the effectiveness of oil bioaugmentation, 

such as using Alpha BioSeaTM to treat the Angolan Palanca crude oil spilled from Mega Borg off 

Texas coast (Mauro and Wynne, 1990) and using Terra-Zyme

TM in enhancing biodegradation of 

a heavy oil spilled from Nakhodka in Japan (Tsutsumi et al., 2000). However, the success of 

these studies was based on either visual observation (i.e. the Mega Borg study) or digital 

photographic image analysis (i.e., the Nakhodka study). No comprehensive monitoring program 

was used to verify the oil was indeed removed through enhanced biodegradation. The two 

products basically contains the same formula of bacteria cultures and nutrients (Hozumi et al., 

2000). The observed visual effects may have been due to physical or chemical processes such as 

surfactant action associated with the products (Swannell et al., 1996). 

It seems that in most environments, indigenous oil-degrading microorganisms are more than 

sufficient to carry out oil biodegradation if nutrient levels and other adverse environmental 

conditions do not limit them. Future research on oil bioaugmentation should focus on 

investigating which ecosystems may be deficient in oil degrading microorganisms and what 

types of oils or important oil components indigenous bacteria may be incapable of degrading. 

31
Biostimulation 

Although laboratory and pilot-scale studies have shown that biostimulation is a promising 

approach in enhancing oil biodegradation, the effectiveness of various types of nutrients and 

delivery strategies still require field demonstration. 

Sveum and Ladousse (1989) investigated the performance of Inipol EAP 22 in different types of 

sediments. The results showed that the oleophilic fertilizer enhanced oil biodegradation in 

coarse-grained sediments but not in fine-grained sediments. 

Researchers from Fisheries and Oceans-Canada (Lee and Levy, 1987; Lee and Levy, 1989; Lee 

and Levy, 1991; Lee and Trembley, 1993; Lee et al., 1995a; and Lee et al., 1997a) conducted a 

series of field tests to investigate the effect of different types of fertilizer and different deliver 

strategies in a low energy, sandy beach or in a salt marsh. Their studies demonstrated that 

biostimulation using periodic addition of inorganic fertilizers (ammonium nitrate and triple super 

phosphate) increased the rate of oil removal from beaches as measured by changes in oil 

composition relative to conserved biomarkers such as C2-chrysenes and/or the decline in the nC17/pristane and n-C18/phytane ratios. In contrast, the addition of the oleophilic fertilizer Inipol 

EAP 22 did not enhance oil degradation in a sandy beach (Lee and Levy, 1987 and 1989). 

Another study involved periodic addition of water-soluble fertilizer granules (ammonium nitrate 

and triple super phosphate) in an attempt to enhance biodegradation of waxy crude oil in a lowenergy, sandy beach and in a salt marsh (Lee and Levy, 1991). Two concentrations of the 

NH4NO3 were tested (0.34 and 1.36 g/L sediment). The oil used was Terra Nova crude at two 

different levels (0.3 and 3.0%). Results from the sandy beach showed that at the lower level of 

oil contamination, no enhancement by fertilizer was achieved. However, at the higher oil 

contamination level, substantial oil degradation occurred in the fertilized plots compared to the 

unfertilized ones. Results in the salt marsh were the exact opposite. Enhancement by fertilizer 

was significant at the 0.3% contamination level, but no enhancement occurred at the 3% oil 

contamination, which was attributed to the penetration of oil into the anaerobic zone where little 

degradation is expected. Studies on the utility and efficacy of various slow-release fertilizer 

formulations also were evaluated (Lee et al., 1993). They demonstrated that the effects of 

environmental factors controlling nutrient delivery from the various formulations under review 

(e.g., sulfur-coated urea) were the key to bioremediation success. Another field study conducted 

by Lee et al. (1995a) compared the performance of inorganic nutrients with organic fish bonemeal fertilizer. These results showed that the organic fertilizer had the greatest effect on 

microbial growth and activity, while the inorganic nutrients were much more effective in crude 

oil degradation. 

All these results suggest that the success of bioremediation is case specific, depending on oil 

properties, the nature of the bioremediation products and the characteristics of the contaminated 

environments. Fortunately, recent studies have shown that the oil biodegradation rate depends 

on the nutrient concentrations in the pore water of the sediments, which could provide important 

guidance for nutrient applications (Bragg et al., 1994, Venosa, 1996). This finding may also 

explain why some earlier trials have failed to demonstrate the effectiveness of nutrient 

32
application since the nutrient concentrations in the interstitial pore water had not been monitored 

and controlled in most of these studies. Venosa et al. (1996) found that maintenance of a 

threshold nitrogen concentration of 1 - 2 mg N/L in the interstitial pore water would result in 

close to maximum hydrocarbon biodegradation in a sandy beach (this will be discussed in detail 

in the section on case studies). Future research on biostimulation probably should focus on the 

determination of the optimal interstitial nutrient concentration and the best strategies to maintain 

this concentration for various environments whenever the degradation is limited by nutrient 

availability. 

In addition to the demonstration of the efficacy of oil degradation, it is also necessary to 

demonstrate that bioremediation does not produce any undesired environmental and ecological 

effects. There have been concerns that enhanced microbial degradation of oil might produce 

toxic metabolic by-products. To address this concern, Lee et al. (1995b) conducted a field study 

using different fertilizers to investigate the effect of bioremediation on the toxicity of Venture 

condensate stranded on sandy beach sediments. The toxicity of the sediments was monitored 

using the Microtox Solid-Phase Test. The results indicated that sediment toxicity was not 

significantly affected by the addition of an inorganic fertilizer (ammonium nitrate and triplesuperphosphate). However, they did observe a slowing of the decrease in toxicity when the 

organic fertilizer (fishbone meal) was applied repeatedly, which was attributed to rapid 

biodegradation of the fertilizer and the production of ammonia that exceeded toxicity threshold 

limits. 

Case studies 

Four representative field studies are described in more detail here. Three types of marine 

shorelines and one freshwater wetland are covered. 

Exxon Valdez 

Following the grounding of the supertanker Exxon Valdez on Bligh Reef in Prince William 

Sound in 1989, U.S. EPA, in conjunction with the Exxon Corporation and the state of Alaska, 

embarked on the largest oil spill bioremediation project ever attempted in the field. Extensive 

field trials at various sites were conducted, which have been well documented in the literature 

(Bragg et al., 1994; Prince, 1993; Prince et al., 1994; Pritchard and Costa, 1991; Swannell et al., 

1996; Venosa, 1998). Important findings and lessons learned from these studies are summarized 

as follows. 

• 	 Seeding of bioaugmentation products failed to demonstrate enhanced oil biodegradation. It 

was found that oil biodegradation on the shoreline of the Prince Williams Sound was limited 

by the concentration of nutrients, particularly nitrogen, and not by the absence of 

hydrocarbon-degrading microorganisms (Pritchard and Costa, 1991; Venosa et al., 1992). 

• 	 Three types of nutrients or fertilizers were tested in the field: a water-soluble inorganic 

fertilizer (23:2 N:P garden fertilizer formulation), a slow-release inorganic fertilizer 

(Customblen), and an oleophilic fertilizer (Inipol EAP 22). Each was shown to be variably 

33
effective. Inipol EAP22 and Customblen were chosen as bioremediation agents, and 

approximately 50,000 kg of nitrogen and 5,000 kg of phosphorus were applied over 120 km 

of the oil contaminated shorelines during 1989 and 1992. Within two weeks after the 

fertilizer application, the area of cobble beach treated with Inipol EAP22 and Customblen 

appeared to be visibly cleaner than the untreated area (Pritchard and Costa, 1991; Pritchard et 

al., 1992). However, it was later found that the oil coating the surfaces of the cobble had 

been lifted and re-deposited within the interstices in the beach surface. 

• 	 Heterogeneous oil distribution on the contaminated beaches made it difficult to determine the 

rates of oil biodegradation using established methods. The changes in the ratios of a 

hydrocarbon component to a conserved internal standard or biomarker were used as the basis 

for determining the oil degradation rate. They also found that previously traditional 

biomarkers such as pristane and phytane degraded rapidly in Alaskan beaches. This 

observation rendered use of such biomarkers ineffective in permitting firm conclusions on 

bioremediation effectiveness. 17α(Η),21β(Η)-hopane, the pentacyclic triterpane containing 

30 carbon atoms, was used instead. Using hopane as the biomarker, Bragg et al. (1994) 

showed that fertilizer application accelerated the rate of oil removal by a factor of 

approximately five-fold compared to natural attenuation. This observation was made, 

however, from samples repeatedly collected from only one site, so the statistical basis 

supporting this conclusion is tenuous. 

• 	 Oil biodegradation rate appeared to be dependent on the nitrogen concentration in the pore 

water of the intertidal sediments, suggesting that on-site monitoring of nutrients in the 

sediment pore waters could provide practical guidance for nutrient applications (Bragg et al., 

1994). 

• 	 According to the EPA/Exxon/State of Alaska joint monitoring program, bioremediation was 

an environmentally sound remediation technique. This was based on the results of testing the 

toxicity of nearshore water to sensitive marine species such as Mysid shrimp, analyzing 

ammonium and nitrate concentrations, evaluating the potential of algal growth, and 

monitoring oil release into nearshore water after the application of fertilizers (Prince et al, 

1994). 

• 	 The results of the fertilizer application following the Exxon Valdez spill generally 

demonstrate that bioremediation may enhance oil biodegradation on certain marine 

shorelines. However, conclusions on the effectiveness of bioremediation in the Exxon 

Valdez study are somewhat questionable, in part because the experimental design was not 

entirely based on sound statistical principles (Venosa, 1998). Major flaws included the lack 

of replication and the attempt to determine too many factors in a limited number of tests, 

resulting in the confounding of different effects. The lessons learned from the Exxon Valdez 

project led to the replacement of “post Exxon Valdez excitement” with more scientificallyvalid approaches (Mearns, 1997). 

34
Delaware Field Study 

The main purposes of this field study were to obtain credible statistical evidence in determining 

if bioremediation with inorganic mineral nutrients and/or microbial inoculation enhances the 

removal of crude oil, to compute the rate at which such enhancement takes place, and to establish 

engineering guidelines on how to bioremediate an oil-contaminated sandy shoreline. 

The trial was conducted on a medium- to coarse-grained sandy beach (environmental sensitivity 

index = 4) at Fowler Beach, Delaware (located midway between Dover and Rehoboth Beach). A 

randomized complete block design was used in the study. Twenty 4 x 9 m plots were set up in 

five replicate blocks. Each block contained four treatments in random order, which included: an 

unoiled control plot, a no-nutrient control plot (natural attenuation), a plot receiving watersoluble nutrients, and a plot receiving water-soluble nutrients supplemented with a natural 

microbial inoculum from the site. Weathered Nigerian Bonny Light crude oil was intentionally 

released onto 15 plots. Nutrients (NaNO3 and Na5P3O10) were applied daily through a sprinkler 

system at a rate designed to achieve a target of 1.5 mgN/L average interstitial pore water 

concentrations. Once a week, 30 L of a suspended mixed population of hydrocarbon-degrading 

bacteria was also added to the inoculum plots. Sand samples were collected every 14 days from 

the 15 oiled plots for oil analysis, and all analytes were normalized to hopane. Nitrate 

concentrations in the interstitial pore water of oiled plots were measured each day. 

Figure 2.3a shows the hopane-normalized concentrations of total target alkanes (i.e., the sum of 

all alkane analytes from n-C10 to n-C35, plus pristane and phytane), while Figure 2.3b shows the 

total target aromatics (i.e., the sum of all groups of PAHs and sulfur heterocyclics analyzable by 

GC/MS and their alkyl-substituted homologues) in the nutrient-treated, inoculum-treated, and 

control plots, all as a function of time. Although substantial hydrocarbon biodegradation 

occurred in the untreated plots, statistically significant differences between treated and untreated 

plots were observed in the biodegradation rates of total alkanes and total aromatic hydrocarbons. 

The results also show that bioaugmentation, even with indigenous organisms, does not stimulate 

further degradation of hydrocarbons beyond simple nutrient addition. The studies further 

demonstrated that maintenance of a threshold nitrogen concentration of 1 - 2 mg N/L in the 

interstitial pore water would permit close to maximum hydrocarbon biodegradation. 

Another important conclusion from this study is that background nutrient concentrations at the 

contaminated site should be a determining factor in the decision to apply bioremediation. The 

background nitrogen concentration at Fowler Beach was high enough to permit close to 

maximum hydrocarbon biodegradation without the need to apply additional fertilizer despite the 

enhancement observed from nutrient addition. The enhanced effect, although statistically 

significant, was not substantial enough to have warranted a decision to implement 

bioremediation on a full-scale basis had there been a real spill at this site. This demonstrates that 

assisted bioremediation might not always be necessary if sufficient nutrients are naturally present 

at a spill site in high enough concentrations to perform natural cleanup. For coastlines having 

low natural input levels of nutrients, bioremediation would be appropriate as an alternative 

cleanup option. 

35
0 10 0 30 0 50 0 70 80 0 100 

0 

2 

4 

6 

8 

10 

12 

14 

16 

n

g 

A

R

O

M 

A

T

I

C

S

n/

g 

h

o

p

a

n 

e 

(b) 

0 

50 

100 

150 

200 

n

g 

A

L

K

A

N

E

S

n/

g 

h

o

p

a

n 

e 

control 

nutrients 

inoculum 

(a) 

Figure 2.3. rst-order declines in (a) total target alkanes and 

(b) total target aromatics 

2 4 6 9

Results of the Delaware field study: fi

36
Bioremediation of a Fine Sediment in England 

The previous two cases have demonstrated that bioremediation can be effective for cobble and 

sandy shorelines, although the extent of the enhancement in effectiveness is determined by the 

natural presence of nutrients. However, much less attention has been given to fine sediment 

marine shorelines such as mudflats. A recent study conducted by Swannell et al. (1999a) was 

intended to fill this gap. One of the objectives of this study was to investigate the potential of 

bioremediation in treating buried oil. 

The field site was located within the Stert Flats on the southwest coast of England. Twelve plots 

were set up on an 80-m stretch of sand composed of 3.2 % mud and 80% particles in the range of 

125-180μm. The experimental area was divided into three replicate blocks. Each block 

consisted of four randomly assigned treatments that consisted of an unoiled control plot, an 

unoiled plot treated with fertilizer alone, an oiled plot with no amendments added, and an oiled 

plot treated with fertilizer. The sediment in each plot was retained in mesh enclosures (0.4 m x 

0.4 m x 0.05m) and buried at a depth of 15 cm. Weathered and emulsified Arabian Light crude 

oil was applied to the appropriate enclosures at 3.7 kg/m2

. Inorganic fertilizer (NaNO3 and 

KH2PO4) was applied using a sprinkling device at rates of 2% of N and 0.2% of P by weight of 

the initial oil concentration, and at a frequency of once a week for the first four weeks and then 

every two weeks thereafter. Samples were taken on Day 7, 49, and 108 for oil analysis, and 

hopane was used as a biomarker. 

The results showed that more oil was degraded in 2 of the 3 fertilized plots than in the controls 

after 108 days. In blocks 1 and 3, the mean total GC resolvable hydrocarbons (TGCRH)/hopane 

ratio decreased by 58.4% and 48.4% respectively, compared to 23.0% and 4.4% in the two oiled 

controls respectively. In block 2, oil degradation was slower, with only 14% decrease of 

TGCRH/hopane ratios in the treated plot vs. no removal in the control plot. Statistical analysis 

demonstrated that differences in the ratio of TGCRH and TPH against hopane between the 

fertilized plots and the controls were highly significant (p<0.0001). Microbiological analyses 

also showed that nutrient addition increased the numbers of hydrocarbon-degraders on the oiled 

plots by ten-fold. This study suggested that bioremediation by nutrient enrichment for the 

treatment of buried oil in fine sediments may be feasible after an oil spill incident. However, 

because of the third replicate failing to confirm the results of the other two, more definitive 

conclusions cannot be made from this study. 

One deficiency of this study was that by aerating the subsurface sediments during the sediment 

burying and periodic excavation of the site due to nutrient application, the potential problem of 

oxygen limitation in this environment could not be evaluated. Oxygen limitation is a major 

concern for application of oil bioremediation in subsurface sediments, anoxic zones of a water 

column, and most fine-grained marine shorelines (Head and Swannell, 1999). Because this 

experimental site was a high-energy beach with a tidal bore, oxygen may not have been a 

limiting factor for this specific fine sediment beach. Conclusions from this study, therefore, 

should only be confined to the Stert Flats and not extrapolated to similar environments. Further 

37
research is still required to determine the effectiveness of bioremediation in other types of fine 

sediments. 

Bioremediation of a Freshwater Wetland in Canada 

A field study was conducted by the U.S. Environmental Protection Agency, University of 

Cincinnati, and Fisheries and Oceans Canada at a freshwater wetland site situated along the St. 

Lawrence River, Ste. Croix, Quebec, Canada. The objective of this study was to determine the 

effectiveness of biostimulation strategies in accelerating restoration of an oil-contaminated 

freshwater wetland site. (Garcia-Blanco et al., 2001b, Venosa et al., 2002). Strategies that were 

evaluated were bioremediation by nutrient enrichment in the presence and absence of vegetative 

growth of the dominant plant species, Scirpus pungens. Twenty 5m x 4m plots were set up in the 

upper intertidal zone of a study site located along the St. Lawrence River, where the water was 

far enough away from the Atlantic to be still fresh, yet was tidally influenced. These plots were 

divided into four replicate blocks. Each block consisted of five randomly assigned treatments, 

which were: (1) oiled, no added nutrients, with intact plants (natural attenuation control), (2) 

oiled, NH4NO3 + Ca(H2PO4)2

.

H2O added, with all vegetative growth cut back to ground surface 

daily to suppress plant growth, (3) oiled, NH4NO3 + Ca(H2PO4)2

.

H2O added, with intact Scirpus 

pungens, (4) oiled, NaNO3 + Ca(H2PO4)2

.

H2O added, with intact Scirpus pungens, and (5) 

unoiled, NH4NO3 + Ca(H2PO4)2

.

H2O added, with intact Scirpus pungens (background control). 

Weathered Mesa light crude oil was released onto each plot earmarked for oiling. The amount of 

oil released was 12 L per plot. Composite core samples were collected after 0, 1, 2, 4, 6, 8, 12, 

16, and 21 weeks for quantification of the remaining oil constituents by gas 

chromatography/mass spectroscopy (GC/MS) operating in the Selected Ion Monitoring mode or 

SIM. To account for differences due to physical washout, all oil constituents were normalized to 

hopane. 

Figure 2.4 illustrates results from the hopane-normalized concentrations of total target alkanes 

and total target PAHs for the four treatments as a function of time. Although the bioremediation 

and phytoremediation treatments achieved slightly better degradation of hydrocarbons than 

natural attenuation, no statistically significant evidence of stimulation through addition of 

nutrients or biodegradation enhancement by vegetation was observed. After 21 weeks, reduction 

of target parent and alkyl-substituted polycyclic aromatic hydrocarbons (PAHs) averaged 32% in 

all treatments. Reduction of total target alkanes was of a similar magnitude. The pattern of 

disappearance of hydrocarbons was characteristic of biodegradation; namely, the lower 

molecular weight alkanes declined to a greater extent than the higher carbon-number alkanes, as 

did the lower molecular weight PAHs compared to the higher molecular weight PAHs. Since 

there was little evidence to support enhancement of biodegradation by nutrient addition with and 

without vegetation, it was suggested that oxygen limitation was most likely the dominant cause 

of the persistence of oil hydrocarbons on the oil-contaminated plots. 

38
600 

500 

400 

300 

200 

100 

0 

125 

100 

75 

50 

25 

0 

0 1 2 4 6 8 12 16 21 

Natural Attenuation 

Bio + Ammonium 

Phyto + Ammonium 

Phyto + Nitrate 

(a) 

(b) 

Natural Attenuation 

Bio + Ammonium 

Phyto + Ammonium 

Phyto + Nitrate 

Time (weeks) 

Figure 2.4. Results of St. Lawrence River field study: concentration declines in (a) total target 

alkanes and (b) total target PAHs ng P

A

Hs / ng hopane ng Alkanes / ng hopane 

39
While the Ste. Croix results indicate that biostimulation might not be an effective strategy to 

mediate the removal of residual oil from the sediments, significant changes in biological 

measures of habitat were observed (Lee et al., 2001a). For example, S. pungens, the dominant 

plant species, was tolerant to the oil, and its growth was significantly enhanced above that of the 

unoiled control by the addition of nutrients. Other biotest organisms (bacteria, Vibrio sp., 

invertebrates, Daphnia, Hyalella, and Viviparus sp.) provided additional evidence of both 

enhanced recovery and potential detrimental effects. The study indicates that on an operational 

scale, natural attenuation may be the most practical treatment option for oxygen-limited 

freshwater wetlands. 

2.5.3 Kinetics of oil bioremediation 

Knowledge of the kinetics of oil biodegradation under different environmental conditions is 

important for assessing the potential fate of targeted compounds, evaluating the efficacy of 

bioremediation, and determining appropriate strategies to enhance oil biodegradation. Oil 

biodegradation rates are difficult to predict due to the complexity of the environment. The rates 

of biodegradation vary greatly among the various components of crude oils and petroleum 

products. The presence of other substrates may affect the degradation rates of the compounds of 

interest. Environmental factors such as temperature, nutrient concentrations, and oxygen tension 

also influence the kinetics of oil degradation. The heterogeneity of oil distribution on shorelines 

or wetland sediments makes kinetics studies even more difficult. 

Very few kinetic studies on oil degradation under field conditions have been conducted. The 

Exxon Valdez monitoring program developed a multiple regression model based on field studies 

conducted by researchers from Exxon (Prince et al., 1994). The best-fitting model was 

expressed as: 

Ch(t) = α[1-p(t)]γ e δr(t)+ωt ε (2.2) 

where Ch(t) is the time-varying hopane-normalized concentration of an analyte, p is the polar 

fraction of the oil, r is the ratio of the average residual nitrogen concentration to oil loading, and 

ε is the assumed multiplicative error term, while α, δ, γ, and ω are fitting parameters determined 

from the multiple regression analysis. This model matched the experimental results in Alaska 

well when the parameters are chosen to fit the data. However, its potential for process 

understanding and prediction is limited because the data set used for the regression was limited 

to only one small, non-replicated area in the field. 

Venosa et al. (1996) developed from field data first-order biodegradation rate constants for 

resolvable alkanes and important two- and three-ring PAH groups present in light crude oil. The 

first order relationship was expressed as: 

 A   A 



e−kt   =  (2.3)  H   H 0 

40 
where (A/H) is the time-varying hopane-normalized concentration of an analyte, (A/H)0 is that 

quantity at time zero, and k is the first-order biodegradation rate constant for an analyte. 

For the field study conducted in Delaware, first-order biodegradation rate coefficients ranged 

from 0.026 to 0.056 day-1 for total resolvable alkanes and from 0.021 to 0.031 day-1 for total 

resolvable PAHs (Venosa et al., 1996). 

Actual 1st-order biodegradation rates are not constant, however. Instead, they are a function of 

the residual nutrient concentration: 

 N  kobs = kmax 





Kn + N 



 (2.4) 

where kobs and kmax (T-1) are the observed and maximum first-order hydrocarbon biodegradation 

rates, respectively, Kn (MnL-3) is the half-saturation concentration for a specific nutrient, and N 

(MnL-3) is the interstitial pore water residual nutrient concentration. Experiments conducted at 

the University of Cincinnati showed that the Kn for nitrate is approximately 0.5 mg N/L 

(unpublished). The model that incorporates Equation 2.3 and 2.4 will be very useful in the 

experimental design and the performance prediction for the bioremediation of oil contaminated 

shorelines. 

Studies have also been conducted to compare oil biodegradation rates obtained in laboratory tests 

to those calculated from the Delaware field study (Venosa et al., 1996, 1997a; Holder et al., 

1999). Venosa et al. (1996, 1997a) found that the degradation rates of all target alkanes and 

aromatics in a light crude oil were close to an order of magnitude lower in the field compared to 

results from the laboratory. However, when the rate data of PAHs were normalized to the highest 

alkyl-substituted homologue in each given PAH series, the first order rate constants in the field 

were nearly identical to rate constants from the lab. These relationships were consistent even 

with different microbial consortia isolated from eight different marine shorelines of the United 

States. Similar results were also observed in a laboratory study using 14 different marine and 

freshwater consortia (Holder et al., 1999). 

Simon et al. (1999) reported some kinetic data derived from a study conducted in a coastal 

wetland contaminated with Arabian light crude oil. First-order biodegradation rate coefficients 

ranging from 0.017 to 0.061 day-1 for total target saturates and from 0.009 to 0.027 day-1 for total 

target aromatics were reported. These rate coefficients were similar to those of Venosa et al. 

(1996). Further research is still required to develop more state-of-art models and to establish a 

database of kinetic parameters for different types of oil under various marine shoreline and 

freshwater wetland environments. 

2.6 Nutrient Hydrodynamics 

Since nutrient addition has been found to be the most effective bioremediation strategy in aerobic 

environments, particularly for marine shorelines, a full understanding of the fate of water soluble 

41 
nutrients on marine beaches and the hydrodynamics controlling their transport and persistence is 

necessary. One of the main challenges associated with biostimulation in oil-contaminated 

coastal areas is maintaining optimum nutrient concentrations in contact with the oil and the 

degrading microorganisms. Oil from offshore spills usually contaminates the upper third of the 

intertidal zone, where the washout rate for water-soluble nutrients can be very high. Various 

oleophilic and slow-release nutrient formulations have been developed to improve the contact 

between oil and nutrients within the environment. However, most slow-release and many 

oleophilic fertilizers rely on dissolution of the nutrients into the aqueous phase before they can 

be used by hydrocarbon degraders (Safferman, 1991). Thus, design of effective oil 

bioremediation strategies and nutrient delivery systems requires an understanding of the 

transport of water-soluble fertilizers in a beach ecosystem. 

Dissolved nutrients are expected to move with the water in the beach sand. Water flow through 

the porous matrix of a beach is driven by a combination of three main factors (Boufadel et al., 

1999b; Wrenn et al., 1997): (1) tides that result in rise and fall of beach groundwater level 

(typically, the water level in a beach tracks the level of the rising tide with only a slight lag, but 

the beach drains much more slowly when the tide ebbs because of resistance from the porous 

matrix), (2) wave action that operates through two main mechanisms (at wave runup, water 

enters the beach and percolates vertically through the unsaturated zone until it reaches the water 

table; at wave rundown, water moves in a predominantly horizontal direction and exits at the 

water line), and (3) flow of fresh groundwater from coastal aquifers, which causes continuous 

horizontal advective flow from the beach face at or near the water line (Glover, 1959). This type 

of groundwater flow can interact with tidal fluctuations to produce complex variations in the 

groundwater level within the beach (See Section 2.6.1). 

2.6.1 Nutrient transport in beaches: a mesocosm study 

Beach hydraulics and nutrient hydrodynamics were investigated through both theoretical and 

experimental approaches by Boufadel et al. (1999 a and b, 1998, 1997). A two-dimensional 

finite element model for water flow and salt transport in saturated and unsaturated porous media 

was developed. The model also considers the effects of salt concentration on water density and 

water viscosity. An experimental wave tank was used to validate the model and to investigate 

cases that cannot be simulated by the numerical model. Tracer tests were carried out to 

investigate the separate and combined effects of tide, waves, and buoyancy on the transport of 

soluble inorganic nutrients in sand beaches. The major findings from these studies were: 

• 	 In the absence of regional seaward groundwater flow, the tide generated a predominantly 

downward and seaward hydraulic gradient that caused the washout to the sea of nutrients 

applied to the top section of beaches. The presence of waves under these conditions 

accelerated the washout, the rate of which was found to increase by about 30% when waves 

were superimposed on the tide. 

• 	 Beach geometry plays a major role in beach hydraulics and hydrodynamics because the flow 

lines are perpendicular to the beach surface. Under tidal action, seawater enters the beach 

42
from above, flowing vertically downward, and may cause the entrapment of less saline water 

in the beach. This finding is very important because systems that rely on continuous or 

intermittent injection of nutrient solutions through trenches or horizontal wells installed in 

the supratidal zone rely on subsurface flow to carry the nutrients through the contaminated 

area (Wise et al., 1994). The approach that was proposed by Wise et al. (1994) assumes that 

nutrients dissolved in the freshwater plume will be brought into contact with the oiled beach 

material periodically by the rising tide, because the freshwater plume should float on top of 

the saltwater. The finding of freshwater trapped between two saltwater wedges indicates that 

subsurface injection of nutrients may not be an effective method for providing nutrients to 

the bioremediation zone when a freshwater plum exists because the impact zone will never 

be exposed to the nutrients. 

• 	 Different nutrient application strategies at low tide were investigated in the mesocosm beach 

since application of nutrients at the beach surface at low tide (Venosa et al., 1996) appears to 

be the best application strategy for biostimulation. The results showed that applying nutrients 

at the beach surface pre-dissolved in water resulted in generally longer residence times and 

larger spreading of the nutrient plume in the top section of the beach compared to applying 

nutrients in granular form at the beach surface and hosing them in with a water spray. 

However, it should be noted that in practice, addition of granular fertilizer may be prudent in 

cases where it is not possible to sprinkle pre-dissolved nutrients as the application method. 

2.6.2 Nutrient transport in beaches: field trials 

To verify the findings from the mesocosm studies and to further investigate nutrient transport 

under field conditions, tracer studies were conducted in the intertidal zone of three different 

marine beaches in Delaware and Maine (Suidan and Wrenn; 2001; Wrenn et al.,1997a & b). 

The Delaware Tracer Study 

The purpose of this study was to characterize the transport of water-soluble nutrients in the 

intertidal region and to estimate their washout rates from the bioremediation zone (i.e., the oilcontaminated area). The study was conducted on a moderate-energy, sandy beach (Slaughter 

Beach) on Delaware Bay. The typical wave heights at this beach were between 15 to 30 cm. A 

conservative tracer (LiNO3) was applied to eight replicate 5 m x 10 m plots in the upper 

intertidal zone at low tide during full moon spring tide and the last-quarter moon neap tide. The 

tops of the plots were placed approximately at the spring high tide line. Sand samples were 

collected for tracer analysis. 

This study showed that the rate of tracer washout from the bioremediation zone was more rapid 

when the tracer was applied at spring tide (when the tidal amplitude is largest) than at neap tide. 

When the conservative tracer (LiNO3) was applied to the beach surface in the upper intertidal 

zone at the full moon spring tide, it was completely removed within one day. When it was 

applied at neap tide, however, the tracer persisted in the bioremediation zone for several days. 

The results indicated that the amount of nutrient remaining in the bioremediation zone was 

highly correlated with the maximum extent to which the treated area had previously been 

43
submerged by water at high tide; submergence resulted in nearly complete removal of dissolved 

compounds from the bioremediation zone. Therefore, the fertilizer release rates should be 

designed to achieve optimal nutrient concentrations while the tide is out. The Delaware tracer 

study indicates that nutrient transport in sandy beaches is driven by tidally influenced hydraulic 

gradients and wave activity. However, it is impossible to clearly separate the influence of tide 

and wave action in this study. 

The Maine Tracer Study 

The main purpose of this field study was to separately evaluate the effect of tide and wave action 

on nutrient transport since the results of the Slaughter Beach field studies could not distinguish 

between these two processes. This was accomplished by comparing the transport rate and 

characteristics of a dissolved conservative tracer (lithium nitrate) on a high-energy beach to those 

on a low-energy beach to determine how waves affect solute transport (Suidan and Wrenn, 2001; 

Wrenn et al., 1997b). The two beaches are located in the town of Scarborough, in southern 

Maine. Scarborough Beach is a high-energy beach that faces the Atlantic Ocean with average 

heights of breaking waves between 0.3 to 1 m during the course of this study, whereas Ferry 

Beach faces a protected harbor with the typical wave height of less than 3 cm. The tidal range at 

both beaches was essentially the same. Dissolved tracer (LiNO3) was applied to four replicate 

plots on each beach at low tide during full moon spring tide and the last-quarter moon neap tide. 

Both water and sand samples were collected for tracer analysis. 

Washout of lithium from the upper intertidal zone during the spring-tide experiment is shown in 

Figure 2.5. The differences between the two beaches are very clear. Whereas lithium was 

completely removed from the entire experimental domain within two days on the high-energy 

beach, more than two weeks were required to achieve the same degree of washout from the lowenergy beach. Washout during the neap-tide experiment was much slower than that observed 

during the spring-tide experiment. Lithium was completely removed from the experimental 

domain on Scarborough Beach within one week, whereas the total mass of lithium was 

essentially unchanged for more than two weeks at Ferry Beach. Slower washout was expected 

during the neap-tide experiment, because only the bottoms of the plots were covered by water at 

high tide during most of the first five or six days. Since plot coverage by the tide was essentially 

the same on the two beaches during the first week of this experiment, the differences in washout 

rate must have been due primarily to wave activity. 

The Maine field study clearly shows that the washout rate of nutrients from the bioremediation 

zone is strongly affected by the wave activity of the contaminated beach. Wave action in the 

upper intertidal zone may cause nutrients from the surface layers of the beach to be diluted 

directly into the water column, resulting in their immediate loss from the bioremediation zone. 

On the other hand, washout due to tidal activity alone is relatively slow, and nutrients will 

probably remain in contact with oiled beach material long enough to effectively stimulate oil 

biodegradation on low-energy beaches. 

44
0 5 10 15 

ti

me (

d

a

ys) 

0 .5 1 1.5 2 .5 .5 4 

ti

me (d

a

ys) 

(a) Ferry 

Beach (b) Scarb

oro

u

gh 

Beach 

0 2 3 3 

(b) Scarb

oro

u

gh 

Beach (a) Ferry 

Beach 

Figure 2.5 Washout of Lithium from the upper intertidal zone during the spring-tide experiment at (a) Ferry Beach and (b) Scarborough Beach 

The mass remaining is shown for (A) i

n t

he total experi

mental domain (B) i

nside t

he plot boundaries, and (C) i

n t

he bioremediation zone 

(i.e., within 30 cm of the surface). 

45

B 

B B B B B 

E 

E E E

E 

B 

E 

B 

B B 

B B B B B B B B 

E 

E 

E E E E E E 

C 

B B 

B B B B B B B 

E 

E 

E E E E E E E 

B 

B B B B B B B B B B 

E 

E 

E E E E E E 

A B 

E 

Spring-1 Spring-2

B 

E 

Spring-1 Spring-2 

E 

E 

B E

E 

E 

3.5 2.5 3 0 

ti

me (

d

a

ys) ti

me (d

a

ys) 

1.5 2 0.5 1 15 0 

B 

B B 

E 

E E 0 

500 

1000 1500 

B E 

0 

500 

1000 1500 

) g ( gni ni ma e r ss ma m ui hti L B E 0 

500 

1000 1500 

0 

100 200 300 400 500 60

0 

0 

100 200 300 400 500 600 

) g ( gni in a me r ssa m m ui h lit 0 100 

200 300 400 500 600 700 

B 

B B 

B 

B 

B 

B 

B 

B 

B 

B 

B B 

B B B 

E 

E 

E 

E 

E 

E 

E 

E 

E 

E 

E E 

E E 

E E 0 

500 

1000 1500 C 

B 

B 

B 

BB B 

B 

B 

B B 

B B B B B B 

E 

E 

E 

E 

E 

E 

E 

E 

E 

E 

E 

E E E E 0 

500 

1000 1500 

) g ( gni ni ma e r ss ma m ui hti L B 

B 

B 

B 

BB 

B 

B 

B 

B 

B 

B 

B 

B 

B B B 

E 

E 

E 

E 

E 

E 

E E 

E 

E 

E 

E 

E 

E 

E E 0 

500 

1000 1500 

A B 

E 

Spring-1 Spring-2

B 

E 

Spring-1 Spring-2 

0 

100 200 300 400 500 60

0 

0 

100 200 300 400 500 600 

) g ( gni in a me r ssa m m ui h lit 0 100 

200 300 400 500 600 700 

B 

B 

E 

E E

B E

5 10 4 
Salinity distribution was also examined in the Maine study. In both beaches, the “sandwiching 

phenomenon” (a layer of lower salinity water located between two higher salinity water layers) 

was observed, which was confirmed by Boufadel et al. (1999b) in the mesocosm study. Both 

these mesocosm and field studies are very helpful in providing guidelines for the optimal 

applications of nutrients on marine beaches, which will be discussed later in this document. 

Although many questions remain unanswered, we have made tremendous progress in 

understanding various aspects of oil bioremediation in the last decade. The development of an 

operational guideline for bioremediation of oil contaminated marine shorelines and freshwater 

wetlands is not only possible but also necessary in ensuring that full-scale cleanup in the future 

proceeds rapidly and efficiently. 

46
Chapter 3 Methods Used in Monitoring Oil Bioremediation 

In order to demonstrate that biodegradation is taking place in the field, the chemistry or 

microbial population must be shown to change in ways that would be predicted if bioremediation 

were occurring (NRC, 1993). Environmental conditions, particularly nutrient concentrations, 

should also be monitored for evaluating the effects of bioremediation. Some of the most 

important methods used in monitoring oil bioremediation will be overviewed in this chapter. 

References will be given for detailed descriptions of methods. 

3.1 Analytical Methods 

3.1.1 Microbiological analysis 

Existing methods for microbial analysis can be classified into cultured-based techniques and 

culture-independent techniques. Commonly used microbial analysis methods in monitoring oil 

bioremediation are summarized in this section and table 3.1. 

3.1.1.1 Enumeration of hydrocarbon-degrading microorganisms: culture based techniques 

Microbial counts are often used to monitor the bioremediation process. In general, the more 

microbes, the more quickly the contaminants will be degraded. Correlating an increase in the 

number of contaminant-degrading bacteria above normal field conditions is one indicator that 

bioremediation is taking place. Analysis of the microbial communities that take part in in-situ 

hydrocarbon biodegradation activities has been a challenge to microbiologists (Macnaughton et 

al., 1999). The reason for this is that most (~90 to 99%) of the species making up competent 

degrading communities do not form colonies when current laboratory-based culture techniques 

are used (Rollins and Colwell, 1986; Rozsak and Colwell, 1987; Wilkinson, 1988). The 

techniques are briefly described below. 

Plate count 

Plate count is a traditional technique, which quantifies the number of bacteria capable of growing 

on a prescribed set of nutrients and substrates in a solid medium, by counting the colonies 

formed (National Research Council, 1993). The general procedure involves (1) making the solid 

medium or gel from a liquid solution with appropriate nutrients and substrates, using a 

solidifying agent like agar, (2) Spreading a sample containing the bacteria of interest thinly over 

the surface of the gel in plates, (3) Incubating the plates, (4) counting the bacterial colonies 

formed. Each colony is assumed to have arisen from a single bacterial cell. 

A number of studies have used hydrocarbon incorporated into either agar-based or silica-based 

media to enumerate of hydrocarbon-degrading microorganisms (Horowitz and Atlas, 1978; 

Sexstone and Atlas, 1977; Walker and Colwell, 1976). However, other researchers reported that 

plate counts are unsuitable for enumerating hydrocarbon-utilizing microorganisms because many 

marine bacteria can grow and produce micro-colonies on small amounts of organic matter 

existing in the solid media, resulting in the counting of non-hydrocarbon utilizers (Atlas, 1981; 

Higashihara, et al., 1978). Plate counts also underestimate the number and diversity of bacteria 

because of the difficulty in enriching viable colonies from environmental samples. Culturable 

47
techniques have been found to be inferior to techniques that do not rely on viable culturing for 

enumeration (Macnaughton et al., 1999). 

Most-probable-number (MPN) procedures 

MPN procedures have been viewed as a more reliable method for enumerating hydrocarbonutilizing microorganisms because such procedures eliminate the need for a solidifying agent and 

permit direct assessment of the ability to actually utilize hydrocarbons (Atlas, 1981; Wrenn and 

Venosa, 1996). MPN procedures use liquid nutrient media in test tubes or microtiter plates and 

hydrocarbons as the sole carbon source. The enumeration is carried out through a statistical 

analysis based on the numbers of a series of diluted liquid samples that show evidence of 

bacterial growth. This evidence of bacterial growth can be established based on turbidity, release 

of 14CO2 from radiolabeled hydrocarbons, disruption of oil sheen, and reduction of dyes (Rice 

and Hemmingsen, 1997). Either statistical tables (Eaton et al., 1995) or a computer program 

(Klee, 1993) can be used to determine the MPN. 

Most existing MPN procedures use crude oil or a refined petroleum product as the selected 

hydrocarbons, which can not distinguish different groups of hydrocarbon degraders. For 

example, the Sheen Screen Method uses dispersion or emulsification of the crude oil substrate to 

identify positive wells (Brown and Braddock, 1990). But these effects are associated primarily 

with growth on aliphatic hydrocarbons (Hommel, 1990). Aliphatic hydrocarbons are of less 

environmental concern than PAHs, however, because they are less toxic and are biodegraded 

more rapidly. Wrenn and Venosa (1996) recently developed an MPN procedure that can 

separately enumerate aliphatic and aromatic hydrocarbon-degrading bacteria. The size of the two 

populations is estimated using separate 96-well microtiter plates. The alkane-degrader MPN 

method uses hexadecane as the selective growth substrate, and positive wells are detected by 

reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20o

C. 

PAH degraders are grown on a mixture of PAHs in separate plates. Positive wells turn yellow to 

greenish brown from accumulation of the partial oxidation products of the aromatic substrates 

after 3 weeks incubation. This method is simple enough for use in the field and provides reliable 

estimates for the density and composition of specific hydrocarbon-degrading populations. 

For a detailed description of existing MPN procedures, readers can refer to Rice and 

Hemmingsen (1997), and Wrenn and Venosa (1996). 

3.1.1.2 Culture-independent population/community techniques 

The main challenge for accurate analysis of hydrocarbon-degraders using existing culture-based 

techniques is that most these species are not able to be cultured (Atlas & Bartha, 1987, 

Macnaughton et al., 1999). The emergent culture-independent molecular techniques have made it 

possible to identify the diversity and composition of uncultivated microbial communities and to 

enumerate bacteria in more precise ways. 

Phospholipid fatty acid (PLFA) analysis 

Phospholipid fatty acid (PLFA) analysis is based on the characteristic “signature” of fatty acids 

present in the membranes of all cells (National Research Council, 1993). The distribution of fatty 

acids is unique and stable. Therefore, it can be used as an identifying index. Determination of 

48
biomass through analysis of the extractable lipids avoids culture bias. This technique also 

provides a quantitative means to measure viable biomass, community composition, and 

nutritional stature (White et al., 1998). 

Phospholipids can be extracted from the sample and the phosphate can be measured by 

colorimetric techniques (Findlay et al., 1989). The results can represent the amounts of viable 

cells and biological activities in the sample. A more powerful PLFA method involves extraction 

and separation of lipid classes into neutral-, glyco-, and polar-lipid fractions, followed by 

quantitative analysis using gas chromatography/mass spectrometry (GC/MS) (Macnaughton et 

al., 1999; White et al., 1998). This procedure can quantitatively determine the characteristics of 

microbial communities. However, PLFA analysis cannot identify species composition. 

Nucleic acid-based molecular techniques 

Nucleic acid-based molecular techniques can identify bacterial species by the unique sequence of 

molecular codes in their genes. One of most useful methods for determining the diversity of 

bacterial communities is denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993). 

The method provides a means of separating the PCR (polymerase chain reaction) products from 

mixed cultures based on the melting properties of the DNA. Usually the 16s rDNA portion of 

the bacterial genome is targeted for PCR amplification, since this region is commonly used for 

bacterial identification. PCR primers can be designed to detect a broad range of bacteria 

(universal) or can be designed just for a specific group of interest. 

The DGGE gel is made of acrylamide, and contains a gradient of formamide and urea, which 

both act to denature, or pull the strands of the DNA apart. The PCR products are loaded onto the 

gel, and a voltage is established across the gel for several hours. As DNA, which carries a net 

negative charge, is carried through the gel, it encounters an increasing gradient of denaturant, 

which causes the DNA chains to separate and the effective size of the molecules increases, 

causing movement through the gel to cease. The end result is a DNA banding pattern, where 

DNA requiring more chemical potential to denature travels farther, and DNA requiring less 

chemical potential to denature stays near the top of the gel. Each DNA band approximately 

corresponds to the presence of one kind of organism in the mixed culture. This banding pattern 

is sometimes referred to as a “community fingerprint,” and allows for a quick approximation of 

number of bacterial species (diversity) present. Bands can be excised for sequencing analysis, 

and sequences can be compared to the Ribosomal Database Project (Maidak et al., 2000) 

containing the 16S rDNA sequences of currently known organisms. 

The use of DGGE for quantitative purposes is still not well established. However, it can be used 

in conjunction with other quantitative methods such as PLFA analysis to provide insight into 

microbial species distribution. The PLFA-DGGE techniques were successfully used in 

determination of microbial population changes during the Delaware field study on oil 

bioremediation (Macnaughton et al., 1999). 

49
Table 3.1 Commonly used microbial analysis methods in monitoring oil bioremediation 

Method Advantages 

Plate counts 

Most probable number (MPN) 

techniques 

Phospholipid fatty acid 

(PLFA) analysis 

Denaturing gradient gel 

electrophoresis (DGGE) 

Well-established and easy to 

perform 

More reliable method for 

enumerating hydrocarbonutilizing microorganisms since 

hydrocarbons are used as the 

sole carbon source. Some of 

the procedures can separately 

enumerate aliphatic and 

aromatic hydrocarbondegrading bacteria 

Eliminate culture bias and 

quantitatively determine 

viable biomass, community 

composition, and nutritional 

stature 

Identify species distribution 

without culture bias 

Counts only organisms viable 

on solid-media and may allow 

growth of non-hydrocarbon 

degraders 

Relatively labor-intensive 

(large number of incubations) 

and time consuming, and may 

still be subject to culture bias 

Require specified knowledge 

and equipment, and can not 

identify species composition 

Also require more specified 

knowledge and equipment, 

and quantitative analysis is 

still not well-established 

Disadvantages 

3.1.2. Chemical analysis of nutrients 

Since oil biodegradation is limited by availability of nutrients in most marine shorelines, 

monitoring nutrients, particularly the nutrient concentrations in pore water, is critical in 

developing proper bioremediation strategies and assessing the effect of oil bioremediation 

(Bragg et al., 1994; Venosa et al., 1996). Important nutrient analyses include measurements of 

ammonium, nitrate, nitrite, and phosphorus. Commonly used methods for these nutrient analyses 

are summarized as follows. 

Sample preparation 

Analysis of nutrients in sediments can either be conducted on site or be frozen and shipped to a 

lab for measurements. Before the analysis, available nitrogen and phosphorus species can be 

extracted from sediments using a 2M solution of KCl or an acidified 0.1% NaCl solution (Page 

et al., 1986; Tan, 1996). Total nitrogen and phosphorus can be liberated from sediment samples 

by persulfate digestion at 121°C. 

50
Ammonia analysis 

The most commonly used methods for ammonia analysis are automated colorimetric methods 

due to their high sensitivity and ease of use. Two major automated colorimetric methods are 

available (1) Automated phenate method (4500-NH3 H, Eaton et al., 1995) (2) Automated 

salicylate-hypochlorite method. The latter was recently developed due to the environmental 

concerns associated with the phenol used by the phenate method (Tan, 1996). The extracted 

ammonia nitrogen can also be measured in the field using an ammonia-selective electrode (4500-

NH3 F, Eaton et al., 1995) or some commercial kits, such as a Chemetrics kit (Chemetrics Inc., 

Calverton, VA) although these methods are less sensitive and more susceptible to interference 

than colorimetric methods. 

Nitrate and nitrite analysis 

Commonly used techniques for nitrate analysis include the ultraviolet spectrophotometric 

method (4500-NO3

-

B, Eaton et al., 1995), automated cadmium reduction method (4500-NO3

-

F, - Eaton et al., 1995) and nitrate electrode method (4500-NO3 D, Eaton et al., 1995). The 

ultraviolet spectrophotometric method is suitable for rapid measurement or screening samples 

that have low organic matter contents. The automated cadmium reduction method is more 

sensitive and suitable for nitrate analysis in various types of water and wastewater. When using 

this method, nitrate is reduced to nitrite by passing through a Cu-Cd reduction column. The 

nitrite is then determined using a colorimetric procedure. Therefore, without the reduction step, 

this method can be used for nitrite analysis. Total nitrogen can also be measured as nitrate by the 

automated cadmium reduction method following oxidation of urea, ammonia, and nitrite by

° potassium persulfate at 121 C (4500-Norg D, Eaton et al., 1995). The nitrate electrode method can 

be used in the field for rapid nitrate analysis although it is less reliable than cadmium reduction 

method. 

Phosphorus analysis 

Phosphorus analysis involves two general procedures: (1) conversion of the phosphorus form of 

interest to dissolved orthophosphate and (2) colorimetric determination of dissolved 

orthophosphate (Eaton et al., 1995). Total phosphorus analysis usually uses persulfate digestion 

procedures (method (4500-P B, Eaton et al., 1995). Various digestion procedures have been 

developed for analysis of Available Phosphorus (Tan, 1996), a variable concept that reflects the 

amount of phosphorus available to plants or microorganisms. The ascorbic acid method (4500-P 

E, Eaton et al., 1995) is recommended for analysis of phosphate in the concentration range of 

0.01 to 6 mg P/L. Commercial kits, such as Hach® phosphate analysis procedures (Hach 

Company, Loveland, CO), are also available for use in the field. 

3.1.3 Chemical analysis of oil and oil constituents 

One of the primary measures of the success of bioremediation treatments is reduction in the 

concentrations of spilled oils and target oil constituents in particular. Various techniques have 

been developed and used in petroleum hydrocarbon analysis, which include gravimetric 

methods, infrared spectroscopy (IR), gas chromatography/flame ionization detection (GC/FID), 

gas chromatography-mass spectrometry (GC/MS), and thin-layer chromatography-flame 

ionization detection (TLC-FID). Oil analysis methods can be generally classified into two 

51 
categories: nonspecific methods to measure total petroleum hydrocarbons (TPHs), and specific 

methods using various chromatographic techniques to quantify target oil constituents. 

Commonly used oil analysis methods in monitoring oil bioremediation are summarized in this 

section and table 3.2. 

3.1.3.1 Total petroleum hydrocarbon (TPH) techniques 

TPH techniques mainly include gravimetric and infrared spectroscopic methods. These 

techniques are widely accepted methods to rapidly quantify the oil due to their simplicity and 

low costs. However, these methods provide little information about oil components, exhibit high 

detection limits and are susceptible to various interferences (Douglas et al 1991, Xie et al., 

1999). Furthermore, they are unable to distinguish between abiotic and biotic losses. 

Gravimetric analysis involves solvent (e.g., dichloromethane) extraction, evaporation, and 

gravimetric measurement (EPA 413.1 and EPA 9071). This method does not distinguish between 

petroleum hydrocarbons and naturally occurring biogenic compounds (such as plant lipids) and 

may result in overestimating TPH. Infrared spectroscopy (IR) involves solvent extraction 

normally using trichlorotrifluoroethane (Freon 113). TPH is subsequently measured by 

comparing the infrared absorption of the extraction liquid against that of a defined hydrocarbon 

mixture (e.g., EPA 418.1 (U.S. EPA, 1992)). Although this technique is a more sensitive 

measure of hydrocarbons than gravimetric methods, it may also overestimate or underestimate 

TPH for a variety of reasons (Xie et al., 1999). Environmental concerns regarding the use of 

Freon as a solvent is also a potential problem for its application (Romero and Ferrer, 1999). 

Fully halogenated solvents such as tetrachloroethene or tetrachloromethane can be substituted for 

Freon. 

Commercial kits, such as Petroflag® test kit (Dexsil, Hamden, CT) and Hach® DR/2000 test kit 

(Hach Company, Loveland, CO), are also available for use in the field with limited reliability 

(Lambert et al., 1999a&b). All TPH techniques are severely affected by the spatial 

heterogeneity. A larger quantity of oil at one spot could give a misleading high TPH value. They 

do not distinguish between abiotic and biotic losses that are important in correctly interpreting 

the data for the fate of the petroleum in the environment. So, one has to be careful in carrying out 

these tests as well as in interpreting the data. A sufficiently large number of samples may help to 

overcome some of the variability. 

3.1.3.2 Analysis of specific oil constituents 

To assess the effect of oil bioremediation, identification and quantification of individual oil 

components and compounds, particularly those constituents that are of significant environmental 

concern , is required. Various chromatographic techniques, particularly GC/FID and GC/MS, are 

widely used to provide specific and sensitive analysis of oil constituents (Douglas et al., 1994; 

Wang et al. 1997). 

Gas chromatography/flame ionization detection (GC/FID) 

GC/FID combines chromatographic separation of hydrocarbon fractions on a capillary GC 

column with quantification by FID (e.g., EPA 8100(U.S. EPA, 1992)). Pretreatment of oiled 

52
sediment samples involves drying of samples, addition of surrogates, and solvent extraction (e.g., 

EPA 3540c (U.S. EPA, 1992)). This method has been mainly used for detection of aliphatic 

hydrocarbons such as individual C10 to C35 n-alkanes, and isoprenoid hydrocarbons (Table 3.3). 

GC/FID can also be used to determine TPHs by the method of internal standards (Douglas et al, 

1994). Total GC-detectable hydrocarbons make up more than 50% of the oil components (Prince 

et al., 1994). However, GC/FID may not be used to identify and quantify PAHs and biomarker 

compounds because it can not clearly separate many of these compounds, especially the 

alkylated PAHs. Since PAHs are of most environmental concerns and biomarker compounds are 

critical in distinguishing between biodegradation from physical weathering processes and 

reducing spatial variability of oil data, measurement of these compounds is very important in 

monitoring oil bioremediation. 

Gas chromatography-mass spectrometry (GC/MS) 

GC/MS, which combines chemical separation by GC and spectral resolution by MS, is the choice 

of methods for specific compound determination, especially for identification and quantification 

of PAHs and biomarkers (Wang et al., 1997). The mass spectrometer is often operated in the 

selected ion monitoring mode (SIM) to further increase sensitivity and selectivity relative to 

conventional full-scan GC/MS. For a detailed description of GC/MS analytical procedures, 

readers can refer to Douglas et al. (1994), EPA Method 8270 (US EPA,1989), and Venosa et al. 

(1996). Typical target alkanes and PAHs analyzed using GC/MS in recent USEPA-sponsored 

projects are listed in Table 3.3 and 3.4 (Purandare, 1999; Venosa et al., 1996). Because the 

distribution of oil on shorelines contaminated by offshore spills can be highly heterogeneous 

(Bragg et al., 1994), the concentrations of all target analytes are often reported relative to a 

conservative biomarker such as 17α(H),21β(H)-hopane (Douglas et al., 1994). Detailed 

discussions on biomarkers will be presented in Section 3.2. 

Thin-layer chromatography flame ionization detection (TLC-FID) 

Thin layer chromatography-flame ionization detection (TLC-FID), which uses a special 

instrument called the Iatroscan, separates hydrocarbons on a Chromarod thin layer based on 

characteristic chemical types or fractions such as aliphatic, aromatic, polar, and asphaltene 

compounds (Goto et al., 1994). This method has advantages in measuring high-boiling-point 

hydrocarbons such as higher molecular weight saturates, aromatics, resins, and asphaltenes, 

some of which may not be detectable by GC or HPLC. Unlike the other analytical techniques, 

which are either too gross (e.g. TPH techniques) or very specific (e.g. GC based methods), the 

TLC-FID can measure the relative percentages of the four major fractions of petroleum in a short 

period of time. This method has been successfully used for monitoring oil bioremediation in a 

wetland environment (Stephens et al., 1999). However, TLC-FID can not identify specific 

compounds and may only be used as a screening tool. Controversy also exists concerning the 

reliability of the methods due to some confusing results, and modifications of the technique have 

been suggested (Cebolla et al., 1998). 

53
Table 3.2 Commonly used methods for petroleum hydrocarbon analysis 

Method Advantages Disadvantages References 

TPH techniques Inexpensive and easy to Low sensitivity and USEPA, 1992 

GC/FID 

GC/MS 

TLC/FID 

perform. Used as quick 

screen tools 

A specific method used 

for detection of aliphatic 

and a limited number of 

aromatic hydrocarbons. 

Highly sensitive and 

selective method for 

identification and 

quantification of a wide 

range of hydrocarbons, 

including PAHs and 

biomarkers 

Quick detection of a wide 

range of oil components, 

including high molecular 

weight saturates, 

aromatics, resin, and 

asphaltenes; can be used 

as an effective screening 

tool. 

selectivity; can not be 

used for identification of 

oil components. Not 

recommended. 

Unable to identify and 

quantify alkylated PAHs 

and biomarkers 

Expensive equipment and 

complicated procedures 

Unable to identify 

specific compounds; 

quantitative analysis is 

still not well-established 

Douglas et al., 

1994; Wang et 

al.,1997 

Douglas et al., 

1994; USEPA, 

1992; Venosa et 

al., 1996; Wang et 

al.,1997 

Stephens et al., 

1999; Cebolla et 

al., 1998 

54
Table 3.3 Target alkanes list (Purandare, 1999) 

Compound Name Misc. 

Info. 

QIon Response Factors 

Reference 

Compounds 

Internal 

Standards 

D26 n-dodecane surrogate 66 D26 n-dodecane D22 ndecane 

QIon: 66 

nC10 57 nC10 

nC11 57 nC11 

nC12 57 nC12 

nC13 57 nC13 

nC14 57 nC14 

nC15 57 nC15 

D36 n-heptadecane surrogate 66 D36 n-heptadecane D34 

n-hexadecane 

QIon: 66 

nC17 57 nC17 

Pristane 57 Pristane 

nC18 57 nC18 

Phytane 57 Phytane 

nC19 57 nC19 

D50 n-tetracosane surrogate 66 D50 n-tetracosane D42 neicosane 

QIon: 66 

nC20 57 nC20 

nC21 57 nC21 

nC22 57 nC22 

nC23 57 nC23 

nC24 57 nC24 

nC25 57 nC25 

nC26 57 nC26 

nC27 57 nC27 

D66 n-dotriacontane surrogate 66 D66-dotriacontane D62 

n-triacontane 

Qion: 66 

nC28 57 nC28 

nC29 57 nC29 

nC30 57 nC30 

nC31 57 nC31 

nC32 57 nC32 

nC33 57 nC33 

nC34 57 nC34 

nC35 57 nC35 

5 –cholestane surrogate 217 5 -cholestane 5 -antrostane 

Ho QIon: 245 pane alkane 191 Hopane 

55
Table 3.4 Target PAHs analyzed by GC/MS (Purandare, 1999) 

Compound Name Misc. 

Info. 

QIon Factors 

Reference 

Compounds 

Internal 

Standards 

D10 1-methyl naphthalene surrogate 152 D10 1-methyl 

naphthalene 

Naphthalene PAH 128 Naphthalene 

C1 naphthalene 142 Naphthalene 

D8-

naphthalene 

QIon: 136 

C2 naphthalene 

2-ring 

alkyl PAHs 156 

C3 naphthalene 170 Naphthalene 

C4 naphthalene 184 Naphthalene 

Fluorene 3 ring PAH 166 Fluorene 

C1 fluorenes 180 Fluorene 

C2 fluorenes 

3 ring 

alkyl PAHs 194 

D10-

anthracene 

QIon: 188 

C3 fluorene 208 Fluorene 

Dibenzothiophene 3 ring PAH Dibenzothiophene 

C1 dibenzothiophene 198 Dibenzothiophene 

C2 dibenzothiophene 

3 ring 

alkyl PAHs 212 

C3 dibenzothiophene 226 Dibenzothiophene 

D10 phenanthrene surrogate 188 D10 phenanthrene 

Phenanthrene PAHs 178 Phenanthrene 

Anthracene 178 Anthracene 

C1 phenanthrenes 192 Phenanthrene 

C2 phenanthrenes 

3 ring 

alkyl PAHs 206 

C3 phenanthrenes 220 Phenanthrene 

C4 phenanthrenes 234 Phenanthrene 

Naphthobenzothiophene ring PAH Dibenzothiophene 

C1 naphthobenzothiophene 248 Dibenzothiophene 

C2 naphthobenzothiophene 

4 ring 

alkyl PAHs 262 

C3 naphthobenzothiophene 276 Dibenzothiophene 

Fluoranthene PAH 202 Fluoranthene 

D10 pyrene surrogate 212 D10 pyrene 

D12-

chrysene 

QIon: 240 

Pyrene PAH 202 Pyrene 

C1 pyrenes 216 Pyrene 

C2 pyrenes 

4 ring 

alky PAHs 230 

Response 

2-ring 

Naphthalene 

Fluorene 

184 

Dibenzothiophene 

3-ring 

Phenanthrene 

4 234 

Dibenzothiophene 

4-ring 

4-ring 

Pyrene 

56
Table 3.4. Target PAHs (Contd.) 

Compound Name Misc. 

Info. 

QIon Response Factors 

Reference 

Compounds 

Internal 

Standards 

Chrysene 4 ring PAH 228 Chrysene D12-

chrysene 

QIon: 240 C1 chrysenes 4 ring 

alkyl PAHs 

242 Chrysene 

C2 chrysenes 256 Chrysene 

C3 chrysenes 270 Chrysene 

C4 chrysenes 284 Chrysene 

benzo(b)fluranthene 5-ring 

PAHs 

252 Benzo(b)fluoranthene D12-

perylene 

QIon: 264 

benzo(k)fluoranthene 252 Benzo(k)fluoranthene 

benzo(e)pyrene 252 Benzo(e)pyrene 

benzo(a)pyrene 252 Benzo(a)pyrene 

indeno(1, 2, 3-cd) pyrene 6-ring 

PAHs 

276 Indeno (1, 2, 3-cd) 

pyrene 

dibenzo(a,h)anthracene 5-ring 

PAHs 

278 Dibenzo 

(a,h)anthracene 

benzo(g,h,i)perylene 6-ring 

PAHs 

276 Benzo(g,h,i)perylene 

3.2 Biomarkers 

As mentioned in Chapter 2, internal biomarkers have been widely used to distinguish between 

biodegradation and the physical or chemical loss of oil from treated plots in bioremediation field 

studies (Bragg et al., 1994; Venosa et al., 1996; Lee et al., 1997b). An ideal biomarker should 

(1) provide source specific information, (2) not be formed during physical, chemical weathering 

and biological processes, (3) be non-biodegradable or relatively resistant to biodegradation on 

time scales relevant to the study or cleanup, (4) be extracted from the sample with similar 

efficiency to the other associated compounds (Douglas et al, 1994; Prince et al., 1994a,b). The 

ideal biomarker would also be subject to the same physical and chemical removal mechanisms as 

the target analytes so that any differences could be attributed to biodegradation. Unfortunately, 

existing biomarkers can rarely meet all these criteria. An overview of commonly used 

biomarkers in monitoring oil bioremediation is given here. Issues to be aware of when using 

biomarkers will be discussed. 

3.2.1 Commonly used biomarkers 

Pristane and phytane 

Hydrocarbon degrading microorganisms usually degrade branched alkanes or isoprenoid 

compounds such as pristane and phytane at lower rates than straight-chain alkanes. Pristane and 

phytane are also subject to the same physical and chemical removal mechanisms as their 

57
corresponding straight-chain alkanes (Wang et al., 1998). Therefore, n-C17:pristane and nC18:phytane ratios have been traditionally used to interpret the extent of biodegradation 

(Gaudlach E.R et al., 1983; Lee and Levy, 1987,1989,1991). However, it was later found in 

Prince William Sound that these compounds were resistant to degradation in the initial time 

period, but they degraded rather rapidly over longer periods of time (Prince 1993, Bragg, et al., 

1994). This phenomenon was also observed in a study on bioremediation of Light Arabian crude 

oil in a freshwater wetland environment as shown in Figure 3.1 (Purandare, 1999). The study 

examined the dry weight normalized pristane, phytane, and hopane concentrations over the 32-

week experimental period. It can be seen that the pristane and phytane showed significant 

degradation, but hopane remained constant throughout the course of the study. These results 

show that the n-alkane:isoprenoid ratio may be useful indications of biodegradation in very short 

term or the earliest part of a study. However, they may substantially underestimate the extent of 

biodegradation in a long run. 

20 

16 

12 

8 

4 

0 

0 weeks 2 weeks 4 weeks 8 weeks 16 weeks 32 weeks 

prystane 

phytane 

hopane 

Weeks of Operation 

Figure 3.1 Change of pristane, phytane and hopane in a freshwater wetland 

Hopanes 

Hopanes, derived from the molecular fossils of prokaryotic and eukaryotic membranes (Peters 

and Moldowan, 1993), are very resistant to biodegradation as shown in Figure 3.1. 

17α(H),21β(H)-hopane (Figure 3.2) has been found to be neither generated nor biodegraded 

during the biodegradation of crude oil on time scales relevant to estimating the cleaning of oil 

spills and therefore has appropriate characteristics to serve as an internal standard for monitoring mg/kg dry weight of sediment 

58
biodegradation of both specific petroleum compounds and total oil in crude oil in the 

environment (Prince et al., 1994). Hopanes have been viewed as the biomarkers of choice 

(Mearns, 1997) since the successful application of 17α(H),21β(H)-hopane for evaluating oil 

bioremediation after the Exxon Valdez spill (Douglas et al., 1994; Prince, et al., 1994a&b). Many 

recent studies have also chosen this hopane as the biomarker (Garcia-Blanco et al., 2001b; 

Purandare, 1999; Swannell et al., 1999a; Venosa et al., 1996). 

Figure 3.2 shows alkane analysis results in soil core samples during the first 21 weeks of the St. 

Lawrence River field study using dry weight soil normalization and hopane normalization 

(Garcia-Blanco et al., 2001b). Due to the substantial heterogeneity of oil distribution in the 

freshwater wetland sediments, large standard deviations (17-72% of the mean concentrations) 

were obtained when using dry weight soil normalization. No convincing conclusions were able 

to be reached based on these data. However, the standard deviation of oil concentrations in the 

sediment samples were much lower (2-15% of the mean oil concentrations) when using hopane 

normalization, which enabled evaluation of oil biodegradation with high levels of statistical 

confidence. 

However, it should be noted that hopanes are also very resistant to those physical and chemical 

weathering processes that affect most target alkanes and aromatics (e.g., dissolution, 

volatilization, and photooxidation). Therefore, although hopane normalization is very useful in 

reducing the variability associated with heterogeneous oil distribution, changes in the hopanenormalized analyte concentrations alone may not be used to verify that biodegradation is the 

primary removal mechanism. Use of hopane normalization to distinguish biodegradation from 

physical loss of oil is valid mostly when effects of dissolution and volatilization are negligible 

(Prince, 1993; Venosa et al., 1996), which is not always true. Other means of verifying 

biodegradation are needed such as the use of alkylated PAH isomers (see next section). 

Alkylated PAH isomers 

Biodegradation can be verified as a removal mechanism by determining the relative degradation 

rates for homologous series of alkylated PAHs. Preferential biodegradation of aliphatic and 

aromatic hydrocarbons based on molecular structure has long been recognized (Jobson et al., 

1972; Walker et al., 1976; Roubal and Atlas, 1978; Fedorak and Westlake, 1981; Elmendorf et 

al., 1994, Wang et al, 1998). This is particularly true for alkylated PAHs, which are biologically 

transformed more slowly as the extent of alkyl substitution increases (Elmendorf et al., 1994; 

Venosa et al., 1997). Biodegradation results in unique characteristic changes in the distributions 

of homologous series of alkylated PAHs. On the other hand, physical weathering does not cause 

the same types of alterations in their relative distributions (Wang et al., 1998). In other words, 

physical weathering causes equal losses in all homologues irrespective of the extent of alkyl 

substitutions. Recent research has also shown that the relative biodegradation rates for alkylated 

homologs of the 2- and 3-ring PAHs were remarkably similar for mixed cultures of hydrocarbondegrading bacteria isolated from a wide variety of sources (Venosa et al., 1997), and they were 

also very similar in the field, despite the much higher absolute rates that were observed in the 

laboratory (Venosa et al., 1996). Therefore, the distribution patterns of alkylated PAHs, when 

used in conjunction with other oil analysis data, can be very useful in accurate assessment of the 

extent and progress of oil biodegradation. 

59
a) Dry soil weight normalized 

1000 

800 

600 

400 

200 

0 

0 1 2 4 6 8 12 16 21 

Natural Attenuation 

Bio + Ammonium 

Phyto + Ammonium 

Phyto + Nitrate 

Unoiled + Ammonium 

Time (weeks) 

b) Hopane normalized 

600 

500 

400 

300 

200 

100 

0 

0 1 2 4 6 8 12 16 21 

Natural Attenuation 

Bio + Ammonium 

Phyto + Ammonium 

Phyto + Nitrate 

Time (weeks) 

Figure 3.2 Comparison of alkanes analysis results in soil core samples from St. Lawrence 

field study using (a) dry soil weight normalization and (b) hopane normalization 

m

g alkanes / m

g h

o

p

ane 

m

g alkanes / Kg 

dry soil 

60
Other Biomarkers 

For refined petroleum products such as diesel fuel and fuel oil #2 that do not contain the hopane, 

C4-phenanthrenes/anthracenes may be substituted (Douglas et al., 1994). These compounds are 

degraded, but very slowly. Lee et al. (1997b) used C2-chrysenes as biomarkers in their studies 

on bioremediation of weathered Venture Condensate because hopane concentration in the oil was 

at or near the detection limits of their instruments and C2-chrysenes are also considered highly 

degradation resistant. C4-chrysenes can also be used if their concentrations are high enough to be 

detectable. 

Table 3.5 Biomarkers and their characteristics 

Biomarkers Limitations Applicable to References 

Pristane 

& Phytane 

Hopanes 

Alkylated 

PAHs isomers 

Phenanthrenes, 

Anthracenes, 

Resistant 

biodegradation 

Loss 

characteristic of 

biodegradation 

Biodegradable 

Can 

distinguish 

biodegradation 

from abiotic 

weathering 

processes 

Monitoring 

early stages of 

biodegradation 

Reducing 

spatial 

variability 

Verification of 

biodegradation 

Refined 

petroleum 

Lee et 

1987&1989 

Prince et , 

1994; 

et al., 1994 

Venosa et al., 

1997a; Wang et 

al., 1997&1998 

Douglas et al., 

1994 

Advantages 

to 

pattern 

not 

al. 

al.

Douglas 

and Chrysenes products 

3.2.2 The effect of contaminant redistribution on observed remediation rates 

Loss of oil due to physical washout and sand redistribution can be significant in an oiled beach. 

Hopane normalization is an effective way to distinguish biodegradation from the effects of the 

physical washout and sediment exchange between the inside and outside of experimental plots 

when all of the oil is initially present inside of the plots and most of the beach is clean, such as in 

a study involving intentional oiling (Venosa et al., 1996). However, it will not work well in a 

study involving small plots set up on a beach contaminated by a “spill of opportunity” or a real 

oil spill. The reason is that when a study is carried out on a beach completely contaminated by an 

oil spill, oil or oiled sand will transport between relatively small treated areas (i.e., experimental 

plots) and large untreated but oiled areas. Since hopane is a conservative biomarker, its 

concentration will be the same inside and outside of the plots (assuming that the beaches were 

61
uniformly oiled initially). Therefore, oiled sand coming into the plots will have the same amount 

of hopane as the sand leaving the plots (assuming that treatment does not result in physical 

removal of bulk oil from sand inside the plots), but the concentrations of target analytes will be 

higher, because the biodegradation rates will be lower in untreated areas. Since the hopane 

concentration inside the plots will not be affected by sand transport, it does not allow us to 

quantify the rate of sand exchange between the inside and outside of the plots. 

A theoretical analysis conducted by Wrenn et al. (1999) illustrates how physical exchange of 

sand between treated and untreated areas of the beach affects the observed biodegradation rate of 

target analytes when small plots are set up on a large beach. For the nutrient-treated plots, the 

rate of change in the analyte concentrations, Atreat , is given by: 

dAtreat Atreat dT  1 dH 1 dT 

dt 

= 

T dt − ktreat Atreat + 

 H dt − 

T dt 

Atreat (3.1) 

For the untreated control plots and in the untreated areas of the beach, the rate of analyte, 

Acon, disappearance is: 

dAcon Acon  dH 

dt 

= 

H 

 dt  − kcon Acon (3.2) 

Where ktreat and kcon are the first-order biodegradation rate coefficients in the presence and 

absence of nutrients; H is the concentration of hopane; and T is the concentration of a 

hypothetical nonbiodegradable tracer that is present in the oil inside of the plots but not outside 

of the plots (e.g., a hydrophobic fluorescent dye that is added to the oiled sand inside the plots at 

the start of treatment). Equation (3.1) describes the rate of change of the analyte concentration 

inside the treated plots due to transport of treated oiled sand out of the plots, biodegradation, and 

transport of untreated oiled sand into the plots. Assuming the rates of physical loss of treated 

oiled sand from inside the plots and loss of oiled sand from the beach to be first-order processes, 

the hopane-normalized analyte concentration inside of the plots at any time can be solved as: 

 Atreat   Ao  (k −kT −ktreat )t (ktreat − kcon ) + (kT − kB )e(kT +ktreat −kcon −kB )t  

 H 

 = 







Ho 



e 



 kT + ktreat − kcon − kB 

 (3.3)

Where kT and kH are the first-order loss coefficients for the nonbiodegradable tracer and hopane, 

respectively. 

Representative results from this model are shown in Figure 3.3 using parameters obtained from 

Delaware field trial (Venosa et al., 1996). It can be seen that the effect of exchange of oiled sand 

between the inside of treated plots and untreated beach is to reduce the observed degradation rate 

relative to the true rate. Sand exchange has no effect on the observed biodegradation rate in the 

control plots, because biodegradation is assumed to occur at the same rates inside and outside of 

those plots. The apparent rate of remediation in the treated plots, however, will decrease while 

relatively large amounts of oil remain even when bioremediation would be capable of achieving 

a complete cleanup if the entire contaminated shoreline were treated. This could lead to the 

62 
incorrect conclusion that, whereas bioremediation can stimulate the initial cleanup rate, it cannot 

restore the contaminated shorelines to acceptable conditions. The analysis shows that, if 

behavior of this type is observed, it is probably an artifact of treating a small fraction of the total 

contaminated area, and more complete remediation would be expected when a larger area is 

treated. Nevertheless, there are other possible explanations for incomplete remediation. These 

include the inability to maintain sufficient nutrients in the bioremediation zone and, especially on 

high-energy beaches, a high loss rate of bacteria from the oiled surfaces (e.g., due to scouring by 

waves). Therefore, a thorough monitoring program is a very important component of this 

research, because data on nutrient concentrations and microbial activity is required to properly 

interpret the results if the cleanup goals are not achieved during the field study. 

50 

40 

30 

20 

10 

0 

0 10 20 30 40 50 60 70 80 90 100 

true degradation rate 

(treated) 

observed degradation rate

(treated) 

control degradation rate 

(true and observed) 

Time (days) 

Figure 3.3 Reduction in the observed oil biodegradation rate due to exchange of oiled sand 

between the inside of treated plots and untreated surrounding beach 

3.3 Sampling in the Field 

Because oil contaminated sites are highly heterogeneous, representative sampling is difficult but 

also extremely important for proper evaluation of bioremediation. Field sampling procedures 

must be designed to achieve statistically valid sampling and to minimize contamination or 

changes in the samples. Variables that affect the representativeness of samples and their methods 

of collection include characteristics of media, concentration distribution of analytes, and bias 

introduced during collection, preparation, and transportation (Lee, 2000). General principles to 

achieving statistically valid sampling in soil environments and solid waste have been welldocumented (Tan, 1996; USEPA, 1992). However, little information is available regarding 

sampling protocols for monitoring oil bioremediation in marine shorelines and freshwater Total PAHs (ng/ng hopane) 

63
wetlands. Major considerations to achieve representative sampling are summarized as follows 

based on the soil-science literature and field experiences from recent oil bioremediation projects. 

3.3.1 Sampling strategies 

Types of sampling 

Sampling methods can be classified into simple random sampling, systematic sampling, stratified 

sampling, and compositing (Tan, 1996). The Simple random sampling or the grab method 

involves collecting samples at random in a sampling area. This method depends completely on 

the luck of the draw without considering the variation of analytes in the sampling field. 

Purandare (1999) found that results of oil analysis in a wetland sediment were highly variable 

and unreliable using this sampling approach during the early stage of his study. Therefore, this 

approach may only be suitable for use in relatively homogeneous systems. Systematic sampling 

involves taking samples based on certain patterns, such as collecting samples in a grid pattern. 

This method will ensure that the entire sampling area is represented in the sample. Stratified 

sampling involves dividing the sampling field into a number of sectors or quadrants and taking 

independent samples in each sector according to the rule of proportionality (e.g., taking more 

samples in more heavily oiled sites). These approaches can often provide more accurate results 

than simple random sampling, because with this method the samples are distributed more evenly 

over the population. Compositing involves the mixing of sampling units to form a single sample, 

which has the advantage of increased accuracy through the use of large numbers of sampling 

units per sample. This approach in combination of stratified sampling has been frequently used 

in recent field studies on oil bioremediation (Venosa et al., 1996; Garcia-Blanco et al., 2001b). 

Depth of sampling 

Sampling depth in oil spill sites mainly depends on the distribution of the analytes of interest, 

especially the depth of oil penetration. Crude oil rarely penetrates coastal sediments to depths of 

greater than one foot (Gundlach, 1987). Penetration of oil in wetland environments will be even 

less deep than in most marine sediments. Purandare (1999) found that oil only penetrated to 2.5 

cm in a wetland sediment in 16 weeks. The top 2 cm layers of the sample cores were then used 

for oil analysis. Therefore, a survey of oil penetration in the contaminated site is critical in 

determination of sampling procedures for a bioremediation application. 

Size of sampling 

The size of the sample required depends on the available resources, the required degree of 

confidence, and the objectives of the analysis (Rupp and Jones, 1993). Generally, the more 

heterogeneous the system, the more intense must be the sampling efforts to reach a given 

accuracy. However, economic considerations often restrict both the quantity and the number of 

samples taken, and a balance should be obtained between the size of samples to be taken for 

required confidence and economic factors. Following expressions are some examples of 

statistical approaches, which can be used to calculate the required number of samples with 

respect to an acceptable error (Peterson and Calvin, 1986; Tan, 1996): 

n = 4σ/E2 (3.4) 

or n = t2

S2/E2 (3.5) 

64
where n is number of samples, t is t-test value, E is acceptable error, S2

 is sum of squares of 

sample deviation, and σ is standard deviation. 

Sample handling and storage 

Sample handling procedures must be designed to minimize cross-contamination or chemical and 

biological changes in the samples. For example, different sets of sampling tools should be used 

for different treatments to avoid cross contamination. Clean and proper containers, such as PVC 

bags and tin cans, should be used for sample storage. Samples, if not being analyzed in the field, 

should be frozen using blue ice or dry ice until they reach their destination. And once they arrive 

the destination, samples should be stored in a freezer at -18 to - 20 o

C until needed. 

3.3.2 Field sampling experiences 

Two examples of well-designed sampling protocols used in definitive bioremediation field 

studies are shown below (Venosa et al., 1996; Garcia-Blanco et al., 2001b). 

Sampling protocols used in the Delaware field bioremediation study (Sandy Beach) 

Each plot was divided into 4 quadrants for sampling purposes, and the sand samples were 

collected at the nodes of a 0.5 m by 0.5 m grid in each quadrant (Figure 3.4). As Figure 3.4 

shows, all sample nodes were at least 0.5 m from the plot boundary on all sides. This buffer 

zone was designed to minimize the impact of edge effects on the observed extent of 

biodegradation. Since the plots were at least 9.5 m long, this provided a minimum of 28 sample 

nodes for each quadrant. 

Samples were composited from 2 randomly selected nodes in each quadrant at 1 or 2 week 

intervals for a period of 14 weeks, but they were never collected from the same node twice. The 

sampling frequency was higher near the beginning of treatment and followed the order of Weeks 

0 (i.e., just before treatment began), 1, 2, 3, 4, 6, 8, 10, 12, and 14. With 10 sample events in this 

study, 20 sample nodes were required per quadrant. Sand samples were collected with hand 

augers to a depth of 15 cm, which was determined based on preliminary oil penetration study. 

Sampling protocols used in St. Lawrence River field study (Freshwater Wetland) 

A randomized sampling plan was designed to eliminate sampling bias. Each plot was divided 

into six sectors, each measuring 1.5 m x 1.0 m, with the 1.5 m dimension parallel to the 

shoreline. Each sector was subdivided into 10 subsampling zones, corresponding to the 

predetermined sampling events. Each subsampling zone had dimensions 50 cm x 30 cm. 

At each sampling event, a 9-cm core sample was collected using a tulip bulb planter from preassigned random subsampling zones from each of the six sectors within each plot. These samples 

were combined into 2 composites (3 predetermined samples per composite). Both composite 

samples were placed into quart size paint cans, frozen, and shipped to the University of 

Cincinnati (OH) for oil analysis. Samples were kept in the freezer (-18 to -20 o

C) until they were 

extracted. Composite 1 served as the sample to be analyzed by GC/MS. Composite 2 was 

analyzed for three of the sampling events to check within-plot variability. The remaining 

composite 2 samples were frozen and archived. The first sampling event (week 0) was carried 

65
out at low tide the day after the application of oil and nutrients. Subsequent sampling events 

were at weeks 1, 2, 4, 6, 8, 12, 16, 21, 48, and 65. 

The sampling designs mentioned above are important because they incorporate replicate plots 

with random placement on the experimental plane. These are essential for permitting proper 

statistical analysis of treatment effects. 

Figure 3.4 Example of an experimental plot in the Delaware field study showing sampling 

quadrants and nodes 

3.4 Monitoring General Site Background Conditions 

Monitoring general site background conditions is very important for properly evaluating effects 

of oil bioremediation. Major background conditions include dissolved oxygen, pore water pH, 

temperature, and salinity. 

66
3.4.1 Oxygen 

Oxygen availability is crucial for rapid bioremediation because hydrocarbon biodegradation is 

primarily an aerobic process. Therefore, the dissolved oxygen (DO) of pore water should be 

monitored on a regular basis. Water samples from the oil-contaminated region of the subsurface 

can be collected through the multi-port sample wells and sealed in DO bottles. Conventional 

methods for DO measurement include iodometric procedures and membrane electrode method 

(Eaton et al., 1995). 

The iodometric technique is the most precise and reliable titrimetric procedure for DO analysis. 

Detailed procedure of this method is described in Standard Methods No. 4500-O B (Eaton et al., 

1995). Various iodometric modifications have been developed. One procedure that is suitable for 

use in the field involves using Hach® high range dissolved oxygen ampoules (Hach Company, 

Loveland, CO). Once the Hach® ampoules are filled and capped and the reaction of the reagents 

with DO in the water sample is complete, the color is stable indefinitely. Therefore, the capped 

ampoules can be transported back to the laboratory trailer where the DO concentration in the 

water samples can be determined with a Hach® colorimeter. (Note: the caps often leak gaseous 

O2 into the samples; so, the samples aren’t really stable indefinitely, even though the color is 

stable as long as additional O2 can be excluded.) 

The membrane electrode method is more suitable for regular field monitoring and in situ DO 

determination. It is also recommended for DO analysis in highly polluted waters and colored 

waters. The general procedure for this method is described in Standard Methods No. 4500-OG 

(Eaton et al., 1995). Detailed analytical procedures may vary depending on the manufacturers of 

the DO probes. 

3.4.2 pH 

Biodegradation of oil is affected by the background pH (Atlas and Bartha, 1992). Oil 

biodegradation can also be severely inhibited by dramatic reductions in pH when ammonia is 

provided as the nitrogen source (Wrenn et al., 1994). The latter is true for closed environments 

such a laboratory flasks where no dilution is possible from continuously changing aqueous 

conditions such as tides. Nonetheless, monitoring pH in the field is of particular importance in 

evaluating the effect of oil biodegradation. 

The pH values of pore-water samples are normally measured using a portable pH meter with a 

combination electrode. The pH can be measured in the field either immediately after the samples 

are collected or by putting pH electrodes directly into water in sampling wells. Sediment pH can 

also be measured in the field by mixing the soil samples with reagent water according to EPA 

method 9045c (US EPA, 1992). 

3.4.3 Temperature 

As discussed in Chapter 2, temperature affects both the properties of spilled oil and the 

biodegradation processes. All the other measurements are also temperature-dependent. 

Temperature profiles in air, water, and sediment should be monitored regularly using appropriate 

67
thermometers. Many instruments for other analyses, such as DO or pH, have built-in 

thermometers, and the temperature should be recorded along with the other measurements. Any 

temperature measurement devices should be calibrated with a National Institute of Standards and 

Technology (NIST)-certified thermometer before field use (Eaton et al., 1995). 

3.4.4 Salinity 

Salinity of the environment may be an important factor in oil bioremediation, particularly in 

estuarine environments or in marine shorelines where regional seaward groundwater flow exists 

(Boufadel, et al., 1999b; Zobell, 1973). 

Salinity of pore water can be measured by either a conductivity method or density methods (e.g., 

Standard Methods No. 2520B or 2520 C; Eaton et al., 1995). The conductivity method is the 

most commonly used method for salinity analysis due to its high sensitivity and ease of 

measurement. Conductivity meters can be installed in the field to monitor salinity profiles in 

marine shorelines. The density method involves using a precise vibrating flow densitometer. 

Water salinity can also be determined rapidly in the field using this method. 

3.5 Monitoring of Biological Impacts 

The public has responded favourably to bioremediation since its implicit goal is that of reducing 

toxic effects by converting organic molecules to cell biomass and other benign materials such as 

carbon dioxide and water (Atlas and Cerniglia, 1995). However, concerns about the net benefit 

of bioremediation strategies remain. This is attributed to lingering questions regarding the 

potential production of toxic metabolic by-products, possible toxic components in the 

formulation of bioremediation agents, and the ineffective degradation of the most toxic 

components of residual oils (Hoff, 1991; Office of Technology Assessment, 1991). 

To date, a single ideal biological method - both sensitive and efficient - for the assessment of 

contaminant impacts to all sediment biota has not been identified. Two separate, yet 

complimentary, approaches have evolved: bioassessment and bioassays. Bioassessments are 

field-based analyses typically characterized by assessing the impacts of the contamination and 

treatment activity on environmental populations such as benthic communities, intertidal flora and 

fauna, etc. They are characterized as having limited experimental controls (Herricks and 

Schaeffer, 1984). Bioassays are laboratory-based tests that incorporate rigorous experimental 

protocols and controls. Both toxicity tests and bioaccumulation studies are bioassays (Chapman, 

1989). 

3.5.1 Bioassessment 

Changes in benthic community structure can be used as a means of assessing ecosystem response 

to contaminated sediments in aquatic ecosystems. Since most contaminants such as crude oil 

within the aquatic ecosystem eventually bind to sediment particles, emphasis on benthic 

organisms (bottom dwelling vertebrates and invertebrates) as a primary means of assessing 

ecosystem response is warranted. Of particular importance are the macrobenthic invertebrates 

(organisms retained on screens of mesh size >0.2 mm) because of their basic longevity, 

68
sedentary lifestyles, proximity to sediments, influence on sedimentary processes, and trophic 

importance. Microinvertebrates such as rotifers and nematodes are of particular ecological 

interest; however, their taxonomy is less well known. Hence, they have not been routinely 

monitored in environmental assessments. 

While there is a vast bioassessment database on the effects of oil spills, the effects of clean up 

techniques have until recently been seldom addressed. Clearly, a database on the effects of 

clean-up operations would have obvious potential for guidance. For example, in a follow up of 

the Exxon Valdez oil spill clean up, Driskell et al. (1996) noted that total abundance, species 

richness, species diversity, and abundance of several major taxa (polychaetes, bivalves, and 

gastropods) were significantly lower in hot-water-washed beaches than in unoiled beaches. 

Infauna at oiled sites that were not hot-water washed rebounded quickly following the 

disturbance. Three years after the spill, recovery of infauna at sites that were cleaned still lagged 

significantly behind the oiled sites. Principal component analysis (PCA), a multivariate 

ordination technique, was used to track site recovery trends. Negative effects were indicated by 

reduction in size or biomass, mortality, and reduced or failed reproductive success. Conversely, 

the possibility of positive impacts was also identified (e.g., when oil tolerant species bloom 

during the period of reduced competition-predation). Changes in epifauna and infauna were also 

used to assess the rates of natural recovery and the impacts of intertidal clean-up activities on the 

coast of Saudi Arabia following the 1991 Gulf oil spill (Watt et al., 1993). 

Macroinvertebrate bioassessment has been limited in field trials evaluating the efficacy of 

bioremediation strategies due to the amount of unrestricted surface area required for sample 

collection. The use of bioassessments in this context will expand with the development of 

bioassay protocols based on bioanalytical techniques (enyzmatic measurements, as well as 

immunoassay and biosensor techniques) aimed at the subcellular or multicellular level of 

biological organization. Application of these kinds of tests should be tailored for both the field 

and laboratory (Lee et al., 1998). 

Bioassessment can readily include potential impacts on vegetation. Field surveys demonstrated 

that the 1991 Gulf War oil spill severely damaged intertidal vegetation along the Saudi Arabian 

Gulf coast (Boer, 1994). Along a 45 km stretch of intertidal mangroves and salt-marshes, 

Salicornia europaea was almost extinct. Dwarf mangrove (Avicennia marina) and salt-marshes 

dominated by Arthrocnemum macrostachyum and Halocnemum strobilaceum were severely 

damaged. Halopeplis perfoliata and Limonium axillare salt-marshes were relatively unaffected. 

It was noted that natural re-establishment of the vegetation would be protracted unless active 

measures were taken to aid recovery. 

A bioassessment of vegetative growth was recently used to document the efficacy of 

bioremediation strategies to enhance the rate of habitat recovery within a tidal freshwater marsh 

located along the St. Lawrence River, Canada (Lee et al., 2001). Scirpus pungens, the dominant 

plant species at the study site was found to be tolerant to the oil, and its growth was significantly 

enhanced above that of the unoiled control by the addition of nutrients (Figure 3.5). 

The aim of oil spill remediation is to restore a site to its pre-spill condition. In this context, 

monitoring the recolonization of impacted areas should be a primary goal in bioassessments. 

69
Colonization and succession describe changes in the numbers and kinds of organisms making up 

the community over time. They provide an integrated measure of a toxicant’s effect on 

immigration, emigration, competition and predation. Colonization is somewhat analogous to 

reproduction in a single species: it reflects the ability of the community to replicate and organize 

itself. Fleeger et al. (1996) showed that unweathered Exxon Valdez crude oil delayed, but did 

not preclude, colonization by meiofauna (harpacticoids) into azoic sediment of Prince William 

Sound. 

To date, emphasis has been placed on the characterization of impacts on the macroinvertebrate 

community and vegetation. Nevertheless, sediment-associated contaminants enter the nonbenthic environment and community through natural processes including resuspension, 

desorption, ingestion of benthic organisms, ingestion of sediment, and adsorption to or uptake 

through membranes during sediment contact. Due to mobility and sampling issues, it is 

inherently much more difficult to work on pelagic organisms such as fish. Nevertheless, given 

the holistic nature of toxicant perturbations on aquatic ecosystems and the multifaceted 

interactions between the water and sediment compartments, consideration should also be given 

to the bioassessment of fish and other nonbenthic community organisms (e. g., bacteria, 

phytoplankton, cladocera, and amphibians). 

Figure 3.5 Minimum and maximum height of the (a) dominant (Scirpus pungens) and (b) 

secondary (Eleocharis palustris) plant species at Week15. Treatment of the oiled plots included 

natural attenuation (Nat. Att.); nutrient amendment with granular ammonium nitrate and super 

triple phosphate (NH4

+

 Intact); nutrient amendment with sodium nitrate instead of ammonium 

nitrate (NO3

-

). Unoiled plots were amended with granular ammonium nitrate and super triple 

phosphate (NH4

+

 Unoiled). 

70
3.5.2 Bioassays 

Bioassays provide a more accurate picture of ecosystem health at a contaminated site than 

chemical analyses because their result is an integration of the interaction that occurs between the 

contaminant and environmental variables. Bioassay endpoints are quantitative measures of 

toxicity. They compliment biological surveys, which describe communities of organisms in the 

field, and chemical analyses, which provide information on the nature of contaminants at a site, 

the magnitude of the remediation problem, and potential methods of treatment. Resource 

managers frequently use bioassays to identify the most toxic areas, thereby helping to prioritize 

sites for more thorough evaluation, including the selection of methods for chemical analyses. 

Sediment toxicity tests are generally classified as “acute” or “chronic”. They are usually 

performed on whole sediment (e.g., solid-phase), suspended sediment, sediment liquid phases 

(pore water, interstitial water), or sediment extracts (elutriates, solvent extracts). In general, 

assays using whole sediment samples are more sensitive than assays using elutriate or pore water 

samples. The American Society of Testing and Materials (ASTM, 1991) currently defines an 

acute toxicity test as a comparative study in which the organisms that are subject to different 

treatments are observed for a short period, usually not constituting a substantial portion of their 

life span. A chronic test is defined as a comparative study in which organisms that are subjected 

to different treatments are observed for a long period or a substantial portion of their life span. 

Acute tests often utilize mortality as the only measure of effect, while chronic tests usually 

include measures of growth, morphology, reproduction, behavioral effects, or other sublethal 

endpoints. 

Plant and animal communities are diverse; their members differ in their sensitivity to toxicants. 

A single species bioassay 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 is required. Accountability for the influence of natural environmental factors in 

sediment bioassays is assisted by the testing of reference and control samples. Reference 

sediment may be defined as sediment collected from the vicinity of a study site, possessing 

similar characteristics to the test sediment, but without anthropogenic contaminants. Sediment 

characteristics, such as particle size distribution and percent organic carbon of the reference 

sediment should simulate, as closely as possible, that of the test sediment. In some cases the 

reference sediment might also show toxicity due to naturally occurring chemical, physical, or 

biological properties. This factor can be addressed by determining the toxicity of control 

sediments (natural or artificially prepared sediments known to be nontoxic) and the use of 

positive controls (a sediment of known toxicity to the test organism under the conditions of the 

test). 

Bioassays have been developed and used extensively since the 1960s for the screening of 

chemicals and regulatory compliance monitoring. Sediment bioassays have been used 

extensively to diagnose the effects of oil spills (Teal, et al., 1992; Gilfillan et al., 1995; Neff and 

Stubblefield, 1995; Randolph et al., 1998). Their application has now been extended to include 

the documentation of effects and success of oil spill countermeasures like bioremediation (Lee et 

al., 1995b; Mearns et al., 1995). While any living organisms can be used in theory, toxicity tests 

with fish and macroinvertebrates have been standardized by environmental agencies to assess the 

71
hazards of industrial wastes to aquatic systems (Blaise et al., 1988). Rapid advances are now 

being made in the development of cost-effective high-performance micro-scale procedures 

involving bacterial, protozoan, microalgal, and microinvertebrate indicators (Wells et al., 1998). 

Furthermore, the high demand for simple, rapid, and practical toxicological procedures has 

resulted in the creation of commercial bioanalytical products such as the Microtox Test (AZUR 

Environmental Inc., USA). 

Major criteria to consider in the selection of species for sediment toxicity testing include: (1) 

their behavior in sediment (habitat, feeding habits, etc.), (2) their sensitivity to test material, (3) 

their ecological and/or economic relevance, (4) their geographical distribution, (5) their 

taxonomic relation to indigenous animals, (6) their acceptability for use in toxicity measurement 

(standardized test method), (7) their availability, and (8) their tolerance to natural sediment 

characteristics such as grain size. 

The response of the test organisms to the toxicant or test sediment is often affected by its life 

stage. Larval or juvenile life stages are generally more sensitive than adults. 

3.5.2.1 Benthic invertebrates 

In terms of benthic invertebrates, amphipods are among the most sensitive of benthic species. 

They are among the first to disappear from benthic communities in sediments impacted by 

pollution (Swartz et al., 1982; Mearns and Word, 1982). They have been used successfully to 

characterize shoreline impacts following oil spill incidents (Teal et al. 1992; Gilfillan et al., 

1995; Wolfe et al., 1996). 

Gilfillan et al. (1995) collected mussels from several locations for tissue hydrocarbon analysis to 

estimate bioavailable hydrocarbon concentrations in epifaunal species. In a newer approach, 

studies of sediment contamination and verification of laboratory bioassays involved controlled in 

situ exposures (caged animals) to expand the level of ecological relevance. In this case, oysters 

were used during a shoreline bioremediation experiment in Delaware Bay to document the loss 

of oil from the study area and to determine how the overall oiling may have impacted offshore 

resources (Mearns et al., 1997). 

3.5.2.2 Microtox 

Simple, sensitive, rapid, cost-effective, reproducible, and practical methods are needed for the 

screening of toxic impacts during oil spill response operations. The Microtox Test, a 

commercial bioassay accepted by regulatory agencies, is based on the measurement of changes 

in light emission by a nonpathogenic, bioluminescent marine bacterium (Vibrio fisheri) upon 

exposure to test samples. The test has been used extensively worldwide over the last 18 years for 

toxicity screening of chemicals, effluents, water and sediment, and for contamination surveys 

and environmental risk assessment. Variations of this test have been applied to time-series 

monitoring of sediment and water toxicity. Ho and Quinn (1993) identified strong rank 

correlations between the Microtox response and polycyclic aromatic fractions of organic extracts 

of sediments. Its application for monitoring the efficacy of oil spill remediation methods has 

72
been proven (Lee et al., 1995b; 1997b). Mueller et al. (1999) quantified the effectiveness of 

intrinsic recovery within an oiled wetland by monitoring the rate of acute toxicity reduction 

using the Microtox 100% Test on a water extractable phase. The observed decrease in toxicity 

followed a pattern similar to the decrease seen in petroleum concentrations by GC/MS total 

target analyte measurements. 

3.5.2.3 Fish 

Due to their economic, recreational, and aesthetic value, fish have been historically selected as a 

primary bioassay organism. Difficulties in using fish as biomonitors of sediment contamination 

arise from their preference for particular sediments or habitats and their residence time in or over 

contaminated areas. Furthermore, their absence in a water body may more directly reflect water 

quality. 

Biochemical and physiological alterations, if severe enough or protracted, can lead to structural 

alterations in organelles, cells, tissue, and organs. Detection of specific alterations using 

anatomical and cytological endpoints may indicate both prior and current exposure to chemical 

contaminants, so histopathology has been instrumental in assessing the toxicological impact of 

contaminated sediments. The documentation of neoplasms in fish and other aquatic organisms 

was perhaps the first use of histopathological indices in ecotoxicology. 

Biomarkers (as distinguished from the oil biomarkers such as hopane discussed earlier) are used 

by resource managers as a means to identify a toxicological response from fish populations. 

Biomarkers can be defined as biochemical, physiological, or pathological responses measured in 

individual organisms on exposure to environmental contaminants, and which also provide 

information concerning sublethal effects arising from such exposures. The family of enzymes 

referred to as cytochrome P-450s (or P450) act on the functional groups of lipophilic substrates 

in a process referred to as mixed function oxidase (MFO) or monooxygenase reactions (Ortiz de 

Montellano, 1986). MFO reactions are induced by polycyclic aromatic hydrocarbons (PAHs) 

and a variety of halogenated hydrocarbons (notably certain chlorinated biphenyls, dibenzofurans, 

and dibenzodioxins). The enzyme system is sensitive to these contaminants at levels 

encountered in the environment. In fish, the most widely employed and readily performed 

techniques are measurements of enzyme activities, particularly aryl hydrocarbon hydroxylase 

(AAH) and ethoxyresorufin O-deethylase (EROD), that are highly associated with the P450 1A 

proteins. 

Hodson et al. (2001) has monitored changes in the bioavailability and toxicity of oil-derived 

PAH to early life stages of fish in a field trial to evaluate the effectiveness of wetland 

bioremediation and phytoremediation strategies. For over 1.5 years, sediments from 

experimental plots were tested by bioassays of MFO (CYP1A) enzyme activity in livers of trout 

as an index of PAH exposure. Oil alone, oil mixed with sediments in the lab, and oiled 

sediments from the experimental plots all caused CYP1A induction relative to unoiled controls, 

indicating the presence and bioavailability of PAH. Induction did not vary markedly among 

treatments, but declined slowly with time. Concomitant chemical analysis suggested that PAHs 

were depleted primarily by weathering or sediment dispersion rather than by bioremediation 

73
treatments. Sediments were also chronically toxic to developing stages of trout and medaka 

(Oryzias latipes), causing increased rates of deformities and mortality. 

3.5.3 Application of bioassays to assess bioremediation in marine environments 

Bioassays were used to document the effectiveness of shoreline bioremediation in accelerating 

toxicity reduction of a sandy shoreline at Fowler Beach, Delaware, USA, that had been oiled 

with weathered Nigerian Bonny light crude (Mearns et al., 1995). The bioassay suite included 

two solid phase (Amphipod Survival, Microtox Solid-Phase) and three pore water tests (Grass 

Shrimp Embryo Survival/Growth, Microtox, Sea Urchin Fertilization). Treatment with nutrients 

(sodium nitrate and sodium tripolyphosphate) or nutrients and oil degrading bacteria (isolated 

from the study site) did not accelerate toxicity reduction. However, results of the high-frequency 

test based on the hatching success of grass shrimp embryo suggested there may have been a 

substantial delay in pore water toxicity reduction due to the addition of the nutrients themselves 

during the first few weeks. The Sea Urchin Fertilization Test was least sensitive. The most 

sensitive tests were the 10-day amphipod and grass shrimp embryo bioassays. 

Bioremediation by nutrient enrichment was investigated as a method of treating a mixture of 

Forties Crude Oil and Heavy Crude Oil stranded on Bullwell Bay, Milford Haven, UK, after the 

grounding of the Sea Empress in 1996 (Swannell et al., 1999b). Experimental results showed 

that the oil was significantly more biodegraded after two months as a result of application of the 

fertilizer. Based on the results of a bioassay that involved monitoring the development of oyster 

embryos, and the results of the Microtox Organic Solvent Basic Test, there was no evidence of 

detrimental effects associated with the bioremediation treatments. 

To date, detrimental effects from nutrient enrichment have not been observed following actual 

field operations (Prince, 1993; Mearns et al., 1997) although the possibility of a future incident 

still exists. As an example, oxygen depletion and production of ammonia from excessive 

applications of a fish-bone meal fertilizer during one field experiment caused detrimental effects 

that included toxicity and the suppression of oil degradation rates (Lee et al., 1995b). 

Furthermore, in a subsequent bioremediation field trial it was reported that a commercial 

bioremediation product suppressed the rates of toxicity reduction as it increased the retention of 

residual oil within the sediments (Lee et al., 1997b). For safety assurance, future operational 

guidelines must include ecotoxicological monitoring protocols. 

74
Chapter 4 TYPES OF AMENDMENTS AND CONSIDERATIONS IN THEIR 

APPLICATION 

The success of oil spill bioremediation depends on our ability to optimize various physical, 

chemical and biological conditions in the contaminated environment. Existing amendments for 

enhancing oil biodegradation in marine shorelines and freshwater wetlands include addition of 

nutrients, addition of microbial cultures or enzymes, phytoremediation, and oxygen 

enhancement. 

4.1 Nutrient Amendment 

As reviewed in Chapter 2, nutrient addition has been proven to be an effective strategy to 

enhance oil biodegradation in various marine shorelines. Theoretically, approximately 150 mg of 

nitrogen and 30 mg phosphorus are consumed in the conversion of 1 g of hydrocarbon to cell 

material (Rosenberg and Ron, 1996). Therefore, a commonly used strategy has been to add 

nutrients at concentrations that approaches a stoichiometric ratio of C:N:P of 100:5:1. Recently, 

the potential application of resource-ratio theory in hydrocarbon biodegradation was discussed 

(Head and Swannell, 1999; Smith et al., 1998). This theory suggests that manipulating the N:P 

ratio may result in the enrichment of different microbial populations, and the optimal N:P ratio 

can be different for degradation of different compounds (such as hydrocarbons mixed in with 

other biogenic compounds in soil). However, the practical use of these ratio-based theories 

remains a challenge. Particularly, in marine shorelines, maintaining a certain nutrient ratio is 

impossible because of the dynamic washout of nutrients resulting from the action of tides and 

waves. A more practical approach is to maintain the concentrations of the limiting nutrient or 

nutrients within the pore water at an optimal range (Bragg et al., 1994; Venosa et al., 1996). 

Commonly used nutrients include water-soluble nutrients, solid slow-release nutrients, and 

oleophilic fertilizers. Each type of nutrient has its advantages and limitations. General 

characteristics of these nutrients and important factors affecting their persistence in the field, 

such as waves and tides, and physical intrusion effects, will be discussed in this section and 

summarized in Table 4.1. More practical issues such as nutrient application strategies will be 

discussed in Chapter 5. 

4.1.1 Water-soluble nutrients 

Commonly used water-soluble nutrient products include mineral nutrient salts (e.g. KNO3, 

NaNO3, NH3NO3, K2HPO4, MgNH4PO4), and many commercial inorganic fertilizers (e.g. the 

23:2 N:P garden fertilizer used in Exxon Valdez case). They are usually applied in the field 

through the spraying of nutrient solutions or spreading of dry granules. This approach has been 

effective in enhancing oil biodegradation in many field trials (Swannell et al., 1996; Venosa et 

al., 1996). Compared to other types of nutrients, water-soluble nutrients are more readily 

available and easier to manipulate to maintain target nutrient concentrations in interstitial pore 

water. Another advantage of this type of nutrient over organic fertilizers is that the use of 

inorganic nutrients eliminates the possible competition of carbon sources. The field study by Lee 

et al. (1995a) indicated that although organic fertilizers had a greater effect on total heterotrophic 

microbial growth and activity, the inorganic nutrients were much more effective in stimulating 

crude oil degradation. 

75
However, water-soluble nutrients also have several potential disadvantages. First, they are more 

likely to be washed away by the actions of tides and waves because of their water-solubility. The 

field study in Maine demonstrated that water-soluble nutrients can be washed out within a single 

tidal cycle in high-energy beaches (Wrenn, 2000, see section 2.6.2). Second, inorganic nutrients, 

ammonia in particular, should be added carefully to avoid reaching toxic levels. Existing field 

trials, however, have not observed acute toxicity to sensitive species resulting from the addition 

of excess water-soluble nutrients (Mearns et al., 1997; Prince et al., 1994). Third, water-soluble 

nutrients may have to be added more frequently than slow release nutrients or organic nutrients, 

resulting in more labor-intensive, costly, and physical intrusive applications. 

4.1.2 Granular nutrients (slow-release) 

Many attempts have been made to design nutrient delivery systems that overcome the washout 

problems characteristic of intertidal environments (Prince, 1993). Use of slow release fertilizers 

is one of the approaches used to provide continuous sources of nutrients to oil contaminated 

areas. Slow release fertilizers are normally in solid forms that consist of inorganic nutrients 

coated with hydrophobic materials like paraffin or vegetable oils. This approach may also cost 

less than adding water-soluble nutrients due to less frequent applications. Slow release fertilizers 

have shown some promises from oil bioremediation studies and applications. For example, 

Olivieri et al.(1976) found that the biodegradation of a crude oil was considerably enhanced by 

addition of a paraffin coated MgNH4PO4. Another slow-release fertilizer, Customblen (vegetable 

oil coated calcium phosphate, ammonium phosphate, and ammonium nitrate), performed well on 

some of the shorelines of Prince William Sound, particularly in combination with an oleophilic 

fertilizer (Atlas, 1995a; Pritchard et al., 1992; Swannell et al., 1996). Lee et al. (1993) also 

showed that oil biodegradation rates increased with the use of a slow release fertilizer (sulfurcoated urea) compared to water-soluble fertilizers. 

However, the major challenge for this technology is control of the release rates so that optimal 

nutrient concentrations can be maintained in the pore water over long time periods. For example, 

if the nutrients are released too quickly, they will be subject to rapid washout and will not act as 

a long-term source. On the other hand, if they are released too slowly, the concentration will 

never build up to a level that is sufficient to support rapid biodegradation rates, and the resulting 

stimulation will be less effective than it could be. The field trials on of the shorelines of Prince 

William Sound showed that on certain beaches, Customblen granules were apparently washed 

away before any significant enhancement of bioremediation was recorded (Swannell et al., 

1996). Several recent studies have shown that a slow release nutrient (Max Bac, a product 

similar to Customblen) failed to demonstrate enhancement of oil degradation because the 

nutrient release rate was too slow to affect oil biodegradation (Croft et al., 1995; Sveum and 

Ramstad, 1995). 

4.1.3 Oleophilic nutrients 

Another approach to overcome the problem of water-soluble nutrients being rapidly washed out 

was to utilize oleophilic organic nutrients (Atlas and Bartha, 1973; Ladousse and Tramier, 1991). 

The rationale for this strategy is that oil biodegradation mainly occurred at the oil-water 

76
interface; since oleophilic fertilizers are able to adhere to oil and provide nutrients at the oilwater interface, enhanced biodegradation should result without the need to increase nutrient 

concentrations in the bulk pore water. A well-known oleophilic fertilizer is Inipol EAP 22, a 

microemulsion containing urea as a nitrogen source, lauryl phosphate (the phosphorus source), 2-

butoxy-1-ethanol as a surfactant, and oleic acid to give the material its hydrophobicity. This 

fertilizer has been subjected to extensive studies under various shoreline conditions and was 

successfully used in oil bioremediation on of the shorelines of Prince William Sound. Other 

oleophilic organic fertilizers include polymerized urea and formaldehyde, and some organic 

fertilizers derived from natural products such as fishmeal (Lee et al., 1995a; Rosenberg et al., 

1992; Sveum and Ramstad, 1995). 

Table 4.1 Major nutrient types used in oil bioremediation 

Type of nutrients Advantages 

Water soluble Readily available Rapidly washed out 

Easy to manipulate by wave and tide 

for target nutrient Labor-intensive, and 

concentrations physical intrusive 

No complicated effect applications 

of organic matter Potential toxic effect 

Slow release Provide continuous Maintaining optimal 

sources of nutrients nutrient release rates 

and may be more cost could be a challenge 

effective than other 

types of nutrients 

Oleophilic Able to adhere to oil Expensive 

Disadvantages 

Effectiveness is 

variable 

Containing organic 

carbon, which may 

compete with oil 

degradation and result 

in undesirable anoxic 

conditions 

Applications in the 

field or field trials 

Alaska (Pritchard et 

al., 1992) 

Delaware (Venosa et 

al., 1996) 

Alaska (Pritchard et 

al., 1992) 

Nova Scotia (Lee et 

al., 1993) 

Alaska (Pritchard et 

al., 1992) 

Nova Scotia (Lee et 

al., 1987, 1989,1995a 

&b) 

and provide nutrients 

at the oil-water 

interface 

The effectiveness of oleophilic fertilizers also depends on the characteristics of the contaminated 

environment such as action of wave and tide, and sediment types. Based on several earlier 

studies, Sveum et al. (1994) indicated that oleophilic fertilizers proved to be more effective than 

water-soluble fertilizers when the spilled oil resided in the intertidal zone. But they have no 

advantages in enhancing oil biodegradation in the supralittoral zone where water transport is 

limited. Inipol EAP 22 was found to be more effective in coarse sediments than in fine sediments 

due to the difficulty in penetration for the oleophilic fertilizer in fine sediments (Sveum and 

77
Ladousse,1989). Variable results have also been produced regarding the persistence of oleophilic 

fertilizers. Some studies showed that Inipol EAP 22 can persist in a sandy beach for a long time 

under simulated tide and wave actions (Santas and Santas, 2000; Swannell et al. 1995). Others 

found that Inipol EAP22 was rapidly washed out before becoming available to hydrocarbondegrading bacteria (Lee and Levy, 1987; Safferman, 1991). Another disadvantage with 

oleophilic fertilizers is that they contain organic carbon which may be biodegraded by 

microorganisms in preference to petroleum hydrocarbons (Lee et al., 1995a; Swannell et al., 

1996), and may also result in undesirable anoxic conditions (Lee et al., 1995b; Sveum and 

Ramstad, 1995). 

In summary, the effectiveness of these various types of nutrients will depend on the 

characteristics of the contaminated environment. Slow-release fertilizers may be ideal nutrient 

sources if the nutrient release rates can be well controlled. Water-soluble fertilizers are likely 

more cost-effective in low-energy and fine-grained shorelines where water transport is limited. 

And oleophilic fertilizers may be more suitable for use in high-energy and coarse-grained 

beaches. However, successful application of bioremediation products will always require 

appropriate testing and evaluation based on the specific conditions of each contaminated site. 

4.2 Microbial Amendments 

Addition of oil-degrading microorganisms or bioaugmentation has been proposed as a 

bioremediation strategy. The rationale for this approach includes the contention that indigenous 

microbial populations may not be capable of degrading the wide range of substrates that are 

present in complex mixtures such as petroleum and that seeding may reduce the lag period 

before bioremediation begins (Forsyth et al., 1995; Leahy and Colwell, 1990). For this approach 

to be successful in the field, the seed microorganisms must be able to degrade most petroleum 

components, maintain genetic stability and viability during storage, survive in foreign and hostile 

environments, effectively compete with indigenous microorganisms, and move through the pores 

of the sediment to the contaminants (Atlas, 1977; Goldstein et al., 1985). 

There are many vendors of bioremediation products, who claim their product (most of them are 

microbial agents) aids the oil biodegradation process. The U.S. EPA has compiled a list of 

bioremediation agents (USEPA, 2000) as part of the National Oil and Hazardous Substances 

Pollution Contingency Plan (NCP) Product Schedule, which is required by the Clean Water Act, 

the Oil Pollution Act of 1990, and the National Contingency Plan. A current list of 

bioremediation agents in NCP schedule is shown in Table 4.2. A product can be listed only when 

its safety and effectiveness have been demonstrated under the conditions of a test protocol 

developed by EPA (NETAC, 1993). However, listing does not mean that the product is 

recommended or certified for use on an oil spill (USEPA, 2000). The efficacy test protocol uses 

laboratory shake flasks to compare the degradation of artificially-weathered crude oil in natural 

seawater with and without a bioremediation product. Similar test protocols for freshwater 

conditions were recently proposed (Haines et al., 1999). 

78
Table 4.2 Bioremediation agents in NCP product schedule (Adapted from USEPA, 2000) 

Type Name or Trademark Manufacture 

Biological additives 

(Microbial Culture or 

Enzyme additives) 

BET BIOPETRO 

BIOGEE HC 

BR (formerly ENVIROZYME BR) 

ENZYT 

(LIQUID/CRYSTA) 

MICRO-BLAZE 

OPPENHEIMER 

FORMULA 

PRISTINE SEA II 

PRP ( Petroleum 

Remediation Product) 

STEP ONE 

SYSTEM E.T. 20 

WMI-2000 

BioEnviro Tech, Tomball, TX 

RMC Bioremediation, Shreveport, LA 

Enviro-Zyme, Inc., Stormville, NY 

Acorn Biotechnical Corporation 

Houston, TX 

Verde Environmental, Inc., Houston, TX 

Oppenheimer Biotechnology, Inc. 

Austin, TX 

Marine Systems, Baton Rouge, LA 

Petrol Rem, Inc., Pittsburgh, PA 

B & S Research, Inc. 

Embarrass, MN 

Quantum Environmental Technologies, Inc. 

(QET), 

Waste Microbes, Inc., Houston, TX 

Nutrient additives INIPOL EAP 22 

(oleophilic) 

LAND AND SEA 

RESTORATION 

OIL SPILL EATER II 

VB591TMWATER, 

VB997TMSOIL, AND 

BINUTRIX 

(partially encapsulated 

Societe, CECA S.A. 

France 

Land and Sea Restoration LLC, San 

Antonio, TX 

Oil Spill Eater International, Corporation 

Dallas, TX 

BioNutraTech, Inc., Houston, TX 

La Jolla, CA 

& oleophilic) 

79
As reviewed in Chapter 2, however, even though the addition of microorganisms may be able to 

enhance oil biodegradation in the laboratory, its effectiveness has not been convincingly 

demonstrated in the field. Actually, most field studies indicated that bioaugmentation is not 

effective in enhancing oil biodegradation in marine shorelines, and nutrient addition or 

biostimulation alone had a greater effect on oil biodegradation than the microbial seeding 

(Jobson et al., 1974; Lee and Levy, 1987; Lee et al., 1997b, Venosa et al., 1996). The failure of 

bioaugmentation in the field may be attributed to the fact that the carrying capacity of most 

environments is likely determined by factors that are not affected by an exogenous source of 

microorganisms (such as predation by protozoans, the oil surface area, or scouring of attached 

biomass by wave activity), and that added bacteria seem to compete poorly with the indigenous 

population (Tagger et al., 1983; Lee and Levy, 1989; Venosa et al., 1992). Therefore, it is 

unlikely that externally added microorganisms will persist in a contaminated beach even when 

they are added in high numbers. In short, those criteria mentioned above for a successful 

colonization are very difficult to be met in the field. 

Fortunately, oil-degrading microorganisms are ubiquitous in the environment, and they can 

increase by many orders of magnitude after being exposed to crude oil (Atlas, 1981; Lee and 

Levy, 1987, Pritchard and Costa, 1991). Therefore, in most environments, there is usually no 

need to add hydrocarbon degraders. In certain circumstances that have not been well defined, 

when the indigenous bacteria are incapable of degrading one or more important contaminants, 

addition of microbial inocula may be considered. Genetically engineered organisms are not 

likely to be used in the near or even distant future. 

4.3 Plant Amendments (phytoremediation) 

Phytoremediation has been defined as the use of green plants and their associated 

microorganisms to degrade, contain, or render harmless environmental contaminants 

(Cunningham et al., 1996). This technique is emerging as a potentially cost-effective option for 

clean-up of soils contaminated with petroleum hydrocarbons (Frick et al., 1999). As 

summarized by Macek et al. (2000), the main advantages of phytoremediation include less 

disruption to the environment, potential to treat a diverse range of contaminants, and high 

probability of public acceptance. Major concerns regarding this technology include dissolution 

and migration of contaminants, limitation by the toxicity of the contaminated environments, and 

it being a relatively slow process. Phytoremediation has been studied in a freshwater 

environment in Quebec, Canada (Garcia-Blanco et al., 2000; Venosa et al., 2002 (submitted)). 

These researchers found that addition of nutrients did not result in enhancement of 

biodegradation of crude oil contaminating the plots, whether or not plants were left intact or 

removed. It appeared that in a wetland environment, oxygen became limiting at depths within a 

few mm from the surface. 

4.3.1 Mechanisms of phytoremediation 

Phytoremediation of petroleum hydrocarbons generally involves three major mechanisms: (1) 

degradation, (2) containment and (3) the transfer of contaminants from soil to the atmosphere 

(Cunningham et al., 1996; Frick et al., 1999). 

80
Degradation can be accomplished by both plants and their associated microorganisms. One of the 

most important processes involved in the degradation is the interaction between plants and 

microorganisms in the rhizosphere (root zone). Plants can stimulate the growth and metabolism 

of soil microorganisms by providing root exudates of carbon, enzymes, nutrients, and oxygen, 

which can result in more than 100-fold increase in microbial counts (Macek et al., 2000). This 

process is also mutual beneficial. The microbes can reduce the phytotoxicity of contaminants so 

that plants can grow in adverse soil conditions. Cometabolism may also play an important role in 

phytodegradation. Ferro et al. (1997) suggested that plant exudates might have served as cometabolites in enhancing the biodegradation of pyrene in the rhizosphere. 

Other major mechanisms of phytoremediation include containment of petroleum hydrocarbons 

and their transfer from the soil to the atmosphere. Containment involves the accumulation of 

contaminants within the plants, adsorption of contaminants onto roots, and binding of 

contaminants in the rhizosphere through enzymatic activities (Cunningham et al., 1996; Frick et 

al., 1999). Plants can also transport volatile petroleum hydrocarbons to the atmosphere through 

leaves and stems. However, these effects are less important than the degradation mechanism in 

phytoremediation of petroleum hydrocarbons (Ferro et al. 1997). 

4.3.2 Considerations in application of oil phytoremediation 

While phytoremediation had been used successfully within the terrestrial environment to 

decontaminate soils (Banks and Schwab, 1993; Schnoor et al., 1995), the technique has not been 

employed as an operational oil spill countermeasure. Until recently, only limited research had 

been carried out on the effectiveness of phytoremediation in freshwater wetlands (Lin and 

Mendelssohn, 1998). Most of the studies were greenhouse experiments rather than field studies. 

Like bioaugmentation, studies on phytoremediation of petroleum hydrocarbons have produced 

mixed results. The effectiveness of phytoremediation is site-specific, which can be affected by 

factors as oil properties, types of plants, and environmental conditions. 

Oil concentrations 

Plants can tolerate oils with certain concentration ranges. When oil concentrations are too high, 

toxic effects will lead to growth inhibition or death of plants. When oil concentrations are too 

low, phytoremediation will not be effective either due to poor bioavailability. Longpre et al. 

(1999) investigated the impact of oil concentrations on a freshwater wetland plant (Scirpus 

pungens) along the shore of the St. Lawrence River. The results showed that the plant growth 

was stimulated in the presence of crude oil at a concentration less than 4.56g/Kg sediment when 

compared to the growth of the control plants. At higher oil concentrations, up to 27.4g/kg 

sediment, growth inhibition or no growth increase was observed. When the oil concentration was 

above 36.4 g/kg sediment, plant growth was significantly reduced. The study concluded that the 

plants were likely to survive and grow in sediments contaminated with crude oil in a range of 

concentrations comparable to oil spill incidents. 

Plant species 

Another important factor in considering phytoremediation is establishment of appropriate plants. 

Lin and Mendelssohn (1996) studied the effect of oil spills on four freshwater marsh plant 

species. Two of them (C. ordoratus, and A. teres) failed to survive in any of the oiled sods and E. 

81
quadrangulata could only persist at oil levels up to 8 L/m2

. In contrast, the growth of Sagittaria 

lancifolia was enhanced in response of oil addition up to 24 L/m2. Generally, legume and grass 

species have been the choices for their potential use in phytoremediation of petroleum 

hydrocarbons (Frick et al., 1999). Legumes are nitrogen-fixing plants, which may have 

advantages in competing with non-legume species in oil-contaminated sediments. Native plants 

should normally be selected since they have better chance to survive or out-compete nonindigenous inocula (Cunningham et al., 1996). 

Environmental factors 

Similar to the environmental factors affecting microbial biodegradation discussed in chapter 2, 

major environmental factors affecting phytoremediation of petroleum hydrocarbons include soil 

types, nutrients, oxygen and temperature. Detailed description can be seen in Cunningham et al., 

1996 and Frick et al., 1999. 

4.3.3 Applications in marine shoreline and freshwater wetlands 

Current applications of phytoremediation in marine shorelines and freshwater wetlands have 

been limited to accelerate recovery and restoration of oiled wetland. For example, mangroves 

were successfully replanted to restore oil-killed mangrove forest in Panama after the 1986 

Refineria Panama oil spill (Teas et al., 1989). Only a few field studies have been carried out on 

the effectiveness of phytoremediation in enhancing oil degradation in marine shorelines and 

freshwater wetlands. 

Lin and Mendelssohn (1998) investigated the effects of biostimulation and phytoremediation in 

enhancing habitat restoration and oil degradation in a coastal wetland environment (greenhouse 

study). They found that application of fertilizer in conjunction with the presence of transplants 

led to much higher oil degradation rates than phytoremediation alone. The results were attributed 

to a higher microbial number and activity induced by the fertilizer. However, it was still not clear 

whether this effect was due to biostimulation of soil microorganisms or due to phytoremediation 

via fertilizer-increased plant biomass. These confounding effects perhaps could have been 

distinguished by adding one more treatment (biostimulation with absence of transplants). 

In 1999 and 2000, a major research study was conducted on the shoreline of the St. Lawrence 

River (Garcia-Blanco et al., 2000; Venosa et al., 2002 (submitted)). The experimental design 

was similar to the one used on the marine shoreline in Delaware Bay (Venosa et al., 1996). There 

were 5 treatments: a no oil control and four oiled treatments. The oiled treatments included a 

natural attenuation control plot with no amendments, a plot receiving ammonium nitrate and 

orthophosphate nutrients but with the wetland plants continually cut back to ground surface to 

suppress photosynthetic activity and growth, a plot receiving the same nutrients as Treatment B 

but with the plants left intact, and a plot similar to Treatment C but with only nitrate (no 

ammonium) serving as the nitrogen source. The no-oil control also received the same nutrients as 

the oiled treatments receiving nutrients. Findings are summarized as follows: (1) alkane 

degraders increased only marginally in all treatments while the PAH degraders were stimulated 

to increase by 3.5 orders of magnitude in response to exposure to crude oil; (2) nitrogen in the 

form of ammonium was partly adsorbed to negatively charged soil particles, partly taken up by 

the root system of the wetland plants, and partly leached into the pore water. Nitrogen in the 

82
form of nitrate leached into the pore water, and some was taken up in the root system (Lee et al., 

2002 (submitted); (3) the primary mechanism of oil mass loss from all the plots, regardless of 

treatment, was physical rather than biological; (4) with respect to biodegradation of total alkanes 

and PAHs during the first 21 weeks of the investigation as measured by GC/MS analysis, only 

about 35% biodegradation occurred in all treatments on average, and no significant differences 

among any of the treatments were observed (p > 0.05); (5) a substantial increase in plant biomass 

was observed due to fertilizer addition; (6) better biodegradation occurred in surface samples in 

plots where the plants had been removed than in any of the core samples because of the oxic 

nature of the surface and the lack of competition for nutrients by the plant species. Enhanced 

oxygen transfer to the rhizosphere by the plants through their roots did not appear to take place, 

at least at the level needed by hydrocarbon degraders to metabolize the oil rapidly. 

The major reason for the lack of biodegradation beyond only about 35% was ascribed to the fact 

that the oil had been raked into the top 2-3 cm of sediment to make sure that penetration had 

occurred. When such oil penetration occurs, little oxygen is available to allow significant 

biodegradation to take place throughout the oiled zone. If oil contaminates only the surface 

where more aerobic conditions exist, and if it does not penetrate deeply into the subsurface, 

better biodegradation should take place, at least theoretically. The major conclusion reached 

from this study was that bioremediation of an oil-contaminated freshwater wetland where 

significant penetration of oil has taken place into the sediment has limited potential for enhanced 

cleanup of the contamination. 

In summary, the effectiveness of phytoremediation in enhancing oil degradation in freshwater 

wetlands is highly site-specific and does not promise to be an effective oil cleanup technique. 

However, it does show promise in accelerating the recovery and restoration of wetland 

environments contaminated with oil and oil products. 

4.4. Oxygen Amendment 

Oxygen usually is not a limiting factor on many sandy beaches. However, oxygen limitation may 

occur in wetlands and fine-grained shorelines as indicated by some field studies (Garcia-Blanco 

et al., 2001b, Lee and Levy, 1991, Purandare, 1999). Under such circumstances, oxygen 

amendment may be considered as a bioremediation strategy. Although oxygen supply has been 

widely used for bioremediation of oil contaminated soils and groundwater, such as at many 

subsurface fuel contaminated sites, this strategy has not been applied to enhance oil degradation 

in marine shoreline and freshwater wetlands. This is because oxygen amendment usually 

involves expensive and environmentally intrusive operations. For bioremediation of a large-scale 

oil spill, use of this approach is probably not practical even when oxygen is a limiting factor. 

However, under certain circumstance that involves high oil contamination in smaller scale and in 

less sensitive habitats, oxygen amendment can still be considered as an alternative for oil 

bioremediation. Commonly used oxygen supply techniques include tilling, forced aeration, and 

chemical methods (Atlas, 1991; Brown and Crosbie, 1994; Riser-Roberts, 1998). These methods 

are summarized below. They are mostly based on studies and practices in soil environments. 

Special attention will be given to their potentials of application in marine shorelines and 

freshwater wetlands. 

83
4.4.1 Tilling 

Tilling has been a conventional physical method to accelerate natural oil removal by exposing 

oiled sediments to a higher level of physical abrasion and biochemical degradation (Owens, 

1998). This technique is also an effective means of aeration for surface layer of sediments. It has 

been successfully used to accelerate biodegradation in landfarming (Atlas, 1991; Jerger et al., 

1994). Traditional tilling machines, such as disk harrows and rototillers, can aerate surface soils 

to a depth of 6 to 24 inches. Sediments deeper than about 2 ft (60 cm) can be aerated by using 

construction equipment, such as a backhoe (Riser-Roberts, 1998). 

Currently, tilling has been recommended as a physical method to accelerate natural weathering 

processes of oil in sandy or coarse-sediment beaches (Owens, 1998). The main purpose of this 

practice is to increase physical abrasion of oils rather than to enhance aeration since oxygen is 

usually not a limiting factor in these environments. However, based on existing experiences in 

landfarming, this technique may have some potential in enhancing oil biodegradation in some 

fine-sediment beaches where oxygen is limited. Tilling is also considered a low-cost technology 

among the available aeration methods (Jerger et al., 1994). 

Major concerns regarding this technique include disturbance of both the natural shape of 

shorelines and local habitats and the potential of releasing of oil and oiled sediment into adjacent 

locations. The experience from the St. Lawrence River field trial also suggests that the tilling of 

surface soil may cause oil penetration deep into the shoreline sediments and may reduce the 

overall oil biodegradation rates if the oil penetrates into anaerobic sediments (Garcia-Blanco et 

al., 2001; Venosa et al., 2002 (submitted); see Section 5.5.2). 

4.4.2 Forced aeration 

Forced aeration techniques, including injection of aerated water, air and pure oxygen, are 

expensive methods and commonly used for enhancing bioremediation in subsurface sediments 

and groundwater contaminated with petroleum hydrocarbons (Brown and Crosbie, 1994; RiserRoberts, 1998). Oil contamination of coastal and wetland environments, however, usually occurs 

near the surface, especially when the contamination is the result of an offshore spill. 

Furthermore, crude oil rarely penetrates coastal sediments to depths of greater than one foot 

(Gundlach, 1987). Therefore, these techniques of subsurface aeration are probably not 

appropriate for use in bioremediation of oil spill in marine shorelines and freshwater wetlands. 

4.4.3 Chemical methods 

Chemical methods involve addition of alternative oxygen sources such as hydrogen peroxide 

(H2O2), or alternative electron acceptors such as nitrate. Hydrogen peroxide can provide oxygen 

at a rate up to two orders of magnitude faster than the forced aeration methods (Brown and 

Norris, 1994). It also requires less equipment and capital cost. However, problems including too 

rapid decomposition, gas blockage, and inefficient use were encountered in some sites when 

using H2O2 (Brown and Norris, 1994). The chemical also can be toxic to microorganisms at high 

concentrations (Riser-Roberts, 1998). 

84
Nitrate has received most attention as an alternative electron acceptor because it is relatively 

inexpensive, very soluble in water, and does not decompose. Since nitrate is also commonly used 

nutrient source for oil biostimulation, addition of nitrate may be a promising option for oil 

bioremediation under oxygen limiting conditions. A potential disadvantage of this method is that 

nitrate may be effective for degradation of fewer classes of compounds than oxygen. It has been 

reported that nitrate-utilizing bacteria can degrade many aromatics but do not degrade aliphatic 

compounds and benzene under denitrifying conditions (Brown et al., 1993). However, others 

recently found that degradation of alkanes could take place under denitrifying conditions (Hess 

et al., 1996). Sulfate is another potentially useful electron acceptor especially in certain marine 

environments, such as salt marshes, where sulfate reduction is one of most important natural 

processes (Mitsch and Gosselink, 2000). Some laboratory studies have shown that PAHs and 

alkanes can be degraded under sulfate-reducing conditions at similar rates to those under aerobic 

conditions in some marine sediment (Caldwell et al., 1998; Coates et al., 1997). However, these 

high oil degradation rates under sulfate-reducing conditions have not been reported or 

demonstrated in the field. 

In summary, the potential of using oxygen amendment for enhancing oil biodegradation in 

marine shorelines and freshwater wetlands is limited. Tilling may be considered as an aeration 

strategy for enhancing oil biodegradation in the upper layer of sediments in less sensitive 

habitats. Nitrate could be a potential alternative electron acceptor for use in a wide range of 

environments, but its use as an effective enhancer of biodegradation is questionable. More 

studies, particularly field trials, are still required to evaluate the effectiveness of these strategies. 

85
Chapter 5 GUIDELINES FOR BIOREMEDIATION OF MARINE SHORELINES 

AND FRESHWATER WETLANDS: DECISION-MAKING AND 

PLANNING 

Existing research and applications have demonstrated that bioremediation is an effective 

technology that can be used to treat certain oil-contaminated environments. Typically, it is used 

as a polishing step after conventional mechanical cleanup options have been applied, although it 

could also be used as a primary response strategy if the spilled oil does not exist as free product 

and if the contaminated area is remote enough not to require immediate cleanup or not accessible 

by mechanical tools. However, one of the major challenges in the application of oil 

bioremediation is lack of guidelines regarding the selection and use of this technology. Although 

extensive research has been conducted on oil bioremediation in the last decade, most existing 

studies have been concentrated on either evaluating the feasibility of bioremediation for dealing 

with oil contamination or testing favored products and methods (Mearns, 1997). Only a few 

limited operational guidelines for bioremediation in marine shorelines have been proposed (Lee, 

1995; Lee and Merlin, 1999; Swannell et al., 1996). The following two chapters will present a 

more detailed and workable guidance for use by spill responders for the bioremediation of 

marine shorelines and freshwater wetlands based on recent field studies and current 

understandings on bioremediation processes. 

As a result of recent field studies (Lee et al, 1997b; Venosa et al., 1996), we now know that there 

is usually little need to add hydrocarbon degrading microorganisms because this approach has 

been shown not to enhance oil degradation more than simple nutrient addition. Therefore, this 

document will only present guidelines for oil bioremediation using biostimulation strategies, 

mainly nutrient addition. 

A general procedure or plan for the selection and application of bioremediation technology is 

illustrated in Figure 5.1. The major steps in a bioremediation selection and response plan include: 

1. Pre-treatment assessment – This step involves the evaluation of whether bioremediation is a 

viable option based on the type of oil that has been spilled, its concentration, the presence of 

hydrocarbon-degrading microorganisms, concentrations of background nutrients, the type of 

shoreline that has been impacted, and other environmental factors (pH, temperature, presence 

of oxygen, remoteness of the site, accessibility of the site and logistics, etc.). 

2. Design of treatment and monitoring plan – After the decision is made to use bioremediation, 

further assessments and planning are needed prior to the application. This involves selection 

of the rate-limiting treatment agents (e.g., nutrients), determination of application strategies 

for the rate-limiting agents, and design of sampling and monitoring plans. 

3. Assessment and termination of treatment – After the treatment is implemented according to 

the plan, assessment of treatment efficacy and determination of appropriate treatment 

endpoints are performed based on chemical, toxicological, and ecological analysis. 

This chapter covers operational guidelines for decision-making in the use of bioremediation and 

describes the planning process for bioremediating marine shorelines and freshwater wetlands. 

86
The next chapter (Chapter 6) will present guidelines for assessment of field results and 

establishment of appropriate treatment endpoints. 

Step 1: 

Pretreatment Assessment 

Step 2: 

Bioremediation Planning 

Step 3: 

Assessment and Termination 

Oil type and 

concentration 

Background Shoreline type

nutrient content 

If bioremediation 

is selected 

Climate, prior oil 

exposure and other 

site characteristics 

nutrient products Nutrient application 

strategy 

Sampling and 

monitoring plan 

Toxicological and ecological 

analysis 

Analysis of oil biodegradation 

and physical loss 

Figure 5.1 Procedures for the selection and application of oil spill bioremediation

87
5.1 Pre-treatment Assessment 

Pretreatment assessment involves some preliminary investigations to assess whether 

bioremediation is a viable option and determination of the rate determining process, which 

include the evaluation of (1) oil types and concentrations, (2) background nutrient content, (3) 

shoreline types, and (4) other environmental factors such as the prevalent climate and prior oil 

exposures. 

5.1.1 Oil type and concentration 

Oil Type 

As reviewed in Chapter 2, the biodegradability of different types of oils and petroleum products 

varies greatly depending on the distribution of oil components. In general, the susceptibility to 

microbial degradation for petroleum hydrocarbons is in the order of n-alkanes > branched 

alkanes > low-molecular-weight aromatics > cyclic alkanes > high molecular-weight aromatics, 

although this pattern is not universal (Perry, 1984). The degradation rate for the same oil 

components may also vary significantly for different oils. It has been found that the rate and 

extent of biodegradation of biodegradable components (e.g. n-alkanes) decreases with the 

increase of non-biodegradable fractions (e.g., resins and asphaltenes) (Uraizee et al., 1998; 

Westlake et al., 1974). Therefore, the heavier crude oils are less likely biodegradable than lighter 

crude oils. McMillen et al. (1995) investigated the biodegradability of 17 crude oils with API 

gravity ranging from 14° to 45°. They concluded that crude oil with greater than 30° API gravity 

can be considered readily biodegradable, and those with less than 20° API gravity (heavier oils) 

are slow to biodegrade. Similar results were obtained by other researchers (Hoff et al., 1995; 

Sugiura, et al., 1997). Wang and Bartha (1990) also investigated the effects of bioremediation on 

residues of fuel spills in soils. The results showed that the treatability by bioremediation for the 

fuel residues are in the order of jet fuel > heating oil > diesel oil. However, more work is still 

required to classify crude oils and refined products with respect to their theoretical amenability to 

cleanup by bioremediation. 

The biodegradation potential of oils also depends on the weathering processes that alter oil 

compositions and properties. For example, evaporation leads to removal of the more toxic, lower 

molecular weight components from the spilled oil. Therefore, there is less need to bioremediate a 

spill of light petroleum products such as gasoline since it would evaporate rapidly. The formation 

of water-in-oil emulsion may increase mass transfer limitation for oxygen and nutrients and 

decrease the oil biodegradation rate. Interactions between oil and various types of shorelines also 

play important roles in oil degradation, which will be discussed later. Field experience also 

suggested that oils that have been subjected to substantial biodegradation might not be amenable 

to bioremediation due to the accumulation of polar components in the oils (Bragg et al., 1994; 

Oudet et al., 1998). 

Oil concentrations 

The concentration of oil is another important consideration in determining whether 

bioremediation is a viable option. Very low concentrations of hydrocarbons in the environment 

may be inefficiently attacked by microbes (Foght and Westlake, 1987). For sites contaminated 

with oils at low concentrations, biodegradation is also less likely to be limited by nutrients or 

88
oxygen. Therefore, bioremediation may not be effective in enhancing biodegradation in these 

cases. Natural attenuation may be a more viable option. 

High concentrations of hydrocarbons may cause inhibition of biodegradation due to toxic effects, 

although the inhibitory concentration varies with oil composition. Therefore, there should be an 

optimum oil concentration range for bioremediation applications, below which degradation is not 

easily stimulated, and above which inhibition occurs. However, this concentration range, 

particularly the maximum concentration of oil amenable to bioremediation, has not been well 

quantified. Field experiences in Prince William Sound, Alaska showed that less than 15 g oil/kg 

sediments could be treated using bioremediation (Swannell et al., 1996). Xu et al. (2001) 

recently investigated the effect of oil concentration in a microcosm study using weathered 

Alaska North Slope crude oil. The results showed that crude oil concentrations as high as 80 g 

oil/kg dry sand were still amenable to biodegradation. Favorable oil concentrations for 

bioremediation are also related to background conditions, such as shoreline types, which will be 

discussed later in this chapter. 

5.1.2 Background nutrient content 

Since nutrient addition has been chosen as the primary strategy to enhance oil biodegradation, 

assessment of background nutrient concentrations becomes critical in determining whether 

bioremediation is a viable option, whether natural attenuation should be considered, and/or 

which nutrient (nitrogen or phosphorus) should be added for oil bioremediation. 

As mentioned in Chapter 2, in marine environments, nutrients are generally limiting due to the 

naturally low nitrogen and phosphorus concentrations in seawater (Floodgate, 1984). Nutrient 

content is more variable in freshwater systems and is normally abundant in freshwater wetlands 

(Cooney, 1984; Mitsch and Gosselink, 1993). However, background nutrients also depend on 

other site characteristics such as local industrial and domestic effluents and agricultural runoff. 

Recent field studies indicate that natural nutrient concentrations in some marine shorelines can 

be high enough to sustain rapid intrinsic rates of biodegradation without human intervention 

(Oudet et al., 1998; Venosa et al., 1996). The field trial in Delaware showed that although 

biostimulation with inorganic mineral nutrients significantly accelerated the rate of hydrocarbon 

biodegradation, the increase in biodegradation rate over the intrinsic rate (i.e. slightly greater 

than twofold for the alkanes and 50% for the PAHs) would not be high enough to warrant a 

recommendation to actively initiate a major, perhaps costly, bioremediation action in the event of 

a large crude oil spill in that area (Venosa, et al., 1996). The high intrinsic biodegradation rate 

was attributed to the high background nutrient concentrations (0.8 mg N/L on average) because 

the Fowler Beach area of Delaware Bay was adjacent to farmland. The relatively high organic 

content of both the Delaware Bay seawater and the underlying geology of the site and the 

presence of a saltwater marsh several hundred meters landward from the beach could also 

account for the high nitrogen levels encountered. The study investigators observed that 

maintenance of a threshold nitrogen concentration of 3-6 mg N/L in the interstitial pore water 

was biostimulatory for hydrocarbon biodegradation. 

89
A similar conclusion was also reached in a field trial to evaluate the influence of a slow-release 

fertilizer on the biodegradation rate of crude oil spilled on interstitial sediments of an estuarine 

environment in the bay of Brest, France (Oudet et al., 1998). Due to the high background levels 

of N and P at the study site, no significant difference in biodegradation rates was detected 

following nutrient addition. It was proposed that bioremediation by nutrient enrichment would be 

of limited use if background interstitial pore water levels of N exceed 1.4 mg/L, which is close to 

the finding from the Delaware study (Venosa et al., 1996). 

Phosphorus is another essential nutrient related to microbial growth. Although no field study of 

critical phosphorus concentrations on marine shoreline and freshwater wetlands has been 

reported, it has been generally accepted that the optimal N:P ratio for microbial growth is in a 

range of 5:1 to 10:1. Therefore, the threshold phosphorus concentration for maintaining optimal 

hydrocarbon degradation can be derived based on this ratio and critical nitrogen concentrations 

obtained from existing field studies. However, further research is still required in determining the 

influence of phosphorus on oil bioremediation under various marine shoreline and freshwater 

wetland environments. 

These results suggest that, in the event of a catastrophic oil spill impacting a shoreline, one of the 

first tasks in pretreatment assessment is to measure the natural nutrient concentrations within the 

interstitial water in that environment. If they are high enough, further investigation is required to 

determine whether such a nutrient loading is typical for that area and season (i.e., determine the 

impact of chronic runoff from nearby agricultural practice and local industrial and domestic 

effluents). The decision to use bioremediation by addition of nutrients should be based on how 

high the natural levels are relative to the optimal or threshold nutrient concentrations. 

5.1.3 Type of shorelines 

The characteristics or type of the contaminated shoreline also play an important role in the 

decision to use bioremediation. This preliminary investigation involves the assessment of the 

need for bioremediation based on wave and tidal energy, the sediment characteristics, and 

geomorphology of the shoreline. 

Shoreline energy and hydrology 

Oil can be removed rather rapidly under high wave and tide influence, typically in rocky 

shorelines. In high-energy environments, bioremediation products are also more difficult to 

apply successfully since they can be washed out rapidly. High wave energy will also scour 

microorganisms attached to the sediment particles, and diminish the net oil biodegradation rate 

that can be achieved. The Maine field study demonstrated that washout rate of nutrients from the 

bioremediation zone will be strongly affected by the wave activity of the contaminated beach. 

However, washout due to tidal activity alone is relatively slow, and nutrients will probably 

remain in contact with oiled beach material long enough to effectively stimulate oil 

biodegradation on low-energy beaches (Suidan and Wrenn, 2001; see Section 2.6.2). 

However, many of the same characteristics that make low-energy beaches favorable for 

bioremediation cleanup from a nutrient persistence perspective might make other conditions 

unfavorable with respect to other important factors. For example, availability of oxygen is more 

90
favorable on high-energy beaches than on low-energy beaches. Aeration mechanisms for nearsurface coastal sediments involve exchange of oxygenated surface water with oxygen-depleted 

pore water by wave-induced pumping and tidal pumping. For low energy beaches, tidal pumping 

is the only likely aeration mechanism, and as a result, the surface sediments are more likely to be 

anoxic than are similar depths on high-energy beaches (Brown and McLachlan, 1990). The 

probability of moisture (or water activity) limitation is also higher on low-energy beaches, 

because wave runup provides water to supratidal sediments on high-energy beaches during neap 

tides (Suidan and Wrenn, 2001). Therefore, it is essential to thoroughly characterize the factors 

that are likely to be rate limiting on each contaminated site before deciding and designing a 

bioremediation response strategy. 

Shoreline substrate 

Although successful bioremediation application and field trials have been carried out on cobble, 

medium sand, fine sand, and some salt marsh shorelines (Bragg et al., 1994; Lee and Levy, 

1991; Swannell et al., 1999a; Venosa et al., 1996), different shoreline substrates or sediment 

types will affect the feasibility and strategies of using bioremediation. In a 7-month field study, 

Lee and Levy (1991) compared the bioremediation of a waxy crude oil on a sandy beach and a 

salt marsh shoreline. Terra Nova crude oil was added at two concentrations, 3% (v/v) and 0.3% 

(v/v) to beach sand and salt marsh sediments retained in in-situ enclosures in a low energy 

environment. The results showed that at the lower oil concentrations (0.3%) within the sand 

beach, oil biodegradation proceeded rapidly in both the fertilized plot and the unfertilized 

control. The application of a bioremediation treatment provided no advantage. However, at the 

higher oil concentrations (3%) on the sandy beach, oil biodegradation rates appeared to be 

nutrient limited and were enhanced by nutrient addition. In contrast, the addition of nutrients to 

the salt marsh sediments containing the lower (0.3%) oil concentration resulted in enhanced rates 

of biodegradation. This additional need for nutrients at the lower oil concentrations is consistent 

with the notion that nutrient demands within a salt marsh environment are higher, due to the size 

of the microbial population within an organic-carbon rich environment. At the higher oil 

concentration (3%) within the salt marsh sediments, insignificant rates of oil degradation were 

reported following fertilization. The results clearly demonstrated that the success of 

bioremediation depends on the characteristics of the shoreline. 

On the sandy beach with low concentrations of oil, neither nutrient nor oxygen was a limiting 

factor. Under these conditions, nutrient enrichment appears to provide little or no benefit, and 

monitored natural attenuation can be considered as an alternative. However, at higher oil levels, 

the microbial community within the sand beach may become nutrient-limited, and 

bioremediation treatment could effectively enhance the rate of oil removal. In the salt marsh 

environment, nutrient addition was only effective at low oil concentrations. Oxygen limitation 

was more likely at higher oil concentrations due to the finer particle size and higher organic 

content of the sediment in these environments. Similar results have been obtained in the field 

study conducted in a freshwater wetland (Garcia-Blanco et al., 2001b; Venosa et al., 2002), 

which also indicated that oxygen availability was likely a major rate-limiting factor in the 

wetland environments. A field study sponsored by EPA and the Department of Fisheries and 

Oceans-Canada was recently conducted on the shoreline of Nova Scotia to further investigate the 

potential of using bioremediation in salt marshes. Guidelines for oil bioremediation in this type 

of shoreline will be available upon completion of the data analysis from this investigation. 

91
5.1.4 Other factors 

Prevalent climate 

Prevalent climate, the ambient temperature in particular, is an important consideration when 

assessing the feasibility of using bioremediation. As discussed in Chapter 2, the ambient 

temperature of an environment affects both the properties of spilled oil and the activity or 

population of microorganisms. At low temperatures, the viscosity of the oil increases, delaying 

the onset of biodegradation (Atlas, 1981), and the volatility of toxic low-molecular-weight 

hydrocarbons is reduced. Although the rates of biodegradation generally decrease with 

decreasing temperature, bioremediation has been tested and applied successfully to enhance oil 

biodegradation in cold arctic, alpine, and Antarctic environments (Margesin and Schineer, 1999). 

This is probably because psychrophilic bacteria are plentiful and generally the dominant species 

in these marine environments (Karrick, 1978). 

A more important consideration regarding the effect of climate or weather on the use of 

bioremediation perhaps is the seasonal factor. Significant seasonal differences in the size of 

hydrocarbon degrader populations have been observed. The numbers of hydrocarbon degraders 

may be much lower during winter than summer in some environments (Atlas, 1981). Oil 

biodegradation slows significantly and even ceases when the contaminated sediments are frozen. 

Therefore, oil bioremediation will be more effective during warmer seasons and probably should 

only be considered during the summer for cold environments such as arctic regions. 

Prior exposure to oil 

Prior exposure of a microbial community to hydrocarbons either from natural sources (e.g. 

chronic seeps and plant derived hydrocarbons) or as a result of pollution (e.g. spills and waste 

disposal) may affect the rate at which subsequent hydrocarbon input can be biodegraded (Leahy 

and Colwell, 1990). Those environments with a history of oil pollution or natural oil inputs have 

been found to have a much higher percentage of hydrocarbon degraders and a generally greater 

potential of hydrocarbon degradation than previously unpolluted areas (Atlas, 1981; Lee and 

Levy, 1987, Pritchard and Costa, 1991). Therefore, for oil bioremediation in environments with 

no prior oil exposure, there may be a lag and adaptation period before any significant oil 

biodegradation occurs. This usually is not a concern since bioremediation itself is a relative slow 

process and typically is used as a polishing step after conventional mechanical cleanup 

operations. In contrast, those environments with prior exposure to oil need a shorter lag period 

before initiation of biodegradation and thus will likely have a higher potential for oil 

biodegradation. Thus, this type of environment is generally considered a favorable condition for 

using bioremediation. 

5.1.5 Summary of pretreatment assessment 

In summary, the following pretreatment assessments should be conducted to determine whether 

bioremediation is a viable option in response to a spill incident: 

• 	 Determine whether the spilled oil is potentially biodegradable – Light petroleum products 

and light crude oils (API gravity > 30°) are relatively biodegradable; products rich in normal 

92
alkanes are relatively biodegradable; heavy crude oils (API gravity < 20°) and residual fuel 

oils, which are high in polar compounds (asphaltenes and resins) are less biodegradable. 

High concentrations of oil may also inhibit biodegradation. 

• 	 Determine whether the nutrient content at the impacted area is likely to be an important 

limiting factor by measuring the background nutrient concentrations within the interstitial 

water in that environment – The decision to use bioremediation by addition of nutrients 

should be based on how high the natural levels are relative to the optimal or threshold 

nutrient concentrations (e.g., > 2 mgN/L on sandy marine shorelines). It should also be 

determined if the natural nutrient concentrations present are typical of the area or sporadic. If 

sporadic, biostimulation may still be appropriate when the nutrient levels fall to limiting 

values; if chronic, biostimulation may not be necessary. 

• 	 Determine whether the shoreline characteristics are favorable for using bioremediation – 

High-energy rocky beaches and some low energy shorelines such as some wetlands are 

considered not likely to be very amenable to nutrient addition. 

• 	 Determine whether climatic or seasonal conditions are favorable for using bioremediation – 

bioremediation will be more effective during warmer seasons, particularly in cold 

environments. Prior exposure to oil will also be a favorable but not a solely determinative 

condition for selecting bioremediation. 

5.2 Selection of Nutrient Products 

After bioremediation is determined to be a potentially effective cleanup option based on the 

preliminary investigations, further assessments and planning are needed before its application. 

The first task is to select appropriate nutrient products through both screening tests and 

assessments based on characteristics of the contaminated site. 

5.2.1 Nutrient selection based on efficacy and toxicity 

To assist response personnel in the selection and use of spill bioremediation agents, it is useful to 

have some simple, standard methods for screening the performance and toxicity of 

bioremediation products as they become available (Blenkinsopp, et al., 1995; Haines et al., 1999, 

Lepo and Cripe, 1998a). One of the most comprehensive examples of such protocols is the tiered 

approach developed by EPA, in cooperation with the National Environmental Technology 

Applications Center (NETAC, 1993; Thomas et al., 1995). Conducting treatability tests using 

micro- or mesocosms is another commonly used approach. 

EPA/NETAC protocols 

The NETAC/EPA protocols consist of five progressive tiers, which increase in environmental 

cost and complexity with each tier of testing (Table 5.1). The approach begins with a Base Tier 

in which basic information on the agent’s toxicity is gathered based on a review of its 

formulation. During this tier, the presence of chemicals or biological components that are 

normally considered unacceptable (i.e. pathogens, carcinogens, or hazardous substances) would 

93
be identified. Tier I provides the basis for a preliminary evaluation of whether an agent could be 

effective and safely applied, which includes a description of how the product will be used, and 

information on previous usage. Tier II provides empirical evidence through the use of laboratory 

shake flask treatability studies to estimate a product’s effectiveness. This tier also provides 

information on the relative changes in aliphatic and aromatic oil constituent concentrations over 

time and the total hydrocarbon degrading microbial activity. Tier III proposes the use of flowthrough microcosm systems to study biodegradation effectiveness. Tier IV is the use of field 

demonstrations to predict a product’s potential effectiveness in the natural environment. Tiers III 

and IV are no longer considered viable options when evaluating a product for use in an oil spill 

due to overwhelming economic considerations. 

It is clear that field studies can provide the most convincing demonstration of the effectiveness of 

oil bioremediation because laboratory studies simply cannot simulate real world conditions such 

as spatial heterogeneity, climate change, and mass transfer limitations. Since conducting a field 

study just to determine that a product might work is unrealistic and economically burdensome, 

the practical approach in selection of nutrient products for the bioremediation of an oil spill 

would be through laboratory tests, microcosm tests in particular, as well as evaluations based on 

existing field study results in similar environmental conditions. 

Table 5.1 Bioremediation product test protocols developed by EPA and in cooperation with 

NETAC. 

Test Levels Description 

Base Tier 

Tier I 

Tier II 

Tier III 

Tier IV 

Collection and analysis of basic information on product safety 

including formulation and acceptability of its chemical or biological 

components 

Feasibility assessment concerning production capabilities, potential 

effectiveness, and safety certification, including a description of how 

the product will be used, and information on previous usage 

Efficacy and safety evaluation using shake flasks to compare the 

degradation of artificially-weathered crude oil in natural seawater with 

and without a bioremediation agent 

Efficacy and safety evaluation using microcosm systems to simulate 

various environments (e.g. open water, beaches, and marshes) 

Efficacy and safety evaluation through a field demonstration 

Nutrient selection through microcosm tests 

The laboratory treatability tests, especially well-designed microcosm tests, are most commonly 

used approaches to determine the type and level of amendments, such as the types of fertilizer 

94
and the optimal nutrient concentrations. A good example of these microcosms is illustrated in 

Figure 5.2, which has been used for various screening and treatability tests (Ahn, 1999, Du et al., 

1999, Xu et al., 2001). These microcosms have at least three advantages over the batch reactors 

that are often used in this type of study: (1) they are connected to respirometers, allowing nondestructive acquisition of kinetic data by continuously recording the oxygen consumption that 

accompanies oil biodegradation; (2) they are open systems that can simulate the nutrient washout 

that will occur in contaminated intertidal zones; and (3) they are designed to simulate tidal 

flushing by filling and draining on a 12-hour cycle, thus simulating the periodic anoxia that can 

occur due to tidal flooding. The potential for oxygen limitation in these reactors is a particularly 

important advantage over more conventional microcosms. Because nutrient concentrations do 

not limit the oil biodegradation rate when oxygen becomes sufficiently depleted, these systems 

will provide more realistic estimates of maximum biodegradation rates than well-aerated shake 

flasks will provide. Also, some fertilizers contain large amounts of readily biodegradable organic 

compounds that can accentuate dissolved oxygen depletion. Although they might be very 

effective in well-aerated microcosms, oxygen availability can limit their effectiveness in the field 

(Lee et al., 1995a). 

Connected to 

Respirometer 

Glass 

Beads 

Oil 

Contaminated 

Sands A 

B 

Timer 

Mineral Salts 

Solution 

Pump

Sampling

Port 

Figure 5.2 Schematic diagram of a beach microcosm for laboratory treatability testing of oil 

spill bioremediation treatment. 

95
Using this microcosm system, Xu et al. (2001) investigated the effect of different nitrogen 

nutrients on the bioremediation of weathered Alaska North Slope crude oil under simulated tidal 

conditions. Three oil concentrations of 5, 20, and 80 g oil/kg dry sand were used. Two types of 

nitrogen nutrients (KNO3 and NH4Cl) were applied at a concentration of 100 mg-N/L. Oil 

biodegradation was evaluated by monitoring CO2 production, oxygen uptake, nitrogen 

consumption, as well as oil constituent analysis. Results indicated that more biomass growth 

occurred in the submerged (sometimes anoxic) portion of the sand, and better oil degradation 

was observed in microcosms to which nitrate-nitrogen was applied. This result suggested that 

nitrate might also have enhanced oil bioremediation by serving as an electron acceptor when 

oxygen was limiting. However, the role of nitrate still requires further investigation. Ramstad 

and Sveum (1995) also found that nitrate had the most pronounced effect in stimulating oil 

degradation when comparing the effect of nitrate, ammonia, and an organic nitrogen product on 

biodegradation of topped Statfjord crude oil in a continuous-flow seawater column system. 

Effect of nutrient type may also depend on the properties of shoreline substrates. Jackson and 

Pardue (1999) found that addition of ammonia as compared to nitrate appeared to more 

effectively simulate degradation of crude oil in salt marsh soils in a microcosm study. The 

ammonia requirement was only 20% of the concentration of nitrate to achieve the same increase 

of degradation. The authors concluded that ammonia was less likely to be lost from the 

microcosms by washout due to its higher adsorptive capacity to sediment organic matter. 

However, in a microcosm study using sandy sediments, Suidan and Wrenn (2000) found that 

there were no significant differences in the nutrient washout rates or the abilities of ammonium 

and nitrate to support oil biodegradation. These results suggest that although cation-exchange 

adsorption may be an important difference between ammonium and nitrate in sediments with 

high cation-exchange capacities (CECs), such as marsh sediments, it is unlikely to be significant 

in sediments with low CECs, such as sand. 

Toxicity and other environmental impacts 

In addition to demonstrating the efficacy of nutrient products in enhancing oil degradation, it is 

also necessary to demonstrate that bioremediation products have low toxicity and do not produce 

any undesired environmental and ecological effects. Various toxicity test protocols have been 

developed (NETAC, 1993; Lepo and Cripe, 1998a, See Section, 3.5). For example, the EPA 

Tier II safety evaluation consists of 7-day toxicity tests with the bioremediation product (without 

oil) in natural seawater using a crustacean (Mysidopsis bahia, mysid), and a fish (Menidia 

beryllina, the inland silverside). Additional Tier II toxicity tests evaluate the potential for 

interaction between the product and the water-soluble-fraction of a weathered crude oil. Indirect 

effects of nutrient products should also be evaluated, which include oxygen depletion through 

increase in organic carbon or eutrophication, and enhanced production of toxic oil degradation 

metabolites (Lepo and Cripe, 1998b). 

So far, no detrimental effects from bioremediation by nutrient enrichment have been observed 

following actual field operations (Prince, 1993; Mearns et al., 1997). However, the possibility 

that harmful effects might occur remains. For example, oxygen depletion and production of 

ammonia from excessive applications of a fish-bone meal fertilizer during a field study caused 

detrimental effects, including a slowing in oil degradation rates and toxicity reduction rates 

96
measured by Microtox Solid-Phase Test (Lee et al., 1995b). For safety reasons, proper 

ecotoxicological assessment is always necessary in selecting nutrient products. 

5.2.2 Environmental factors affecting nutrient selection 

Nutrient selection also depends on environmental factors such as temperature, shoreline energy, 

and substrate. A field study conducted by Lee et al. (1993) indicated that the effectiveness of 

specific nutrient formulations might be influenced by temperature conditions. The study 

investigated the efficacy of water-soluble inorganic fertilizers (ammonium nitrate and triple 

superphosphate) and a slow release fertilizer (sulfur-coated urea) to enhance the biodegradation 

of a waxy crude oil in a low energy shoreline environment. The results showed that at temperate 

conditions above 15°C, the slow-release fertilizer appeared to be more effective in retaining 

elevated nutrient concentrations within the sediments and more effective in enhancing oil 

degradation than water-soluble fertilizers. However, lower temperatures were found to reduce 

the permeability of the coating on the slow-release fertilizer and suppressed nutrient release 

rates. Water-soluble fertilizers such as ammonium nitrate were then recommended under these 

temperature conditions. 

The action of wave, tide, and sediment type will also affect the selection of nutrients. Some 

studies suggested that oleophilic fertilizers might be more suitable for use in high-energy and 

coarse-grained beaches (Sveum et al., 1994; Sveum and Ladousse, 1989; See Chapter 4), 

although stronger evidence is needed to confirm this suggestion. Therefore, for optimal 

effectiveness, the nutrient selection should always take into account the environmental 

conditions, the type of contaminated shoreline, and the methods of application, which will be 

discussed later in this chapter (Lee et al., 1993; Prince, 1993; Swannell et al., 1996). 

5.3 Determination of the Optimal Nutrient Loading and Application Strategy 

After the initial selection of nutrient products that meet the requirements of efficacy and safety, 

the next step is to determine the proper nutrient loading and the best nutrient application 

strategies. Major considerations in this task include the determination of optimal nutrient 

concentration, frequency of addition, and methods of addition. Finally, selection of appropriate 

nutrient products should also be conducted in conjunction with this process. 

5.3.1 Concentration of nutrients needed for optimal biostimulation 

Since oil biodegradation largely takes place at the interface between oil and water, the 

effectiveness of biostimulation depends on the nutrient concentration in the interstitial pore water 

of oily sediments (Bragg et al., 1994; Venosa et al., 1996). The nutrient concentration should be 

maintained at a high enough level to support maximum oil biodegradation based on the kinetics 

of nutrient consumption. Higher concentrations will not only provide no added benefit but also 

may lead to potentially detrimental ecological and toxicological impacts. 

Studies on optimal nutrient concentrations have been conducted both in the laboratory and in the 

field. Boufadel et al., (1999a) investigated the optimal nitrate concentration for alkane 

biodegradation in continuous flow beach microcosms (Figure 5.2) using heptadecane as a model 

97
alkane immobilized onto sand particles at a loading of 2g heptadecane/kg sand. They determined 

that a continuous supply of approximately 2.5 mg N/L supported maximum heptadecane 

biodegradation rates. Du et al. (1999) also investigated the optimal nitrogen concentration for oil 

biodegradation using weathered Alaska North Slope crude oil in the same microcosms with an 

oil loading of 5g/kg sand. The results showed that nitrate concentrations below approximately 10 

mg N/L limited the rate of oil biodegradation. The higher nutrient requirement was attributed to 

the more complex substrate (crude oil) compared to the pure heptadecane of Boufadel et al. 

(1999a). The more complex substrate (crude oil) of Du et al. (1999) also likely selected a 

different population of degraders than those that grew on the pure heptadecane (Boufadel et al., 

1999a), which might have contributed to the different growth rate characteristics observed. 

Ahn (1999) further studied the effect of nitrate concentrations under tidal flow conditions instead 

of continuous flow. He used the same beach microcosms as Du et al. (1999) filled with sand 

loaded with weathered Alaska North Slope crude oil at 5g/kg sand. A nutrient solution with 

nitrate concentrations ranging from 6.25 to 400 mg N/L was supplied semi-diurnally to simulate 

tidal flow. The results indicated that the optimum nitrate concentration for maximum oil 

biodegradation rate was over 25 mg N/L. Some laboratory studies have reported that greater than 

100 mg N/L was required to stimulate maximum biodegradation rates (Atlas and Bartha, 1992; 

Reisfeld et al., 1972), but this observation probably reflects a stoichiometric rather than a kinetic 

requirement, since these experiments were conducted in closed batch reactors. 

Compared to the results from laboratory studies, nutrient concentrations that supported high oil 

biodegradation rates were found to be lower in field studies. For example, the field tests that 

were conducted after the Exxon Valdez oil spill in Prince William Sound, Alaska showed that the 

rate of oil biodegradation was accelerated by average interstitial nitrogen concentrations of about 

1.5 mg N/L (Bragg et al., 1994). A similar result was obtained from the study conducted in the 

Bay of Brest, France, in which nitrogen was not a limiting factor when the interstitial pore water 

concentrations exceeded 1.4 mg N/L (Oudet et al., 1998). The Delaware field trial also showed 

that the background nitrate concentration (0.8 mg N/L) was sufficient to support fairly rapid 

natural (but not maximal) rates of alkane and PAH biodegradation (Venosa et al., 1996). 

Increasing the average nitrate concentration in the bioremediation zone of the experimental plots 

to between 3 and 6 mg N/L resulted in a moderate increase in the oil biodegradation rate. 

Observations from the referenced field studies suggest that concentrations of approximately 1 to 

2 mg/L of available nitrogen in the interstitial pore water is sufficient to meet the minimum 

nutrient requirement of the oil degrading microorganisms for the approximately 6-hour exposure 

time to the contaminated substrate during a tidal cycle. However, laboratory microcosm results 

as well as the Delaware field study suggest that higher concentrations of nitrogen can lead to 

accelerated hydrocarbon biodegradation rates. Since the minimum nitrogen concentration needed 

to satisfy the nitrogen demand in a tidal cycle is 1 to 2 mg N/L, and since concentrations of 

nitrogen in pore water that lead closer to maximum rates of biodegradation can be several-fold to 

as much as an order of magnitude higher, it is recommended that biostimulation of oil impacted 

beaches should occur when nitrogen concentrations of at least 2 to as much as to 5-10 mg N/L 

are maintained in the pore water with the decision on higher concentrations to be based on a 

broader analysis of cost, environmental impact, and practicality. In practice, a safety factor 

should be used to achieve target concentrations, which will depend on anticipated nutrient 

98
washout rates, selected nutrient types, and application methods. For example, in the Delaware 

study, since nitrate in the interstitial pore water was quickly diluted to background levels 

whenever the incoming tide completely submerged the plots, water-soluble nutrients were 

applied every day using a sprinkler system. A 100-fold safety factor to account for dilution was 

used to achieve the 3-6 mg/L average interstitial pore water concentrations experienced at 

Delaware. A lower safety factor may be needed when using slow release nutrients. 

5.3.2 Nutrient application strategies 

Once the optimal nutrient concentrations have been determined, the next task is to design 

nutrient application strategies, which include nutrient application frequency and delivery 

methods. 

Frequency of nutrient addition 

The frequency of nutrient addition to maintain the optimal concentration in the interstitial pore 

water mainly depends on shoreline types or nutrient washout rates. On marine shorelines, 

contamination of coastal areas by oil from offshore spills usually occurs in the intertidal zone 

where the washout of dissolved nutrients can be extremely rapid. Oleophilic and slow-release 

formulations have been developed to maintain nutrients in contact with the oil, but most of these 

rely on dissolution of the nutrients into the aqueous phase before they can be used by 

hydrocarbon degraders (Safferman, 1991). Therefore, understanding the transport of dissolved 

compounds in intertidal environments is critical in designing nutrient addition strategies, no 

matter what type of fertilizer is used. 

The Maine field study on nutrient hydrodynamics (See Section 2.6) has demonstrated that during 

spring tide, nutrients can be completely removed from a high-energy beach within a single tidal 

cycle. But it may take more than two weeks to achieve the same degree of washout from a lowenergy beach. Washout during the neap tide can be much slower because the bioremediation 

zone will be only partially covered by water in this period. Since nutrients may be completely 

washed out from high-energy beaches within a few days, and remain in low energy beaches for 

several weeks, the optimal frequency of nutrient application should be based on observations of 

the prevalent tidal and wave conditions in the bioremediation zone. For example, a daily nutrient 

application may be needed for a high-energy beach during spring tide. But weekly or monthly 

additions may be sufficient for low-energy beaches when the nutrients are applied during neap 

tide. Nutrient sampling, particularly in beach pore water, must also be coordinated with nutrient 

application to ensure that the nutrients become distributed throughout the contaminated area and 

that target concentrations are being achieved. The frequency of nutrient addition should be 

adjusted based on the nutrient monitoring results. 

Methods of nutrient addition 

Nutrient application methods should be determined based on the characteristics of the 

contaminated environment, physical nature of the selected nutrients, and the cost of the 

application. 

Shoreline energy and geometry are important factors in determination of nutrient application 

methods. The study in Maine suggested that surface application of nutrients may be ineffective 

99
on high-energy beaches because wave action in the upper intertidal zone may cause nutrients 

from the surface layers of the beach to be diluted directly into the water column, resulting in their 

immediate loss from the bioremediation zone. Daily application of water-soluble nutrients onto 

the beach surface at low tide could be a feasible approach (Venosa et al., 1996), although this 

method is highly labor-intensive. Nutrients that are released from slow-release or oleophilic 

formulations will probably behave similarly to water-soluble nutrients with respect to nutrient 

washout. Formulations with good long-term release characteristics probably will never achieve 

optimal nutrient concentrations in environments with high washout rates. Therefore, they will not 

be effective on high-energy beaches unless the release rate is designed to be high enough to 

achieve adequate nutrient concentrations while the tide is out. 

Another potentially effective strategy is the subsurface application of nutrients onto high-energy 

beaches. Wise et al. (1994) found that application of nutrients through a trench or subsurface 

drain placed above the high-tide level, rather than directly on the beach by sprinklers, would 

result in significantly longer retention times. However, since nutrients move downward and 

seaward during transport through the intertidal zone of sandy beaches, nutrient application 

strategies that rely on subsurface introduction must provide some mechanism for insuring that 

the nutrients reach the oil-contaminated area near the surface. The approach that was proposed 

by Wise et al. (1994) assumes that nutrients dissolved in the freshwater plume will be brought 

into contact with the oiled beach material periodically by the rising tide because the freshwater 

plume should float on top of the saltwater. However, the finding of freshwater trapped between 

two saltwater wedges by Boufadel et al. (1999) indicates that subsurface injection of nutrients 

above the high tide level would likely not be an effective method for providing nutrients to the 

bioremediation zone. 

Compared to high-energy shorelines, application of nutrients on low-energy beaches is much less 

problematic. Since washout due to tidal activity alone is relatively slow (Suidan and Wrenn, 

2001), surface application of nutrients is an effective and economical bioremediation strategy on 

low-energy beaches. 

The physical forms of fertilizers are also critical in determination of appropriate nutrient 

application methods. Generally, available fertilizers can be classified into four types in terms of 

their physical forms: (1) slow release fertilizer briquettes, (2) dry granular fertilizers, (3) liquid 

oleophilic fertilizers, and (4) water-soluble inorganic nutrient solutions (Glaser et al., 1991; 

Swannell et al., 1996). The application of the briquette forms is problematic in regards to 

buoyancy of the briquettes and redistribution by tide and wave action (Glaser et al., 1991). The 

method used during the Exxon Valdez spill involved packing the briquettes in mesh bags tethered 

to steel bars driven into the beach subsurface. The poor distribution problem occurs by 

channeling of nutrients vertically down the beach rather than lateral spreading. 

Dry granular fertilizers can be slow-release (e.g., Customblen in Alaska) or water-soluble, solid 

granules (e.g., prilled ammonium nitrate). Granular fertilizers are easier and more flexible to 

apply using commercially available whirlybird-type hand spreaders. Although this type of 

fertilizer is also subject to washout by wave and tidal action, dry granular fertilizers are probably 

the most cost-effective way to control nutrient concentrations. Liquid oleophilic nutrients, such 

as Inipol EAP 22, are also relatively easy to apply by using hand-held or backpack sprayers. One 

100
of the problems when using Inipol EAP 22 during the Exxon Valdez spill cleanup was its low 

pour point (11°C), which led to clogging of spraying nozzles and poor uniformity of application 

(Glaser et al., 1991). The problem was later resolved by warming the product. This type of 

fertilizer is significantly more expensive than granular fertilizers. Water-soluble nutrient 

solutions are normally delivered to the beach by a sprinkler system after dissolving nutrient salts 

in a local water source. Although this type of nutrient may be easier to manipulate to maintain 

target concentrations in interstitial pore water, its application may require more complicated 

equipment such as large mixing tanks, pumps, and sprinklers. 

Based on current experiences and understandings, application of dry granular fertilizer to the 

impact zone at low tide is probably the most cost-effective way to control nutrient 

concentrations. 

5.4 Sampling and Monitoring Plan 

To properly evaluate the progress of bioremediation, a comprehensive and statistically valid 

sampling and monitoring plan should be developed before the application of bioremediation. The 

sampling and monitoring plan should include important efficiency and toxicity variables, 

environmental conditions, and sampling strategies. 

5.4.1 Important variables 

Important variables to be monitored in an oil bioremediation project include limiting factors for 

oil biodegradation (e.g., interstitial nutrient and oxygen concentrations), evidence of oil 

biodegradation (e.g., concentrations of oil and its components), microbial activity (e.g., bacterial 

numbers), environmental effects (e.g., ecotoxicity levels) and other environmental conditions 

(e.g., temperature and pH). A comprehensive monitoring plan proposed for a bioremediation 

field study is listed in Table 5.2. A monitoring plan for a full-scale bioremediation application 

should be similar to this and at least should include those critical measurements. 

Since oil biodegradation in the field is usually limited by availability of nutrients, nutrient 

analysis, particularly the nutrient concentrations in the pore water, is one of the most important 

measurements in developing proper nutrient addition strategies and assessing the effect of oil 

bioremediation. The frequency of nutrient sampling must be coordinated with nutrient 

application, making certain that the treatment is reaching and penetrating the impact zone, target 

concentrations of nutrients are being achieved, and toxic nutrient levels are not being reached. 

Otherwise, nutrient application strategies should be adjusted accordingly. The location from 

which nutrient samples are collected is also important. Recent research on solute transport in the 

intertidal zone has shown that nutrients can remain in the beach subsurface for much longer time 

periods than in the bioremediation zone (Wrenn et al., 1997a). Nutrient concentration profiles 

along the depth of the oil-contaminated region can be monitored by using multi-port sample 

wells or sand samples collected from the oil-contaminated region. 

The sampling depth should be determined based on the preliminary survey of oil distribution. It 

can be established by determining the maximum depth of oil penetration, then adding a safety 

factor, which will be chosen based on the observed variation in oiled depth, to ensure that the 

101
samples will encompass the entire oiled depth throughout the project. The safety factor will be 

modified if observations during the bioremediation application suggest that the depth of oil 

penetration has changed. 

The success of oil bioremediation will be judged by its ability to reduce the concentration and 

environmental impact of oil in the field. As discussed in Chapter 3, to effectively monitor 

biodegradation under highly heterogeneous conditions, it is necessary that concentrations of 

specific analytes (i.e., target alkanes and PAHs) within the oil be measured using 

chromatographic techniques (e.g., GC/MS) and are reported relative to a conservative biomarker 

such as hopane. On the other hand, from an operational perspective, more rapid and less costly 

analytical procedures are also needed to satisfy regulators and responders on a more real time, 

continual basis. Existing TPH technologies are generally not reliable and have little biological 

significance (See Chapter 3). TLC-FID seems to be a promising screening tool for monitoring oil 

biodegradation (Stephens et al., 1999). 

Table 5.2 Monitoring plan for a bioremediation field study 

Analysis Matrix Recommended Methods

*dissolved nitrogen Sediment (interstitial 

pore water) 

extract in acidified 0.1% NaCl 

4500-Norg D (persulfate oxidation to NO3 

-

) 

4500-NO3 

- F (automated Cd-reduction) 

dissolved phosphorus Sediment (interstitial 

pore water) 

extract in acidified 0.1% NaCl 

4500-P B.5 (persulfate oxidation) 

4500-P E (ascorbic acid method) 

*residual oil Sediment extract into methylene chloride 

analyze components by GC/MS-SIM 

dissolved oxygen Aqueous Hach® high range assay 

pore-water pH Aqueous potentiometric with combination electrode 

microbial populations Sediment MPN for alkane and PAH degraders 

metabolic activity Sediment CO2 production from sand slurries 

*toxicity of residual oil Sediment Microtox® Solid-Phase Test 

toxicity of residual oil Sediment 10-day amphipod survival bioassay 

toxicity of pore-water Aqueous Microtox® Acute Test 

toxicity of pore-water Aqueous sea-urchin fertilization bioassay 

bioaccumulation potential SPMDs** 2-week adsorption into SPMDs 

beach profile surveying using fixed markers (e.g., wells, 

plot boundary markers) in intertidal zone 

beach profile surveying relative to fixed benchmarks in 

the supratidal zone 

* Critical measurements 

** Semi-permeable membrane devices 

102 
In addition to monitoring the treatment efficacy for oil degradation, the bioremediation 

monitoring plan should also incorporate reliable ecotoxicological endpoints to document 

treatment effectiveness for toxicity reduction. Commonly used ecotoxicity monitoring 

techniques, such as the Microtox® assay and an invertebrate survival bioassay, have been 

summarized in chapter 3. These microscale bioassays may provide an operational endpoint 

indicator for bioremediation activities on the basis of toxicity reduction (Lee et al., 1995b). 

Considerations for selecting an appropriate bioremediation endpoint based on both oil 

degradation and toxicity reduction will be presented in detail in the next chapter. 

Other important variables in a comprehensive monitoring plan include site background 

conditions (e.g., oxygen, pH and temperature) and shoreline profiles. Oxygen availability is 

crucial for rapid bioremediation, because hydrocarbon biodegradation is primarily an aerobic 

process. Therefore, dissolved oxygen (DO) in the pore water should be monitored on a regular 

basis. The frequency of DO sampling should also be coordinated with nutrient application, 

particularly when organic nutrients are used (Lee et al., 1995b; Sveum and Ramstad, 1995; See 

Section 4.1.3), to insure that anoxic conditions do not result. When oxygen does become limited, 

the nutrient dosage and application frequency should be adjusted accordingly. Alternatively, 

oxygen amendment strategies, such as tilling or addition of chemical oxygen sources, may be 

considered, although these approaches are likely to be expensive and potentially environmentally 

hazardous (see Chapter 4). 

Measurement of pH in the pore water is also important in monitoring oil bioremediation. 

Biodegradation of oil in marine environments is optimal at a pH of about 8 (Atlas and Bartha, 

1992). The pH of seawater is usually around 8.5, which is adequate to support rapid oil 

biodegradation. 

5.4.2 Statistical considerations in the sampling plan 

To ensure that monitored results reflect the reality in a highly heterogeneous environment, it is 

important that a bioremediation sampling plan be designed according to valid statistical 

principles such as randomization, replication, and proper control. 

To minimize bias, a random sampling plan should be used to evaluate treatment effects and their 

variance within the bioremediation zone. For samples with a high degree of spatial 

heterogeneity, which will be the case for most oil spill sites, stratified sampling strategies should 

be used. For example, the sampling field on a marine shoreline can be divided into a number of 

sectors or quadrants based on the homogeneity of geomorphology within each sector (e.g., upper 

and lower intertidal zones), and independent samples should be taken in each sector according to 

the rule of proportionality (e.g., taking more samples in more heavily oiled sites). 

Although economic factors may be restrictive, efforts should be made to ensure that an adequate 

number of samples are taken to reach a given accuracy and confidence. Power analysis should be 

used to assist in the determination of sample replications required in a monitoring plan. A 

statistical power test was performed in the Delaware field study to determine the number of 

replicates that would be needed in future studies to detect significant treatment differences under 

103
similar conditions (Venosa et al., 1996). The study indicated that the required replicates to detect 

treatment effects depend on expected variance and expected treatment differences. For example, 

if oil distribution and shoreline characteristics are highly heterogeneous, variance will be high, 

thus requiring more replicates to detect significant treatment effects. If background nutrients are 

high, treatment differences will be low, and more replicates will also be required. By comparing 

three shoreline assessment designs used for the Exxon Valdez oil spill, Gilfillan et al. (1999) also 

proposed several strategies to increase power (i.e., the probability that significant differences 

between two or more treatments are detected when indeed they exist). One of the approaches to 

increase power is to select sampling sites from only the most heavily oiled locations. This 

strategy may not be feasible for assessing the oil degradation within the whole bioremediation 

zone, although it may be useful for evaluating the effect of bioremediation on ecological 

recovery since the ecological injury most likely occurs at the heavily oiled locations. 

A control area normally refers to a set-aside untreated site, which has similar physical and 

biological conditions as the treated site. Although on-scene coordinators prefer not to leave oiled 

sites uncleaned, it is difficult to assess the true impact of a treatment without control or set-aside 

areas (Hoff and Shigenaka, 1999). When selecting control areas, one must consider not only the 

similarity of the conditions but also the effect of sand and nutrient exchanges between the treated 

and untreated areas (See Section 6.1.2). 

5.5 Considerations for Freshwater Wetland Bioremediation 

Guidelines proposed in earlier sections are mostly derived from studies and practices on marine 

shorelines. However, freshwater conditions or habitats may differ sufficiently from marine 

situations so that a simple transfer of response strategies may not be necessarily the most 

appropriate. Special considerations for oil bioremediation in freshwater wetlands are summarized 

here based on current understandings, particularly the findings of the St. Lawrence River field 

study (Garcia-Blanco et al., 2001b; Venosa et al., 2002; Lee et al., 2001a). 

5.5.1 Characteristics of freshwater wetlands 

Wetlands occupy the interface between terrestrial and aquatic systems. They have been defined 

by EPA and the U.S. Army Corps of Engineers as: “Those areas that are inundated or saturated 

by surface or groundwater at a frequency or duration to support, and that under normal 

conditions do support, a prevalence of vegetation typically adapted for life in saturated soil 

conditions. Wetlands generally include swamps, marshes, bogs, and similar areas.” (EPA, 40 

CFR 230.3 and Corps, 33 CFR 328.3). Wetlands normally should have the following three 

characteristics or diagnostic parameters: (1) at least periodically, the land supports predominantly 

hydrophytes (i.e., plants adapted to the flooded conditions), (2) the substrate is predominantly 

undrained hydric soil (i.e., a soil with unique physical and chemical characteristics, such as 

highly reduced conditions, due to repeated and prolonged saturation), and (3) the substrate is 

saturated with water or covered by shallow water for a significant part of the growing season 

each year (Greene, 2000; Mitsch and Gosselink, 1993). 

Wetland ecosystems have enormous ecological and environmental value, contributing to aquifer 

recharge, water quality improvement, flood mitigation, and shoreline erosion protection. They 

104
also provide unique and extensive habitats for a wide variety of flora and fauna. Furthermore, 

wetlands play an important role in the global cycles of nitrogen, sulfur, methane, and carbon 

dioxide (Mitsch and Gosselink, 1993). 

The lower 48 states contained an estimated 100 million acres (400,000 square kilometers) of 

wetlands in the mid-1980s, an area about the size of California, among which freshwater 

wetlands are estimated to make up 80 to 95 percent (Mitsch and Gosselink, 1993, Greene, 2000). 

Freshwater wetlands also cover extended areas of Alaska and Canada. Their proximity to areas 

of human activity makes them susceptible to contamination by petroleum hydrocarbons, via 

leakage, runoff or spill. Necessary measures need to be taken to protect these ecosystems since 

they are among the environments most sensitive to oil and clean-up activities (Hayes et al., 1995, 

1997). Furthermore, there are reports that the application of traditional oil spill cleanup 

techniques in wetland habitats caused more damage than the oil itself (Baker et al., 1993). When 

looking for both inexpensive and environmentally friendly technologies for wetland cleanup and 

restoration, bioremediation and phytoremediation have potential for being the most suitable 

options (Atlas, 1995; Cunningham et al, 1996). 

The limiting conditions for oil biodegradation in freshwater wetlands may be significantly 

different from most marine environments. In terms of nutrient supply, freshwater wetlands can 

be divided into eutrophic wetlands (e.g., tidal marsh) and oligotrophic wetlands (e.g., cypress 

domes (Mitsch and Gosselink, 1993). Many freshwater wetlands also exhibit a seasonal pattern 

of uptake and release of nutrients. During the growing season (i.e., late spring and summer), 

there is a high rate of nutrient uptake by vegetation from both the water and sediments. And 

when higher plants die in the fall, a substantial portion of nutrients will be released to the water 

and sediments. The amount of inorganic nutrients or nutrients available for oil biodegradation 

also depends on many other processes, such as nutrient mineralization, denitrification, anaerobic 

release of phosphorus, and wetland hydrodynamics (Mitsch and Gosselink, 2000). 

When wetland soils are inundated with water, oxygen diffusion rates through the soil are 

drastically reduced. Available oxygen in the soil and in the interstitial water is quickly depleted 

through metabolism by aerobic organisms. Below a few centimeters—and sometimes only a few 

millimeters—of the soil surface, the environment becomes anaerobic (Gambrell and Patrick, 

1978). When the metabolic demand for oxygen by soil organisms exceeds that of supply, the 

redox potential in the soil drops and other ions (nitrate, manganese, iron, sulfate, and carbon 

dioxide) are used as electron acceptors (Mitsch and Gosselink, 1993). Therefore, in freshwater 

wetland environments, petroleum biodegradation is likely to be limited by oxygen availability. 

Because wetland sediments are generally more fine-grained and, more importantly, saturated 

with water, the extent of oil penetration is expected to be much lower in freshwater wetlands 

than on a porous sandy marine beach. In a microcosm study, Purandare (1999) investigated the 

penetration of weathered light Arabian crude oil in freshwater wetland sediment under two 

different water levels. For the case of low water level, where the sediment was saturated but not 

covered with water, the oil was found to penetrate only about 2.5 cm in 16 weeks. For the case of 

high water level, where the water level was 10 cm above the sediment surface, most of the oil 

was floating on the surface of the water and the penetration depth in the sediment for some 

settled and dissolved oil was also about 2.5cm during this study. The depth of oil penetration in 

105
the case of the St. Lawrence River study was higher (about 9 cm), due to the initial raking of the 

wetland sediments after they were oiled. This depth is still much lower than the depth of oil 

penetration that occurs in marine sandy beaches (up to 30 cm, Gundlach, 1987) and cobble 

beaches (up to 1 m, Wolfe et al., 1994). 

Another important feature of wetlands is that at least periodically, the land supports 

predominantly hydrophytes, or plants “growing in water, soil, or on a substrate that is at least 

periodically deficient of oxygen as a result of excessive water content.” (Greene, 2000). These 

wetland plants may play important roles in oil bioremediation and wetland restoration. On the 

one hand, they may be involved in degradation, containment, and transfer of petroleum 

hydrocarbons from the soil to the atmosphere (Frick, et al, 1999). On the other hand, these 

wetland plants may also compete with hydrocarbon-degrading microorganisms for nutrients. 

5.5.2. Bioremediation strategies in freshwater wetlands 

Although the same decision-making and planning principles that were described earlier in this 

chapter for bioremediation of marine shorelines should also apply to freshwater wetland 

environments, the feasible bioremediation strategies are likely to be different due to the distinct 

characteristics of wetlands. The potential effectiveness of different amendments is discussed as 

follows mainly based on the findings of St. Lawrence River field study (Garcia-Blanco et al., 

2001b; Venosa et al. 2002; Lee et al., 2001a). 

Nutrient amendment 

Since nutrients could be limited in wetland sediments during the growing season in particular, 

addition of nutrients would seem to have some potential for enhancing oil biodegradation in such 

an environment. However, the results from the St. Lawrence River field study (See Section 2.5) 

showed that no significant enhancement was observed in terms of the oil biodegradation 

following biostimulation through addition of nutrients (either ammonium or nitrate). After 21 

weeks, reduction of target parent and alkyl-substituted PAHs averaged 32% in all treatments. 

Reduction of target alkanes was of similar magnitude. The removal of PAHs in nutrient-amended 

plots was only slightly better than natural attenuation after 64 weeks of treatment. Oil analysis 

from the top 2 cm sediment samples showed that the plots amended with ammonium nitrate and 

with Scirpus pungens plants cut back demonstrated a significant enhancement in target 

hydrocarbon reduction over natural attenuation as well as all other treatments. This suggests that 

biostimulation may be effective only in the top layer of the soil, where aerobic conditions are 

greater, and when hydrocarbon-degrading microorganisms do not have to compete for nutrients 

with the growing wetland plants. 

Another potential problem with respect to the use of biostimulation in wetlands is that some 

plant communities may be sensitive to nutrient additions. Repeated and excessive nutrient 

additions may alter the nature of the wetland ecosystem as indicated by the effects of chronic 

nutrient additions to the Everglades in Florida (Davis, 1994). 

Oxygen amendment 

Since oxygen has been found the most likely limiting factor in oil biodegradation in freshwater 

environments, oxygen amendment may be considered. However, an appropriate technology for 

106
increasing the oxygen concentration in such environments, other than reliance on the wetland 

plants themselves to pump oxygen down to the rhizosphere through the root system, has yet to be 

developed. Existing oxygen amendment technologies developed in terrestrial environments, such 

as tilling, forced aeration, and chemical methods (See Section 4.4), are not likely to be costeffective for bioremediation of freshwater wetlands since they often involve expensive and 

environmentally intrusive practices. 

During the St. Lawrence River field trial (Garcia-Blanco et al., 2001b; Venosa et al., 2002), after 

the first nutrient and oil applications, the top 1-2 cm surface soil in all plots was manually raked 

using cast iron rakes. This was done to minimize loss of oil from the plots due to tidal action and 

to uniformly incorporate the nutrients and the oil into the soil. However, the oil analysis results 

suggested that the tilling of surface soil might have slowed the overall oil biodegradation rates by 

enhancing oil penetration deep into the anaerobic sediments. Based on these observations, 

surface tilling will not be an effective strategy for increasing the oxygen concentration in 

freshwater wetlands (although this was not the intent of the raking). The slightly better but 

statistically insignificant performance in both alkane and PAH degradation with addition of 

nitrate compared to ammonium after 64 weeks of treatment implied that nitrate may have served 

as an alternative electron acceptor in enhancing oil biodegradation when oxygen was limiting. 

However, the limited increase in biodegradation rate over natural attenuation would not warrant 

a recommendation to use nitrate as an oxygen amendment agent in such an environment. 

Phytoremediation 

Since plants cover wetlands at least periodically, the use of phytoremediation becomes a natural 

option for wetland cleanup and restoration. Phytoremediation is emerging as a potentially viable 

technology for cleanup of soils contaminated with petroleum hydrocarbons (Frick et al., 1999 

See Section 4.3). However, this technique has not been used as a wetland oil spill 

countermeasure. Only limited studies have been carried out on the effectiveness of 

phytoremediation in enhancing oil degradation in marine shorelines and freshwater wetlands. Lin 

and Mendelssohn (1998) found in a greenhouse study that application of fertilizers in 

conjunction with the presence of transplants (S. alterniflora and S. patens) significantly enhanced 

oil degradation in a coastal wetland environment. In the case of freshwater wetlands, the St. 

Lawrence River study suggested that although application of fertilizers in conjunction with the 

presence of a wetland plant (Scirpus pungens) may not significantly enhance oil degradation, it 

could enhance habitat recovery through the stimulation of vigorous vegetative growth and 

reduction of sediment toxicity and oil bioavailability (Lee et al., 2001a). The effectiveness of oil 

phytoremediation in freshwater wetland environments still requires further study. 

Natural attenuation 

Natural attenuation has been defined as the reliance on natural processes to achieve site-specific 

remedial objectives (USEPA, 1999b). This approach has been increasingly recognized as a 

possible viable option for oil spill cleanup with more understanding gained over the past decade 

about the advantages and disadvantages of active treatment versus natural attenuation (Owens et 

al., 1999). As indicated by Sell et al. (1995), the decision-making should focus more on a 

preference for natural attenuation except when a large amount of viscous oil is present, where 

natural removal will be slow, or when non-ecological factors are of greater importance. 

107
The St. Lawrence River Study demonstrated that the availability of oxygen, not nutrients, is 

likely to be the limiting factor for oil biodegradation in freshwater wetlands. However, no 

feasible technique is currently available for increasing oxygen concentration under such an 

environment. As a result of this study, natural attenuation is recommended as the most costeffective strategy for oil spill cleanup in freshwater wetlands when the oil concentration is not 

high enough (e.g., less than 30 g/kg soil; Longpre et al., 1999) to destroy wetland vegetation. 

108
Chapter 6 GUIDELINES FOR ASSESSMENT OF FIELD RESULTS AND 

TERMINATION OF TREATMENT 

After the treatment is implemented according to the bioremediation plan, the next or final step in 

an oil bioremediation project is to assess the treatment efficacy and terminate the bioremediation 

action at appropriate treatment endpoints. Key questions to be answered in this task include 

“what are the measurements of oil bioremediation success?” and “how clean is clean?” or “when 

should bioremediation efforts be terminated?”. Actually, these issues should be dealt with during 

the bioremediation planning stage, when proper treatment objectives, strategies, and sampling 

protocols should be established. On the other hand, more definite answers to these questions can 

only be reached during the bioremediation actions and based on the findings of comprehensive 

monitoring programs. Cost-benefit analysis (e.g., net environmental benefit analysis, Baker, 

1995&1999) and sometimes political considerations should also be taken into account in this 

process, however, which are beyond the scope of this document. From a technological point of 

view, the measurements of bioremediation success and establishment of operational endpoints 

should be based on both the efficacy of oil biodegradation and the evidence of ecotoxicity 

reduction and ecological recovery, each of which will be discussed in this chapter. 

6.1 Assessment of Oil Biodegradation Efficacy 

6.1.1 Verification of oil biodegradation 

Evidence for the effectiveness of oil bioremediation in terms of oil biodegradation should 

include: (1) faster disappearance of oil in treated areas than in untreated areas, and (2) a 

demonstration that biodegradation was the main reason for the increased rate of oil 

disappearance. As described earlier in this document, assessing the effectiveness of oil 

bioremediation in oil spill sites is difficult due to the heterogeneous conditions of contaminated 

sites and lack of control over the oil distribution. Nevertheless, the success of bioremediation can 

be verified through well-designed monitoring programs and proper data interpretation. 

Distinguishing biodegradation from abiotic loss 

Oil constituents can be lost from a shoreline by physical washout, dissolution, volatilization, and 

biodegradation. To demonstrate the effectiveness of a bioremediation treatment, biodegradation 

should be identified as the main mechanism for the increased rate of oil disappearance. As 

described in Chapter 3, to effectively distinguish biodegradation from abiotic loss, specific oil 

components or analytes should be analyzed using GC/MS techniques and then these analytes 

should be normalized to a conserved biomarker, such as hopanes and chrysenes. This approach 

has been successfully used to distinguish between biodegradation and the physical or chemical 

loss of oil in recent bioremediation field studies (Bragg et al., 1994; Venosa et al., 1996; Lee et 

al., 1997b). It should also be noted, however, that hopane normalization is most useful for 

reducing the variability associated with heterogeneous oil distribution. The use of hopane 

normalization to distinguish biodegradation from physical loss of oil is valid mostly when losses 

due to dissolution and volatilization are negligible. Biodegradation can also be verified as the 

main removal mechanism by examining the relationships between the degradation rates and the 

substrate structure such as the relative degradation rates for homologous series of alkylated 

PAHs. These relationships, when used in conjunction with other oil analysis data, can be very 

109
useful in accurate assessment of the extent and progress of oil biodegradation (Venosa et al., 

1997a; Wang et al., 1998; See section 3.3). 

In addition to the demonstration of oil biodegradation based on chemical analysis, the 

effectiveness of oil bioremediation can also be verified by monitoring the changes in growth and 

activity of oil degrading microorganisms. Growth of hydrocarbon degrading bacteria can be 

determined by Most Probable Number (MPN) techniques, particularly the procedure proposed by 

Wrenn and Venosa (1996), which can separately enumerate aliphatic and aromatic hydrocarbondegrading bacteria and is simple enough for use in the field. Because many viable 

microorganisms are unculturable (Atlas & Bartha, 1987, Macnaughton et al., 1999), the 

emergent culture-independent molecular techniques, such as the PLFA-DGGE techniques (See 

section 3.1.1.2), are becoming important tools to identify the diversity and composition of 

uncultivated microbial communities and to enumerate bacteria in more precise ways. Other 

useful tools in monitoring biological activities include in-situ measurement of microbial CO2 

production by the use of respirometric or radiorespirometric methods (Sugai et al., 1997; 

Swannell et al., 1997). 

Assessing treatment significance 

To show that a treatment increases the rate of oil biodegradation, the concentrations of the target 

analytes (e.g., hopane normalized total resolvable alkanes or aromatics) should be significantly 

lower in treated than untreated areas within the time frame of bioremediation applications (e.g., 

several months to a year). Convincing demonstration of an increased rate of oil degradation 

requires taking a sufficient number of true replicate samples that are randomly interspersed 

throughout the sampling domain (see section 5.4). The field results should then be interpreted 

using proper statistical analysis (Venosa et al., 1996), including analyzing field data using 

standard statistical models and analysis of variance (ANOVA) techniques, which are usually 

done at each sampling event. In addition to the ANOVAs, the entire data set should be analyzed 

by non-linear regression analysis to estimate the rate of decline in biodegradable analytes for 

each treatment over the entire course of the bioremediation treatment. As demonstrated in the 

Delaware field trial (Venosa et al., 1996), the interpretation of treatment effectiveness will be 

affected by reaction kinetics, background nutrient concentration, the variance of analytical 

results, the number of replicates, and statistical significance required to demonstrate the 

differences between treated and control areas. 

An example of a likely treatment effect under different kinetic conditions is shown in Figure 6.1. 

The performance of oil biodegradation expected on an oiled shoreline can be estimated based on 

the first-order kinetic models described in Section 2.5.3. In the Delaware field study, first-order 

biodegradation rate coefficients ranging between 0.026 and 0.056 day-1 for total resolvable 

alkanes and 0.021 to 0.031 day-1 for total resolvable aromatics were observed (Venosa et al., 

1996). These first-order biodegradation rate coefficients are also a function of the nutrient 

concentration (See equation 2.4). The half-saturation concentration Kn for nitrate is 

approximately 0.5 mg N/L (Boufadel et al., unpublished). In this example, the background 

nitrogen concentration is assumed to be 0.1 mg N/L. The assumption of low background nutrient 

concentrations is reasonable since this is one of the prerequisites that bioremediation actions 

should be selected. Two cases of oil biodegradation rates (i.e., 0.026 and 0.056 day-1) in the 

treated areas are examined, which covers both the high (treated) and low (natural attenuation) 

110
ends of what was observed in the Delaware study (Venosa et al., 1996). A theoretical control rate 

of 0.0093 day-1 was assumed based on a background pore water nitrogen concentration of 0.1 

mg/L and a Kn of 0.5 mg/L. This is the natural attenuation rate that would have occurred had the 

background nitrogen concentration been 0.1 mg/L rather than the actual 0.8 mg/L. 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

0 10 20 30 40 50 60 70 80 90 100 

ktreat2 = 0.056 day

-1 

kcontrol = 0.0093 day-1 

ktreat1 = 0.026 day

-1 

Time (days) 

Figure 6.1 Influences of biodegradation rates a on detectability of treatment effect 

It can be seen from Figure 6.1 that differences between treated and control plots are prominent 

very early in the project period when first order decay coefficient was 0.056 day-1. Differences 

were also evident when the decay rate was only 0.026 day-1, but they were not as prominent. 

Thus, had the background nitrogen concentration been closer to 0.1 mg/L, the overall conclusion 

from the study would have been to recommend bioremediation since a substantial enhancement 

would have been evident with nutrient addition. This would have been true even considering the 

aromatic fraction, which had a lower decay rate but still higher than the theoretical control. 

To determine the effect of variance on the ability to detect differences between treatments, 

Figure 6.2 was developed. This figure was based on the power analysis conducted by Venosa et 

al. (1996). Using 5 replicates and at a statistical power of 80% (i.e., the probability that 

significant differences between two or more treatments are detected when they actually exist), 

the minimum detectible difference clearly increases linearly with variance. Thus, if the variance 

doubles, the ability to detect a difference between treatments lessens inversely. Figure 6.3 was 

developed to show how the minimum detectable difference varies at a constant variance but at an 

increasing number of replicate plots. Obviously, the fewer the number of replicate plots 

established, the more difficult it is to detect statistically significant treatment differences. These 

figures point out why it is so important to minimize variance by hopane normalization and 

increasing the number of replicate plots. 

Total Alkanes (

n

g/ng hopane) 

111
Figure 6.2 Minimum detectible treatment difference as a function of variance. 

150 

140 

130 

120 

110 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 2 4 6 8 10 12 

number of replicates 

minimum detectable difference, ng/ng hopane 

Figure 6.3. Minimum detectible treatment difference as a function of number of replicate plots. 

112
6.1.2 Assessment of physical loss 

To better evaluate oil bioremediation performance, one should be able to distinguish between 

physical loss and biodegradative loss. This requires a mass balance. However, little information 

is available in this regard for most oil bioremediation field studies and applications due to the 

lack of comprehensive monitoring programs, reliable measurement tools, and proper data 

interpretation. 

So far, the most complete mass balance of any major oil spill was the Exxon Valdez incident. In 

the early 1990s, Wolfe et al. (1994) undertook a comprehensive monitoring program. They 

estimated that about 3 years after the spill, approximately 20% of the spilled oil had evaporated 

and undergone photolysis in the atmosphere; approximately 14% was recovered and disposed of; 

approximately 2% remained on intertidal shorelines and 13% in subtidal zones. Approximately 

30% of the spilled oil was biodegraded in the water column, and nearly 20% was biodegraded on 

the shorelines. However, the paper did not provide a mass balance for the 113 km of shorelines 

in Prince William Sound where bioremediation applications (nutrient additions) were performed. 

A methodology was proposed by Venosa et al. (1996) to conduct a mass balance and to 

distinguish biodegradation from physical loss of oil in the Delaware field study. As mentioned 

earlier, spilled oils can be lost from a shoreline by physical washout, dissolution, volatilization, 

and biodegradation. The method assumes that a nonbiodegradable component of oil (namely, 

hopane) can be used to estimate the first three loss rates, and that the actual biodegradation rate 

of an analyte can be estimated from the difference between its total loss rate and its physical loss 

rate. For this approach to be valid the physical washout rate of the oil must be dominant, and the 

oil loss due to dissolution and volatilization must be negligible. Volatilization and dissolution 

can cause some preferential loss of oil components particularly in the early stage of an oil spill 

(see Chapter 2). However, these factors are unlikely to be important during the bioremediation 

treatment since bioremediation is typically used as a polishing step after conventional 

mechanical cleanup options have been applied and is often initiated weeks to months after an oil 

spill. Wolfe et al. (1994) reported that evaporation was no longer an important loss mechanism 

three months after the Exxon Valdez spill. 

Figure 6.4 shows the overall first order disappearance of hopane and total extractable organic 

matter (EOM) based on the results of the Delaware study. Over 90% of the spilled oil was 

removed from the shoreline through physical washout based on the rate of hopane loss. The 

EOM first-order rate coefficient was higher than the hopane disappearance rate. The difference 

in loss rates between hopane and EOM was attributed to biodegradation because EOM includes 

both biodegradable and nonbiodegradable components. However, because EOM and other total 

petroleum hydrocarbon (TPH) measurements are not sensitive enough, no differences between 

bioremediation treatments and control could be determined using this approach in the study. This 

observation suggests that losses due to bioremediation may not be detectable using TPH analysis. 

This again demonstrates that the success of bioremediation should be judged by analyzing 

variables of biological significance, such as the reduction of concentrations of oil components of 

ecological concern (e.g., PAHs), toxicity of the oil, and ultimately the ability to accelerate the 

recovery of the oil contaminated ecosystem. 

113
1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

0 10 20 30 40 50 60 70 80 90 100 

Hopane: k = 0.025 

EOM: k = 0.034 

Physical loss of oil 

Total oil loss 

Biodegradation 

Time, days 

Figure 6.4 Fate of oil disappearance from shoreline during the Delaware study 

Another important consideration in assessing physical loss of oil and bioremediation success in a 

marine shoreline is the effect of shoreline substrate transport. Suidan and Wrenn (2001) found 

that substantial sand transport occurs over short time scales on marine shorelines, particularly on 

high-energy beaches, although the net amount of shoreline sediment did not change significantly 

during the tracer study. Sediment transport will affect oil transport since oiled sand will move 

with the bulk sand. As discussed in Section 3.2.2, the physical redistribution of oil between the 

inside and outside of experimental plots can affect interpretation of results from bioremediation 

field studies that are conducted on shorelines contaminated by real oil spills. Studies of this type 

often involve treatment of oiled sand in small plots that are surrounded by large areas of 

untreated, contaminated beach. Because the treated sand will become mixed with untreated sand 

from surrounding areas of the beach by wave-induced sediment transport, the apparent 

effectiveness of the treatment will be reduced. Hopane normalization cannot correct for this 

underestimation of the treatment effect since the hopane concentration inside the plots will not be 

affected by sand transport. However, this effect will be less important in cases of an actual 

bioremediation application when the entire contaminated shoreline is treated. The potential effect 

of shoreline substrate transport on data interpretation in these cases, however, will be to 

overestimate the treatment effect due to the sand transport from large treated areas to small 

untreated control areas. Therefore, the control area set aside for assessing oil bioremediation 

should be either large enough to reduce this effect or relatively isolated from the treated area to C/C0 

114
minimize the exchange of sand and nutrients between the two areas. The influence of shoreline 

substrate transport should always be taken into account in both the design of the sampling plan 

and the interpretation of data from the field, particularly for high-energy beaches. 

6.1.3 Operational endpoints based on oil biodegradation 

Bioremediation endpoints for many soil and groundwater sites contaminated with petroleum 

products are often selected based on predetermined remediation target concentrations, such as 

10-10,000 mg/kg TPH and 0.1-500 mg/kg BTEX, which are adopted by various regulatory 

agencies (King, et al., 1998; Salanitro et al., 1997). However, for reasons discussed previously in 

regard to the inadequacies of TPH analysis, such targets are not appropriate for protecting the 

environment. It is a good idea, however, for parties involved in a remediation project to have 

some measurable endpoints for management and regulatory purposes. Based on existing 

experiences, the following criteria are suggested in determining bioremediation endpoints with 

respect to oil biodegradation. 

• 	 Bioremediation treatment should be terminated when the extent of oil degradation tends to 

level off based on oil analysis results. Cost-benefit analysis should be used in establishing 

target bioremediation levels. It is unrealistic and uneconomical to remove all traces of oil 

hydrocarbons using bioremediation technologies. 

• 	 The concentrations of target oil analytes can also be used as endpoint indicators, particularly 

when the treatment is highly effective. Emphasis should be given to those chemicals of 

environmental concerns, such as PAHs. The target concentrations should be agreed upon in 

the treatment plan and can be determined based on existing standards used for other 

environments (e.g., oil contaminated surface and subsurface soils, Bell et al., 1994). 

• 	 The change in oil composition may also help to establish the bioremediation endpoint. As oil 

becomes more biodegraded, the fraction of less biodegradable components (e.g., resins and 

asphaltenes) in the oil become enriched. Studies following the Exxon Valdez spill showed 

that oil biodegradation slowed substantially when the polar content of the North Slope crude 

oil reached 60-70% of the total mass (Bragg et al.,1994). Therefore, the polar fraction as a 

percent of the total oil mass remaining may potentially serve as an endpoint indicator 

although more research is still required to establish quantitative criteria. If rapid removal of 

the resin and asphaltene fractions of the oil is the desired endpoint, the only way to achieve 

this is by excavation and hauling to a contained or secure facility. That is because removal of 

these constituents in nature is known to occur only through dispersion and dilution. The rate 

of these processes can be very site specific and depend heavily on the type of substrate (i.e., 

cobble, sand, etc.) and wave energy. 

6.2 Environmental Assessments 

Bioremediation is among the least intrusive of the current operational physical and chemical oil 

spill countermeasure options available. Nevertheless, apprehension remains about the 

environmental impact of bioremediation agents released into the environment (Hoff, 1993; 

Holloway, 1991; Lee et al., 1995a; Office of Technology Assessment, 1991). In addition to 

115
potential effects on wildlife and humans, there is concern that the by-products of enhanced oil 

biodegradation may be more toxic than the parent compounds. For general acceptance of 

bioremediation as an oil spill countermeasure, we must demonstrate that it does not induce 

negative effects that suppress the rates of natural habitat recovery. Environmental assessment 

should be an integral part of guidelines governing the application of bioremediation treatments to 

ensure protection of the environment. 

Both ecosystem structure and function must be considered in environmental assessments. 

Ecosystem structure is studied by examining species abundance, biomass, and diversity and other 

components at one point in time. Bioassessment field surveys (Chapter 3.5.1) provide this basic 

information on ecosystem structure. Ecosystem function describes the dynamics or changes in 

the system over time. Information on ecosystem function is provided by the quantification of 

rates of biological processes like production, respiration, mineralization, and nutrient 

regeneration. In addition, bioassays (Chapter 3.5.2) provide a means of quantifying the potential 

effects of toxicants on ecosystem function. 

6.2.1 Operational guidance from environmental assessments for treatment application 

Controlled studies suggest that during remedial operations, optimal rates of degradation in 

sediments can be achieved by sustaining elevated interstitial nutrient levels that do not elicit an 

adverse effect (Lee et al., 1993; Swannell et al., 1996; Lee et al., 1997b; Venosa et al., 1996; 

Boufadel et al., 1999b). Bioassessment field surveys can be used to guard against detrimental 

effects such as the stimulation of toxic algal blooms associated with eutrophication. 

Environmental assessments can also provide guidance for bioremediation by pinpointing the 

optimal time for the onset of treatment. It has become clear in numerous studies that treatments 

have limited or no success as long as the residual oil is retaining its most toxic compounds, 

which are typically low molecular weight compounds that are removed through natural 

weathering processes. In such a case, it is better to wait one to two weeks before treatment to 

allow for toxicity levels to decline, which can be delineated by time-series monitoring of 

sediment or water toxicity using bioassays. 

The results of toxicity tests have been used to explain the mode of action and performance results 

of commercial bioremediation agents containing biostimulation (nutrients) and bioaugmentation 

(bacterial inocula) properties. Lee et al. (1997b) observed that increases in microbial activity 

following treatment are not necessarily correlated with toxicity reduction or habitat recovery. 

Natural attenuation was more effective because the bioremediation agent inhibited the physical 

loss of residual oil from the sediments. 

Standard bioassay test protocols are now being developed by regulatory agencies for toxicity 

evaluation of oil spill bioremediation agents (Thomas et al., 1995; Blenkinsopp et al., 1995). 

Field application of bioremediation products should be limited to those that have passed 

regulatory screening procedures for performance and toxicity. For a conservative approach, it is 

recommended that feasibility studies be conducted prior to full-scale operations. This can be 

accomplished by conducting contaminant biodegradability studies in the laboratory and 

monitoring the effectiveness of the proposed treatment on several untreated but oiled shoreline 

116
segments. If the desired treatment endpoint (reduction of residual oil concentration or enhanced 

rate of habitat recovery) has been identified, chemical analysis and bioassays can be used to 

quantify the efficacy of bioremediation treatments. 

6.3 Case Study: Environmental Assessment of Bioremediation Treatments in a Tidal 

Freshwater Marsh 

Since environmental assessment is a relatively new approach in evaluating the effectiveness of 

oil bioremediation treatments, a case study is presented in detail here to help spill responders 

better understand and conduct this type of assessment. To evaluate the efficacy of nutrient 

amendment and phytoremediation as bioremediation strategies, a controlled oil spill field trial 

was recently conducted in a tidal freshwater marsh on a shoreline of the St. Lawrence River, 

Canada to determine if nutrient enrichment would enhance the rates of residual oil loss and 

habitat recovery (Blanco-Garcia et al., 2001b; Venosa et al., 2002; Lee et al., 2001a). The 

experimental design and bioremediation performance with respect to oil biodegradation has been 

reviewed in Chapter 2 (See Section 2.5.2). Environmental assessment of the extent of habitat 

recovery, which included a suite of bioassays for the identification of possible detrimental 

treatment effects (e.g. toxicity of the bioremediation agent or oil degradation by-products), is 

described as follows. 

6.3.1 Alterations in ecosystem structure 

Vegetative recovery of the predominant plant species (Scirpus pungens) was monitored by 

determining changes in species composition (predominant S. pungens and secondary Eleocharis 

palustris), total biomass, height, and percent cover. During the first growing season S. pungens 

tolerated the experimental oil concentrations, but suffered oil-induced growth inhibition (Figure 

6.5), which is consistent with the results of recent greenhouse studies (Longpré et al., 1999). 

The effects of growth inhibition by oil were still evident within natural attenuation plots at Week 

65. The enhanced recovery of the vegetation in nutrient amended plots during the second field 

season could not be attributed to the concentration of nutrients in the porewater, as they had 

diminished to background levels before the previous winter. Analysis of nitrogen content within 

the roots of plants suggested that the abundant growth was attributable to the recycling of 

organic nitrogen stored by these plants over the first season. 

With the reduction of residual oil concentrations (primarily by physical removal) and regrowth 

of the predominant vegetative species, extensive recolonization by indigenous invertebrate 

species such as the mystery snail (V. georgianus) was also observed during the second field 

season in all experimental plots (Week 45). 

117
Nat. 

Attn. 

NH4 

+ NO3 - NH4 + 

Unoiled 

0 

20 

40 

60 

80 

100 

120 

140 

Figure 6.5 Average height of the predominant plant species (Scirpus pungens) at Week 15 in 

three oiled treatments (Nat. Attn., NH4 

+

, NO3 

-

) and the unoiled but fertilized (NH4 

+

) treatment. 

6.3.2 Alterations in ecosystem function 

The environmental impact of contaminants in aquatic ecosystems cannot be assessed accurately 

with a single species bioassay, which cannot represent the range of sensitivity of all biota. 

address this issue, a test battery with organisms from several trophic levels was used: (1) 

microbial response; (2) Microtox solid phase; (3) algal solid phase; (4) cladoceran survival; (5) 

amphipod survival; (6) gastropod survival/histopathology; and (7) acute and chronic effects in 

fish. 

6.3.2.1 Microbial response 

Concentrations of bacteria are up to seven orders of magnitude higher in the surface sediments 

than in the water column. microbial activity makes sediments the most 

active site for transformations of organic carbon, nitrogen, phosphorus, magnesium, and sulfur. 

If the processes of decomposition, mineralization, and nutrient regeneration are disturbed by 

contaminants, the nature of the ecosystem will be changed. portance of these and 

other processes concentrated in the sediments, relatively few assessments have focused on 

functional changes attributable to sediment-associated contaminants. 

Detailed studies on the microbial response to bioremediation treatments within experimental 

enclosures were conducted by Greer et al. (2000). 

showed a slight increase during the first 4 weeks following oil and fertilizer application. 

increase was clearly due to the fertilizer, as evident from the contrasting population densities in 

untreated areas which remained relatively unchanged throughout the monitoring period. Patterns 

of ineralization nd 

microorganisms can be used as an indication of in-situ biodegradation of petroleum (Braddock et 

al., 1996). icrobial populations demonstrated a rapid and sustained increase in 

naphthalene mineralization activity in the plots that were both oiled and fertilized. Activity was 

somewhat lower in unfertilized/oiled and fertilized-only plots. ineralization 

To 

This concentration of

Despite the im

The viable bacterial population density 

The 

hydrocarbon m activity a distribution of hydrocarbon-degrading 

The m

Hexadecane m

118
activity increased in response to fertilizer application, especially ammonium nitrate, in 

comparison to sodium nitrate: activity in the unfertilized/oiled plots and unoiled reference 

control areas remained relatively low. Laboratory assays to monitor various pathways in the 

nitrogen cycle (nitrification, denitrification, nitrogen fixation) have been developed (Pritchard 

and Bourquin, 1985) and can be used to assess changes in function due to contaminant stress. 

Field and laboratory evaluation of nitrogen metabolism indicated significant denitrification 

activity in sediments following fertilizer application, which was not adversely affected by oiling. 

In contrast to the results of chemical analysis that showed no treatment effect on oil 

biodegradation rates, the results demonstrated that the application of fertilizers stimulated the 

activities of indigenous hydrocarbon degrading and denitrifying bacteria, and the presence of oil 

did not have a detrimental effect on these activities. 

6.3.2.2 Microtox solid phase test 

In the Microtox

® Solid Phase Test (Microbics Corporation 1995), the bacterium, Vibrio fisheri, 

was exposed to oiled sediment. A significant decrease in bioluminescence relative to water-only

controls was indicative of sediment toxicity. Toxicity levels were calculated as the concentration of

sample that would result in a 50% reduction in luminescence (‘effective concentration,’ EC50). To 

account for interferences from differences in sample grain size distribution, turbidity, and to a lesser 

extent, color of the sample dilutions, sample test results were compared with results from unoiled 

sediments from the immediate study area. 

Oil toxicity was evident on comparison of oiled with unoiled plots (Figure 6.6). While the 

fertilized plots showed a trend towards a reduction in toxicity, there was no such evidence in the 

natural attenuation plots over 65 weeks. Major treatment effects were not observed in the first 

field season. However, in the second field season the relative toxicity levels in nutrient amended 

plots were similar to values of the unoiled controls. 

6.3.2.3 Algal solid phase assay 

An algal solid-phase assay (ASPA) was used to assess the toxicity of sediments recovered from 

the experimental test plots (Blaise and Ménard, 1998). The endpoint for this assay is based on the 

concentration of sediment causing 50% inhibition (IC50) of esterase enzyme activity in 

Selenastrum capricornutum due to toxicants. This test is an excellent biomarker for 

environmental assessments, as esterases are a key group of ubiquitous enzymes found in both 

plants and animals (Dorsey et al. 1989 ; Gala and Giesy 1990). 

Results of the algal solid-phase assay (Figure 6.7) showed that there was no toxicity in the 

unoiled reference plots throughout the course of the experiment (IC50 > 7%), and that nutrient 

additions to unoiled sediment appear to cause no detrimental effects. For the first 21 weeks, 

elevated levels of toxicity were observed in all the oiled plots. Significant reduction in sediment 

toxicity was not observed until the second field season. At this point, a marked decrease in 

toxicity was observed for the oiled plots amended with nutrients (either ammonium nitrate or 

sodium nitrate). Nutrient amended plots were deemed non toxic by Week 65. While nutrient 

treatments were terminated at the end of the first field season, treatments resulted in significant 

positive effects that were not observed until the second field season. This lag suggests that 

toxicity reduction may be correlated with enhanced vegetative growth associated with nutrient 

enrichment. 

119
30

25 

20 

15 

10 

5 

0 

- Nat. NH4

+ NH4

+ NO3 Unoiled 

Attn. Cut NH4

+ 

Treatments 

Figure 6.6. Time-series changes in sediment toxicity as quantified by the Microtox Solid 

Phase Test. Error bars = 1 standard deviation 

8 

EC50 (ppt) 

012468

1216214965 

012468

12

16214965 

012468

1216214965 

012468

12

16214965 

012468

1216214965 

7 

6 

5 

4 

3 

2 

1 

0 

not toxic 

0 1 6 12214965 0 1 6 1221 49 65 0 1 6 1221 49 65 0 1 6 12 21 4965 0 1 6 12214965 - Nat. Attn. NH4

+

 Cut Plants NH4

+ NO3 Unoiled NH4

+ 

Treatments 

Figure 6.7. Algal solid phase assay (ASPA) as IC50 for sediment samples collected at Weeks 0, 

1, 6, 12, 21, 49 and 65. Bars represent means ± standard deviation. 

120
6.3.2.4 Cladoceran survival test 

The cladoceran Daphnia magna (‘water flea’) was used to assess the toxicity of elutriates from

sediment samples (Environment Canada, 1990). Treatment of unoiled plots with ammonium

nitrate and super triple phosphate appeared to cause negligible impacts on the survival of D. 

magna (Figure 6.8a). In contrast, immediate, but limited, toxic responses were observed in all 

oiled plots. Ammonium nitrate and triple super phosphate additions appeared to reduce residual 

oil toxicity to background levels by Week 2. Amendments with sodium nitrate appeared less 

effective. Since the sensitivity of this assay appeared to be limited — the effect of residual oil 

was deemed negligible by Week 6 — this assay was excluded from the second field season. 

6.3.2.5 Amphipod survival test 

The Amphipod Test measured the effects of sediment samples on survival of sediment-dwelling 

Hyalella azteca neonates, 2-9 days old (Environment Canada, 1997). Both the mean percent 

survival and the mean weight of animals in each treatment were compared with mean percent 

survival and mean weight of amphipods in reference control sediments to determine if the 

treatments caused a significant decrease in organism survival or growth. 

Hyalella azteca mortality (Figure 6.8b) was a more sensitive bioassay endpoint (i.e. higher 

response) than Daphnia mortality (Figure 6.8a). This may be due to species differences and the 

fact that the Amphipod Survival Test is a direct-contact sediment test. Oil derived toxicants 

within the sediment may not have been effectively transferred to the elutriate used in the 

cladoceran test. Furthermore, in a laboratory test, the uptake of sediment-associated anthracene 

by the freshwater amphipod Hyalella azteca was reported to occur at a rate much higher than 

predicted. It was concluded that selective feeding of H. azteca on smaller particles results in a 

diet of fines containing the highest organic matter concentration and, hence, contaminant levels 

(Landrum and Scavis, 1983). H. azteca mortality was consistently lower in the unoiled nutrient 

amended plot. High mortality was observed during the first week in all oiled plots. The presence 

or absence of plants appeared to have no significant effect. 

At the latter part of the first field season there appeared to be a pronounced increase in sediment 

toxicity in the oiled plots amended with prilled ammonium nitrate. The sensitivity of H. azteca 

to ammonia (LC50 = 14.9 mg-N/L) was verified in the laboratory. Many samples, notably 

Weeks 12 and 21, had overlying water ammonia levels exceeding the established toxicity limits, 

with the highest values corresponding to the oiled plots amended with ammonium nitrate. The 

fertilized unoiled site had lower elevated ammonia levels. This and a possible synergistic effect 

between oil and elevated NH4

+

 concentrations may explain the observation of little or no toxic 

response observed in the fertilized unoiled plots (Figure 6.8b). In response to a reduction in 

residual oil concentrations, percentage mortality was near background levels by the second field 

season. The results of this bioassay suggested that the experimental treatments offered little 

advantage on an operational scale. 

121
50 

40 

30 

20 

10 

0 

120 

100 

80 

60 

40 

20 

0 

b) 

0126 12 21 49 65 0 126 12 21 49 65 01 26 12 21 49 65 01 2 6 12 21 49 65 012 6 12 21 49 65 

- Nat. NH4+ NH4+ NO3 Unoiled 

Attn. Cut NH4

+ 

Treatments 

Figure 6.8 Time-series changes in sediment toxicity as quantified by: a) Cladoceran Survival 

Test; b) Amphipod Survival Test. Error bars = 1 standard deviation 

6.3.2.6 Gastropod survival/histopathology 

Although many organisms have been used as sentinels or biomonitors of environmental 

contaminants (LeBlanc and Bain, 1997), there is still a need to identify and exploit alternative 

species that are sensitive and amenable to ecotoxicological testing. Molluscs are abundant and 

widely distributed and their use as in situ biomonitors has been on the rise (Lagadic and Caquet, 

1998). In this study (Lee et al., 2001b), gastropod survival and histopathology assays were 

conducted with the mystery snail, Viviparus georgianus. They were specifically selected for use 

as an in situ biomonitor as they feed on sediment detritus, algae and decaying organic matter 

within the wetland. Snails (n = 50/treatment/sampling time) were caged within 20 × 20 × 22 cm 

open mesh polypropylene baskets moored to the sediment surface of experimental plots, and in 

designated ‘untreated control’ areas within the vicinity of the plots. Cages were recovered after 

30, 60 and 90 days of exposure to evaluate effects on survival at the end of the second field 

% M

ortality 

% M

ortality

a) 

122
season (Week 65). Healthy snails were also exposed for a 30 day period to test sediments 

recovered from the plots, under laboratory conditions, to determine survival rates. 

Significant growth (p < 0.001) of the snails was observed during the study, but no significant 

differences (p > 0.05) were observed in tissue weight, shell size or shell thickness between the 

experimental treatments. Under adverse conditions V. georgianus is known to retract into its 

shell and show no motility for prolonged periods, so animal vitality was assessed by the presence 

of an operculum. No surviving snails were found in any of the continuous exposure cages after 

three months (Figure 6.9). To factor out the influence of stress from the caging of animals for 

extended periods, data at each sampling event were normalized to unoiled control plots adjacent 

to the test blocks, which received no oil or nutrient amendments (4%, 47% and 100% mortality 

at 30, 60 and 90 days, respectively). The only oiled treatment that gave a higher percentage 

survival after 30 days exposure was the ammonium nitrate amended plots with intact plants. The 

other treatments were more toxic than the natural attenuation plots. Elevated nutrient 

concentrations for 60 days did not reduce the toxic effects of the oiled sediments to V. 

georgianus. Indeed, exposure to the treatment probably exacerbated stress. 

100 

75 

50 

25 

0 

NH4 

NH4 

+ 

NO3 

-

Unoiled, NH4 

+ 

Unoiled 

Natural Attenuation 

+, Cut Plants 

30 60 

Exposure Time (days) 

90

Figure 6.9 Percent survival of the mystery snail, Viviparus georgianus, following exposure 

to test treatments for 30, 60 and 90 days. Results normalized to survival in unoiled sediment 

without nutrient amendment. 

Toxicity of nitrogenous compounds at high levels has been noted with various organisms. 

Among invertebrates, ammonia toxicity was assessed with a freshwater snail (Hickey and 

Vickers 1994). Acute values were derived for the snail to be 0.15 g/m ammonia compared to the 

EPA value of 0.52 for salmonids, and they found that the snail was more sensitive than the 

normally accepted sensitive species such as mayflies and stoneflies. After 30 days of continuous 

exposure, the highest mortality was reported in the fertilized plots with plants cut back. Direct 

observations suggested that these animals suffered from harsh conditions in the absence of 

natural ground cover. All treatments containing nutrients with or without oil caused higher 

mortalities than no oil or oil alone. Laboratory exposures using sediment recovered from the 

experimental plots at Week 65 (which had less oil, and of lower bioavailability) showed that the 

application of treatments in the first season had insignificant long-term effects (Figure 6.10). 

123
Based on the results of the amphipod and snail bioassays, bioremediation using nutrients in situ 

will need to take the above into consideration. 

100 

80 

60 

40 

20 

0 

Nat. NH4

+ NH4+ NO3- Unoiled 

Attn. Cut NH4

+ 

Figure 6.10 Percent survival of the mystery snail, Viviparus georgianus under laboratory 

conditions following 30 days of exposure to sediments sampled at Week 65. Error bars = 1 

standard deviation 

When organisms are exposed to xenobiotics, cellular changes have been observed to occur. 

Evaluation of histological changes can provide important information as to the stress of the 

organism and mode of action of pollutants. Specimens of live snails were preserved for 

histological analysis. All tissues showed degenerative changes over time in snails exposed to 

fertilizers. Pathological changes were most obvious in epithelial cells lining such tissues as gills, 

intestine and digestive gland, but some degenerative changes were also observed in other organs 

such as gonads. The most dramatic changes were observed in snails from plots treated with 

sodium nitrate (Figure 6.11). Effects of treatment on reproductive success of V. georgianus were 

noted. Gravid females contained on average 10 young (range 3 to 15) visible with the naked eye. 

At 1-week post oil exposure, gravid females showed some degenerating embryos in all but the 

control treatments. This was also true for some of the 1-month post exposure gravid females, but 

number of gravid females with macroscopic embryos decreased. 

6.3.2.7 Acute and chronic effects on fish 

Experience with the Exxon Valdez demonstrated that oil deposition in shoals where pink salmon 

(Oncorhynchus gorbuscha) and Pacific herring (Clupea pallasi) spawn, caused blue sac disease 

(BSD) of newly hatched larvae (Hose et al., 1996; Marty et al., 1997). BSD is characterized by 

yolk sac and pericardial edema, hemorrhaging, deformities, and induction of mixed function 

oxygenase enzymes (Marty et al., 1997; Billiard et al., 1999; Fragoso et al., 1998). As a 

consequence, there is considerable interest in the application of bioremediation strategies that 

accelerate the removal of oil from intertidal beaches, which provide the nursery habitat for many 

fish species. The success of these technologies should be judged not simply on how quickly oil 

disappears, but on a demonstrated reduction in risk, i.e. on how quickly toxicity (hazard) and 

exposure to oil are reduced. % S

urvival 

124
a 

b 

Figure 6.11. Representative sections through the intestine of Viviparus showing normal villi 

seen in control snails (a) and degenerated villus with hemocytic infiltration seen in snails from 

the sodium nitrate treatment after 1 month exposure (b). Bar = 75 μm. 

Establishing the exposure of fish to oil is difficult. While chemical measures of oil in sediments, 

water and tissues are routine, there is no guarantee that fish accumulate oil or its components 

equally or in proportion to environmental concentrations. Further, many of the components of 

oil (e.g. alkanes, polynuclear aromatic hydrocarbons (PAH)), are metabolized by fish, so that 

chemical analyses of fish tissues may not represent the true dose or dose rate. 

Assessing hazard is equally difficult. Oil is a complex mixture, and many of its components 

have different modes of toxicity. At acutely lethal concentrations, mortality is rapid and is likely 

due to narcosis caused by monoaromatics (benzenes, toluenes, xylenes, etc.) and alkanes. At 

lower concentrations (Billiard et al., 1999), chronic toxicity (BSD) may be linked to the 

concentrations of alkyl PAH such as 7-isopropyl-1-methylphenanthrene (retene, a C-4 

phenanthrene). This mechanism may involve metabolism of alkyl PAH to more toxic forms by 

CYP1A enzymes. Toxicity and rate of excretion of phenanthrene and retene can be modulated 

by inhibiting or inducing CYP1A activity (Hawkins et al., 2000). Delayed responses, such as the 

long-term onset of cancer, may also follow brief exposures to pro-carcinogens such as 

benzo(a)pyrene (BaP). In this case, the mechanism involves oxygenation of BaP by CYP1A 

enzymes to carcinogenic diols and epoxides, which cause genotoxicity by forming adducts with 

bases of DNA (Varanasi et al., 1989). 

In this case study, bioassays of CYP1A induction in juvenile rainbow trout (Oncorhynchus 

mykiss), BSD in embryo-larval stages (ELS) of trout, and reproductive developmental studies 

125
with Japanese medaka (Oryzias latipes) were used to evaluate the presence, bioavailability, and 

toxicity of PAH (and parent mixture of oil) in contaminated beach sediments (Hodson et al., 

2001). Changes in bioavailability and toxicity with time were monitored by analysis of timeseries samples. 

The key to sediment assessment is bioavailability. Although sediments might contain relatively 

high concentrations of toxic compounds, this condition does not necessarily lead to adverse 

effects on organisms living in the sediments (Payne et al., 1988). The only means of measuring 

bioavailability is by measuring or determining biological response. Such testing has often 

involved measures of bioaccumulation (the ability of an organism to accumulate contaminants in 

tissues. However, because bioaccumulation is a phenomenon, not an effect (and can be 

relatively expensive due to costly chemical analyses), emphasis has shifted towards indicative 

endpoints that are based on sediment toxicity tests, which are effects-based and relatively 

inexpensive. 

Bioavailability was assessed by the extent of CYP1A induction in fingerling trout after a 4 d 

exposure to sediments. S-9 fractions were prepared from liver homogenates and activity of 

ethoxyresorufin-o-deethylase (EROD, CYP1A enzyme) was measured by a kinetic microplate 

fluorescence method following the protocols outlined by Hodson et al. (1996). Each bioassay 

included negative controls (water only), unoiled sediment controls, and positive water controls 

(fish exposed to ß-naphthoflavone, a model inducer). Results indicated that one day after oiling, 

EROD activity of fish exposed to oiled sediments was on average 25-fold higher than that in the 

control sediments (range = 13-35-fold). Over a 17 month period, there was about an 80% 

decline in induction potency by oiled sediments sampled from the beach. The decline in EROD 

activity paralleled declines in total hydrocarbons, total PAH, and total alkyl-PAH, measured by 

GC/MS with correlation coefficients between log of EROD activity and log of hydrocarbon 

concentrations of 0.96 or higher (Hodson et al., 2001). 

Blue sac disease of trout was assessed by exposing 50 eyed-eggs (about 15 d post fertilization) to 

sediments until they had hatched, resorbed the majority of their yolk sac, and begun to swim up 

(Zambon et al., 2000). After 32 d of exposure, larvae were removed and the % survival and 

prevalence of the symptoms of BSD (edema, hemorrhaging, deformities) were measured 

(Guiney et al., 1997). Results showed that trout embryos and larvae exposed to oiled sediments 

from Ste. Croix exhibited a low prevalence of symptoms of BSD, although rates were higher 

than observed for fish exposed to the reference sediments. All oil-exposed fish examined 

histologically demonstrated intense staining for CYP1A protein, indicating a significant 

exposure to CYP1A-inducing compounds such as PAH. These preliminary results suggested 

that symptoms of BSD were more frequent in fish exposed to oiled than to un-oiled sediments, 

indicating a risk to early life stages of species that spawn on tidal beaches. 

Medaka larvae exposed to oiled sediments for 90 days (maturity) experienced a higher mortality 

rate (42.5± 4.2%) than those exposed to un-oiled sediments (9.1±7.4). The growth of surviving 

medaka in the oiled sediment treatments was impaired (Figure 6.12), as shown by their smaller 

size compared to controls (20.6±1.3 vs 16.4±3.3 mm). In addition, nearly all medaka from the 

oiled sediment treatments had deformed or missing fins. Preliminary histological examination of 

126
fixed medaka indicated a degenerative liver lesion, spongiosis hepatis, in >95% of the fish from 

the oiled-sediments, but not in controls. There was also a low incidence of male medaka (<10%) 

with intersex gonads (testis-ova) in fish exposed to oiled sediments. 

24 

20 

16 

12 

U n -o ile d O ile d S e d im e n t

W a t e r s e d im e n t (E ) (P o o le d A-D) 

±S t d . D e v . 

±S t d . E r r . 

Me a n 

a 

a 

b 

Figure 6.12. Effect of exposure to Ste. Croix oiled sediment (Oct. 1999) on growth of medaka. 

Treatments sharing the same letters are not statistically different. 

The laboratory fish bioassay represents a ‘worst-case’ scenario as the fish could not avoid 

exposure. Furthermore, mixing of the sediments in the test chambers might have disrupted the 

top-most sediment layer (that might have been depleted of oil in the field by weathering) and 

exposed more oily layers of sediment. The ratio of water to sediment is also fixed, which is very 

different from the situation in well-flushed tidal beaches. Finally, the test organisms were not 

beach spawning species of fish such as smelt, capelin, and herring. 

Semi-permeable membrane devices (SPMDs) were used to assess in-situ changes in the 

bioavailability and toxicity of residual oil components following treatment. These passive in-situ 

samplers, developed by Huckins et al. (1990), contain purified triolein, a substance that 

constitutes a major fraction of the neutral lipid of fish. When immersed in water, SPMDs absorb 

non-ionic, organic chemicals having a log Kow>1, a size <1 Å, a molecular weight of about 600 

or less, and possibly neutral organo-metal complexes. These characteristics correspond to that of 

known mixed-function oxygenase (MFO) inducing compounds, including PAHs. The 

operational advantage of SPMDs is that they can be deployed within the environment to provide 

an integrated sample over time. This characteristic makes the assay highly advantageous for 

field use. In terms of ecological relevance, the diffusion of dissolved neutral organic chemicals 

into the triolein through the pores of the polyethylene membrane within SPMDs simulates the 

diffusion of compounds across a live fish gill membrane. The lipid can be analyzed by 

traditional chemical techniques to provide a list of chemicals absorbed, their concentrations in Total Length (mm) 

127
the SPMDs, and, by back calculation, their concentrations in water or sediment. In this study 

SPMDs were used as concentrating devices for biological testing with the Microtox Assay. 

SPMD units (12.5 cm) enclosed in protective cases were deployed at the water sediment 

interface at predetermined intervals and recovered for analysis after one week of exposure. 

SPMD concentrated samples were diluted with organic solvents for toxicological analysis by 

Microtox. The results showed that by 4 weeks, the toxicity of sediments within the oiled plots 

had declined to the levels of the unoiled but fertilized control as the result of a reduction in 

bioavailability (Johnson et al., 2000). 

6.4 Ecotoxicological Tests for Risk Assessment 

The application of in-situ bioremediation operations is not expected to generate a large volume 

of waste materials like ex-situ operations do. If the program is effective, any residual 

hydrocarbons will elicit little or no biological effect due to physical, chemical, and biological 

processes that reduce their bioavailability. As illustrated in the case study, sediment quality can 

be assessed by a number of methods that tend to fit into one of five categories: sediment 

chemistry, sediment toxicity, community structure, tissue chemistry, and pathology. Ideally, all 

five components would be utilized to assess sediment quality. However, in reality, 

environmental managers are faced with limitations in both resources and time. They must 

optimize use of their resources by selecting the information that will have the greatest utility. 

Thus, monitoring programs should be focused on the measurement of variables that allow 

quantification of treatment success against a pre-defined endpoint. Project coordinators must 

strive to strike a balance between level of effort and type or quality of information needed to 

make effective decisions. 

While detrimental effects have not been linked to the application of bioremediation strategies 

based on nutrient enrichment in actual spill response operations (Mearns et al., 1997; Prince, 

1993), the results of recent field trials clearly demonstrate that the possibility exists. In the case 

study presented (Section 6.3), improper application of bioremediation agents (the addition of 

excess fertilizer) was detrimental to the environment. For example, synergistic effects between 

ammonia and the test oil were observed in the Amphipod Survival Test. However, the nutrient 

additions had no effect on the unicellular algal species, Selenastrum capricornutum, and actually 

enhanced the growth and productivity of the dominant plant species within the oiled plots 

relative to those that received no treatment. Rapid recovery of vegetation is critical within an 

impacted wetland for erosion control. Depending on the desired endpoint of the remedial 

operation, a balance must be made among the many positive (e.g. enhanced recovery of 

vegetation) and negative effects of treatment (e.g. changes in productivity, species composition, 

and diversity among the remaining wetland plants). 

With refinement, bioassays can be used in oil spill operations to provide real-time guidance to 

the treatment operations (e.g. determining the optimal nutrient concentration that does not elicit a 

detrimental effect), to verify the success of countermeasures and to quantify the extent of habitat 

recovery. The demonstration of species dependent responses in previous investigations and the 

current case study suggest that future environmental risk assessments be based on a multispecies, multi-trophic level test battery approach. The results of the ecotoxicological tests should 

128
be used to build an ecological risk assessment — an estimate of the probability of harm to the 

aquatic environment derived from the synthesis of results of separate exposure and effects 

components in a scientific manner (Gentile et al., 1989). It is often stated that the objective of 

oil spill countermeasures are to return an impacted site to its immediate pre-spill condition. This 

is an unrealistic goal as the environment is a dynamic rather than a static system. On an 

operational scale, our goal should be to return the structure and function of an ecosystem to 

within the limits of pre-defined, acceptable criteria. 

6.5 Ecotoxicological Tests to Identify Operational Endpoints 

The effectiveness of oil spill countermeasures ultimately must be judged by their ability to 

reduce injury to aquatic life. The worse case scenario would be the use of a response method 

that is not effective in reducing exposure and increases injury to aquatic life. In bioremediation 

operations the application of ecotoxicological monitoring protocols may be used to verify the 

efficacy for toxicity reduction over that of no treatment. 

Bioremediation treatments should be terminated when it is deemed that: (1) treatments offer no 

operational advantage over natural recovery, (2) the contaminant concentrations and toxicity 

values are reduced to acceptable levels, or (3) detrimental effects from the treatment strategy are 

identified. Cost-benefit analysis should be considered in the decision of the acceptable level. It 

is futile to expect bioremediation techniques to remove all traces of residual hydrocarbons. In 

terms of ecological relevance, declaration of habitat recovery can be made when toxicity limits 

are within regulatory guidelines and there is evidence for the return of the original community 

structure (Lee et al., 1995b; Mearns et al., 1995). 

129
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